Gene expression profile in keratinocytes from atopic

Transcript

EDITORIALS
G ITAL DERMATOL VENEREOL 2005;140:459-60
Gene expression profile in keratinocytes
from atopic dermatitis patients.
The perception of a basic scientist
I. BERTINI, A. QUATTRONE
H
igh density microarray technology represents the
first and more powerful instrumental tool of the
new post-genomic science, systems biology, and is
expected to provide a useful new method of analysis
for the current open problem of medical genetics, the
identification of the genomic lesions underlying susceptibility to complex diseases. The study by Pastore
et al. reported in this issue of Giornale Italiano di
Dermatologia e Venereologia represents a paradigmatic example of the power and limitations of this
approach, with the description of its application to
atopic dermatitis (AD). AD is a chronic pruritic disease
of the skin belonging to the group of allergic disorders (which includes asthma, allergic rhinitis and food
allergy) with a large prevalence, up to 15% among
children of western countries.1 A complex interaction
between environmental factors and genetic susceptibility results in the clinical expression of the disorder,
with genetic influence playing a pivotal role.2 Segregation analysis in families predicts the presence of a
few genetic lesions with moderate effects, even if standard positional cloning approaches have proven to be
unsuccessful until now in the identification of AD susceptibility genes, as happened for asthma. The alternative classical genetic approach to complex diseases,
association with candidate susceptibility genes, has
IN THIS ISSUE SEE PAGE 475
Address reprint requests to: Prof. I. Bertini, Centro Europeo di Risonanza
Magnetica e Dipartimento di Chimica, Università degli Study of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino (Italy). E-mail:
[email protected]
Vol. 140 - N. 5
Magnetic Resonance Center, Department of Chemistry
University of Florence, Florence, Italy
produced a list of about 2 dozens of possibly related
polymorphic variants1 but is generally affected by an
high degree of uncertainty, mainly deriving from the
average small size of the studied populations which
implies low statistical power. In parallel with genetic
studies, clinical and molecular analysis of AD pathology has evidenced the key role played in the disease mechanisms by epithelial skin cells, especially epidermal keratinocytes, with an abnormal pattern of production of
cytokines and chemokines which could trigger an sustain
chronic inflammation. The difficulties in the traditional
genetic analysis and the identification of a specific hystological compartment primarily responsible for the disease effects make of AD an ideal subject for expression
profiling, which, applied on a pure population of cells,
is expected to provide valuable information on the second level of analysis of a perturbed cellular network
after the genome, the population of cellular mRNA transcripts or transcriptome.
Pastore et al. have applied to the expression profiling of AD epidermal keratinocytes a careful experimental design, involving multiple sampling with
individual analysis of diseased and normal skin biopsies, the isolation of pure populations of epidermal
keratinocytes and the use of the available gold standard technology for microarray analysis, the
GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA
459
BERTINI
GENE EXPRESSION PROFILE IN KERATINOCYTES FROM ATOPIC DERMATITIS PATIENTS
tic samples, and in the of complex diseases as AD
could be an effect of the presence of different clusters
of genomic lesions in different affected individuals.
Other expression profiling approaches to AD pathogenesis have reported recently such individual variability.3, 4 Overall, the study by Pastore et al. underscores the complexities intrinsic to the application
of systems biology-based approaches to the study of
diseases, like AD, in which environmental factors
interact differentially in different individuals to produce and sustain the pathological state.
While suggesting caution in the interpretation of
the data coming from this type of studies, we have to
stress that these new genome-based methods are the
only possible alternative to the classical genetic analysis for complex diseases, and have already provided
fundamental information greatly helping in the identification of the subtle genetic lesions underlying susceptibility to these diseases.
M
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Affymetrix system. As anticipated, their conclusions
are important both for the deriving clues on AD pathogenesis and for the critical points emerged. About
200 genes (1.6% of the total analyzed) were found to
be significantly differentially expressed in the analysis, with an equal distribution in upregulated and
downregulated genes. They were clustered by function in transcription factors, signal transducers, cell
cycle regulators, enzymes involved in various cell
activities. The known activities of these genes could
suggest as a signalling lesion up-regulation of the
pathway leading to activation of the AP-1 transcriptional complex, but subsequent validation of 10 differentially expressed genes by real-time RT-PCR, a
methodology routinely used to confirm microarrayderived quantitative determinations, questioned the
profiling results. Of the 10 verified genes, the authors
obtained opposite results for 2, undetectability for
other 2 genes and variable interindividual levels for
5 genes, therefore confirming only 1 gene, cathepsin
O, out of the 10 tested. This marked lack of concordance between the 2 methodologies could be at least
partially due to the average absolute low expression
levels of the genes detected as changed by microarray profiling, in a window potentially affected by
instrumental noise. This problem could be overcome
by the use in microarray profiling of stimulated keratinocyte cultures, which should enhance possible
differences in the genetic programs leading to the
establishing of chronic inflammation. The individual
variability is instead a well-known factor affecting
expression profiling experiments, especially in biop-
460
References
1. Hoffjan S, Epplen JT. The genetics of atopic dermatitis: recent findings and future options. J Mol Med 2005;83:682-92.
2. Abramovits W. Atopic dermatitis. J Am Acad Dermatol 2005;53 (1
Suppl 1):S86-93.
3. Sugiura H, Ebise H, Tazawa T, Tanaka K, Sugiura Y, Uehara M et al.
Large-scale DNA microarray analysis of atopic skin lesions shows
overexpression of an epidermal differentiation gene cluster in the
alternative pathway and lack of protective gene expression in the
cornified envelope. Br J Dermatol 2005;152:146-9.
4. Nomura I, Gao B, Boguniewicz M, Darst MA, Travers JB, Leung
DY. Distinct patterns of gene expression in the skin lesions of atopic
dermatitis and psoriasis: a gene microarray analysis. J Allergy Clin
Immunol 2003;112:1195-202.
Ottobre 2005
Hemangiomas of infancy: to treat or not to treat
R. RUIZ-MALDONADO
I
n this issue of the Italian Journal of Dermatology, C.
Gelmetti, I. Moglia and L. Restano publish their experience in the characterization of hemangiomas and vascular malformations using magnetic resonance imaging
(MRI) in 20 pediatric patients. Their conclusion is that
“MRI alone is not sufficient to provide the necessary
information for the diagnosis and treatment of all vascular anomalies”. Another conclusion could be that for
the time being, the best available technology needs to be
complemented by excellent clinical knowledge in order
to give the patient the best possible treatment.
Clinical knowledge and clinical-pathological correlation have provided the basis for the understanding we have at present of vascular anomalies. It is not
widely known, but at the Pediatric Dermatology
Department of the National Institute of Pediatrics in
Mexico City, the concept of vascular new growth and
vascular malformation was currently in use in the seventies, last century.
In 1980, 2 years before Mulliken and Glowacki´s
publication on the classification of vascular lesions,1
in the book “Themes of Pediatric Dermatology” we
wrote: “Two are the types of blood vessels tumors
most frequent in children: vascular new- growths and
vascular malformations”.2
Vascular new-growths are currently known as hemanIN THIS ISSUE SEE PAGE 491
Address reprint requests to: R. Ruiz-Maldonado, MD, Professor of
Dermatology and Pediatric Dermatology, Department of Dermatology,
National Institute of Pediatrics, Mexico City, Mexico. E-mail: [email protected]
Vol. 140 - N. 5
Department of Dermatology, National Institute of Pediatrics
Mexico City, Mexico
giomas of infancy or infantile hemangiomas and are
characterized by post-natal development, and initial
rapid growth followed by spontaneous regression during the first years of life. This simplistic description of
the natural history of the most frequent benign tumor in
childhood was first published by Lister in 1938.3 For
more than 50 years this paradigm has been at the origin
of frequent parental dissatisfaction and treating physician frustration. Hemangiomas of infancy do not always
behave by the book. They may be present at birth, do not
regress when they should, and have a number of local
and systemic complications that may require early recognition and prompt intervention.
Even in relatively small hemangiomas that comply
with their expected evolution, the fact that the face is
their most common location requires tons of patience
to explain the parents that the tumor will regress and
that the final cosmetic result will be good. Parents are
often not fully convinced and look for remedies, sometimes given by healers, that may have regrettable consequences.
To treat or not to treat an uncomplicated hemangioma of infancy should be based not only in the personal opinion of the specialist that may be a qualified
pediatrician, dermatologist, surgeon etc. Whenever
possible the decision should be additionally supported by evidence based medicine, meaning by that “The
461
RUIZ-MALDONADO
HEMANGIOMAS OF INFANCY: TO TREAT OR NOT TO TREAT
conscientious, explicit and judicious use of current
best evidence in making decisions about the care of
individual patients”.4
Treatment of non-complicated hemangiomas of
infancy is not for all patients. The final decision should
be taken by the well informed parents. Studies like
the one from Gelmetti el al. may, in individual cases,
be a strong argument to decide the best management.
A frequent argument supporting the treatment of small
uncomplicated hemangiomas of infancy is that they may
grow and attain large proportions. In fact, the vast majority of hemangiomas of infancy are small and remain
small, initially growing in volume but not in surface.
Most large hemangiomas are initially multi-focal.
If the decision is to treat a small, uncomplicated
hemangioma, the next step is to know which are the
therapeutic options? Among medical treatments systemic corticosteroids, with all its well known side
effects is the only proved effective therapy.5 Intralesional injection of corticosteroids is equivalent to its
systemic administration. The injected corticosteroid
remains in the vascular tumor no more than a few seconds before going to the general blood circulation.
When dealing with potentially complicated or clearely complicated hemangiomas the question is not to
treat or not to treat but to decide which is, among the
plethora of available resources the best possible treatment. Here again the combination of personal expertise and of evidence based medicine should guide the
decision. Unfortunately even for the most frequent
complication of hemangiomas of infancy, ulceration,
there is lack of consensus: antibiotics, pulsed dye laser,
occlusive dressings (petrolatum, polyurethane), corticosteroids, interferon, recombined human platelet
derived growth factor-BB, all claim good results sometimes based on a single case experience.
We know that hemangiomas of infancy are not only
an esthetic nuisance, even if small they can in certain
locations as the tip of the nose cause destruction of the
collumela, orbital tumors even if small may cause
reduced vision (amblyopia). Other possible complications are ptosis and proptosis, strabismus, myopia, and
tear duct obstruction. The alterations in the so-called
PHACE syndrome associating large segmental facial
hemangiomas with cardiovascular, central nervous system (Dandy-Walker malformation) and eye alterations
were first reported since 1978 by Pascual-Castroviejo 6
and redescribed by Frieden and Cohen in 1996.7 Lumbosacral hemangiomas may be markers for tethered
462
spinal cord and spinal dysraphism. The use of MRI in
segmental hemangiomas is the most sensitive means
to detect central nervous system alterations.8
Segmental hemangiomas in the beard area may associate respiratory tract hemangiomas. Multiple hemangiomas of infancy, from 2 or 3 to hundreds may be
associated to visceral involvement, more frequently
of the liver, and complicate with congestive heart failure resulting in high mortality.
The above complications require a high degree of
suspicion in order to be diagnosed, a deep knowledge
of the therapeutic resources, and a critical analysis of
the literature in order to provide each individual patient
with the best available management.
Serious evaluation of therapeutic resources should
be prospective and have a control group. These requirements, important in all therapeutic research, become
fundamental when the condition under treatment is
self-healing as is the case for hemangiomas of infancy either cutaneous or extra-cutaneous, including the
Kasabach-Merritt phenomenon.
Returning to the original question “To treat or not to
treat hemangiomas of infancy” in conclusion I may say
that it must be kept in mind that we are not dealing with
a vascular tumor but with a patient that is unique in his
biology, psychology, and sociology. The management
should be adapted to the patient and not vice-versa. Some
patients will require treatment and some will not.
References
1. Mulliken JB, Glowacki J. Hemangiomas and vascular malformations
in infants and children: a lassification based on endothelial cell characteristics. Plast Reconst Surg 1982;12:412-20.
2. Ruiz-Maldonado R. Hemangiomas cutaneos. In: Ruiz-Maldonado R,
Saul Amado, Ibarra-Duran G, Tamayo -Sanchez L editors. Temas de
Dermatología Pediatrica. Mexico: Francisco Mendez Cervantes;1980.
p. 185-93.
3. Lister WA.The natural history of strawberry nevi.Lancet 1938;1:1429-34.
4. Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS.
Evidence based medicine: what it is and what it isn’t. Br Med J
1996;312:71-2.
5. Bennett ML, Fleisher AB Jr, Chamlin SL, Frieden IJ. Oral corticosteroids use is effective for cutaneous hemangiomas: an evidencebased evaluation. Arch Dermatol 2001;137:1208-13.
6. Pascual-Castroviejo I, Pascual-Pascual SI, Velazquez-Fragua R., Garcia-Guereta L, Lopez-Gutierrez JC, Viano J et al. [Cutaneous hemangiomas and vascular malformations and associated pathology (PascualCastroviejo type II syndrome). Study of 41 patients.] Rev Neurol
2005;41:223-36. Spanish.
7. Frieden IJ, Reese V, Cohen D. PHACE syndrome: the association of
posterior fossa brain malformations, hemangiomas, arterial anomalies, coarctetion of the aorta, and cardiac defects, and eye abnormalities. Arch Dermatol 1996;132:307-11.
8. Bruckner AL, Frieden I. Hemangiomas of infancy. J Am Acad Dermatol 2003;48:477-93 ; quiz 494-6.
Ottobre 2005
Hair growth and angiogenesis
R. PAUS, 1 L. MECKLENBURG 2
D
uring the last 2 decades, the molecular controls,
clinical implications and therapeutic modulation
of angiogenesis have been among the most central, and
most hotly debated, topics of biomedicine. While landmark progress was made in defining the - unexpectedly complex -controls of angiogenesis and vasculogenesis, the initial enthusiasm that anti-angiogenic
therapies will quickly usher, in a new area in cancer
therapy, has given way to a more cautious, sobering
assessment of what can realistically be achieved in
clinical practise to-date. Dermatological research has
greatly profited from this new focus on angiogenesis,
not only in areas where angiogenesis is of evident
importance (skin development, wound healing, vascular malformations and neoplasms, tumor angiogenesis), but also where this is less self-evident, e.g. during the pathogenesis of psoriasis, cutaneous autoimmune disease and UV-light-induced skin damage.1-5
It used to be a much-reverberated dogma that, in
adult mammalian tissues and under physiological conditions, angiogenesis essentially does not occur at all,
if one excepts the ovarian and endometrial cycles and
the lactating mammary gland from this rule.6, 7 Circumstantial evidence, however, has long questioned this
dogma, and has suggested that angiogenesis does occur
in adult mammalian skin under physiological condiIN THIS ISSUE SEE PAGE 497
Address reprint requests to: R. Paus, Klinik für Dermatologie und Venerologie, Universitätsklinikum Schleswig-Holstein / Campus Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
E-mail: [email protected]
Vol. 140 - N. 5
1Department of Dermatology
University Hospital Schleswig-Holstein
Campus Lübeck, University of Lübeck, Lübeck, Germany
2Department of Pathobiology
College of Veterinary Medicine
Texas A&M University, College Station, TX, USA
tions, namely during the cyclic growth and regression
of the hair follicle: already decades ago, prominent
rearrangements of the skin vasculature and skin perfusion were noted to be associated with hair follicle
cycling,8-10 i.e. the cyclic transformations of the hair follicle from periods of massive growth (anagen), via
rapid, apoptosis-driven organ involution (catagen) to
a state of relative quiescence (telogen).11
The appearance and arrangement of perifollicular
blood vessels, as visualized by dye-injection, had been
found to change dramatically during synchronized
hair follicle cycling from the growth stage of the hair
cycle (anagen) to relative resting (telogen) in rabbits.8
The dermis around anagen hair follicles was noted to
be more vascular than that around telogen follicles,
and proliferating endothelial cells inside the follicular
dermal papilla were noted only during anagen.12 Finally, the epithelial hair bulb of anagen, but not of telogen
follicles, possesses angiogenic properties under experimental conditions.13 Subsequently, it became appreciated that outer root sheath keratinocytes and dermal
papilla fibroblasts express a key modulator of angiogenesis, vascular endothelial growth factor (VEGF),
both in vivo and in vitro.14-17
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PAUS
HAIR GROWTH AND ANGIOGENESIS
However, none of these studies had presented formal
proof that anagen development of hair follicles truly is
associated with genuine, physiological angiogenesis,
and not just mere hair-cycle-associated changes in
vessel perfusion and caliber.18 The theoretical possibility existed that preexisting blood vessels and anastomoses just collapsed during catagen/telogen, to be
reopened again during anagen, thus merely simulating
angiogenesis and blood vessel regression.
The situation was clarified only recently by Mecklenburg et al.,19 with the help of morphometric and
functional assays in the C57BL/6 mouse model for
hair research, using a monoclonal antibody against
PECAM-1/CD31 as a specific marker for endothelial
cells. This study showed that the cutaneous microvasculature undergoes substantial rearrangements during
the murine hair cycle, including a significant anagenassociated increase in the microvessel density (MVD),
i.e., the number of microvessels per microscopic field.
MVD constantly increased during anagen development, and declined again during telogen. This was
associated with a significant increase in the number of
endothelial cell nuclei, and the demonstration of proliferating (Ki-67+) and mitotic endothelial cells only
during anagen. Moreover, systemic administration of
a fumagillin-derived angiogenesis inhibitor signifcantly retarded experimentally induced anagen development.19
Taken together, this study had provided the first
convincing evidence that synchronized hair follicle
cycling in mice is indeed associated with genuine
angiogenesis, and already suggested that this is followed by vascular regression when hair follicles reenter into catagen and telogen. Subsequent studies by
Yano et al. confirmed and elegantly extended this line
of research, strongly supporting the general concept
that hair follicle cycling is coupled to major remodelling of the skin vasculature.20, 21 These authors found
perifollicular angiogenesis to be temporally and spatially correlated with an upregulation of VEGF transcripts by outer root sheath keratinocytes. Targeted
overexpression of VEGF in the hair follicle epithelium
of mice significantly enhanced perifollicular vascularization, accelerated hair regrowth after depilation,
and increased the size of both hair follicles and hair
shafts. Instead, systemic treatment with a neutralizing anti-VEGF antibody retarded hair growth retardation and reduced hair follicle size.20 Conversely,
mice with a targeted deletion of VEGF in follicular
464
keratinocytes exhibited a decreased MVD, and showed
accelerated spontaneous catagen development.22
The study by Yano et al.20 offered the first direct
evidence that improved follicle vascularization can
indeed promote hair growth and can increase hair follicle volume and hair shaft diameter in genetically
engineered mice. This further encouraged the previously voiced concept that VEGF may be exploited as
a hair growth stimulatory agent.14-16 However, while
this is an intellectually pleasing concept, it deserves to
be inserted here as a note of caution that unequivocal
evidence that the mere upregulation of VEGF expression and synthesis in human scalp hair follicles in vivo
really has any significant, cosmetically revelant effects
on hair growth, hair loss and/or hair shaft diameter is
still missing. In fact, treating mice continuously with
recombinant exogenous murine VEGF subcutaneously
failed to modulate either MVD or catagen development.22
Yano et al. went-on to show apoptosis-driven blood
vessel regression during hair follicle regression (catagen).21 We were able to confirm this finding, and could
show that, during the catagen-associated regression
of the perifollicular vasculature, endothelial cell apoptosis is complemented by shedding of degenerating
endothelial cells into the vessel lumen.22 One of the
many factors regulating catagen-associated vascular
regression may be the natural angiogenesis inhibitor
thromospondin-1 (TSP-1). In murine skin, the angiogenesis inhibitor thrombospondin-1 (TSP-1) was
shown to be absent from hair bulb and dermal papilla
cells during early to mid-anagen, while it became highly upregulated throughout catagen.21 In contrast, TSP1 deficient mice showed a significantly prolonged anagen phase, associated with increased perifollicular
vascularization and vascular proliferation, while anagen was delayed in transgenic mice that expressed
high levels of TSP-1 in outer root sheath keratinocytes.21
Since the factors required for promoting angiogenesis can differ rather substantially between tissues,
species and biological settings, it is by no means a
trivial challenge to dissect which key factors and their
cognate receptors drive anagen-associated angiogenesis and catagen-associated blood vessel regression.
Besides VEGF and TSP-1, many other factors are now
recognized as important players in the control of cutaneous angiogenesis and blood vessel remodelling,
such as platelet-derived growth factor,23 fibroblast
Ottobre 2005
PAUS
growth factor 2, 24 hepatocyte growth factor/scatter factor (HGF/SF),25, 26 angiopoietin and angiopoietin-like
proteins,27 as well as angiogenesis inhibitors such as
angiostatin 28 and endostatin.29
Since HGF/SF and its receptor, c-Met, are expressed
in a strikingly hair cycle-dependent manner, with
HGF/SF expression being exclusively confined to the
dermal papilla of anagen hair follicles,30 HGF and cMet certainly deserve careful scrutiny as a potential key
modulator of hair cycle-dependent blood vessel remodelling. Most recent work from our laboratory suggests
that outer root sheath keratinocytes in mice not only
express VEGF, but also angiopoietin-1 and angiopoietin-2, and their transcript levels as well as those of their
endothelial cell receptor (Tie-2) significantly change
during synchronized hair follicle cycling. This is also
true for the 2 VEGF receptors on endothelial cells,
Flt-1 and Flk-1.22
It is on this background that the intriguing study by
Cianfarani et al. presented in this issue of the Giornale
introduces yet another player in the hair follicle-associated “angiogenesis control circus” of murine skin:
placental growth factor (PlGF). This dimeric glycoprotein is a member of the VEGF family and binds to
the VEGF receptor Flk-1. It synergizes with VEGF
and angiopoietin-2 in the control of angiogenesis in a
number of pathological conditions, including in
tumorangiogenesis in the outer root sheath of mouse
skin.31 Cianfarani et al. now report that PlGF is upregulated in anagen mouse hair follicles, and this apparently at a time before the morphological evidence for
perifollicular angiogenesis that was assessed here
reached its maximum. The authors conclude from this
that PlGF plays a role in anagen-associated angiogenesis. This postulate is consistant with a recent report
that mice overexpressing PlGF under the control of
the Keratin-14 promoter showed a increase in subcutaneous vascularization.32
Likely, angiogenesis and vascular remodelling during hair follicle cycling are controlled by tightly coordinated switches in the local signalling milieu, in which
all of the factors mentioned above (VEGF, angiopoietins, TSP-1, possibly also PDGF, HGF and FGF-2) and now also PlGF - all may operate as important
players. However, the relative hierarchy of signalling
events, remain to be identified, and redundant signals
need to be distinguished from indispensable ones,
before any convincing therapeutic concepts can be
woven out of these recent insighs. Also, we must face
Vol. 140 - N. 5
the possibility that, so far, we have seen nothing but the
proverbial tip of the iceberg of factors that govern hair
cycle-associated angiogenesis and vascular remodelling in human skin and that the underlying controls
are even more complex than we are beginning to grasp.
Moreover, the natural inhibitory controls that prevent
overshooting angiogenesis, the maintenance signals
that uphold vascular integrity and function, and the
deconstruction signals that drive vascular regression
during catagen, all remain to be defined more comprehensively. Certainly, we are still quite far away
from understanding the molecular basis of the master
switches that dictate and coordinate these changes in
angiogenic signalling in murine - not to mention
human! - skin.
While this is bad news for anyone looking for fast
answers, the paper by Cianfarni et al. nicely underscores at least this fact: the hair follicle and its cyclic
growth and regression activity, which seems to drive
subsequent changes in the perifollicular vasculature,
offers a superb, highly instructive research model for
dissecting both the morphological characteristics and
the functionally predominant molecular controls of
angiogenesis and vascular remodelling in adult mammalian skin. This model deserves to be discovered and
systematically exploited by everyone with a serious
interest in understanding the signaling concert that
drives physiological angiogenesis and vascular remodelling in vivo.
As to the new therapeutic concepts that may be distilled from work such as it is presented in this issue of
the Giornale, another note of caution appears in order.
Despite circumstantial evidence that the vasculature can
also be affected in some forms of alopecia,14 there is
currently no convincing evidence available that the
most common causes of hair loss (androgenetic alopecia, telogen effluvium, alopecia areata) result from
primary abnormalities in the perifollicular vasculature. In addition, the popular belief that just improving
a hair follicle’s blood supply will automatically promote hair growth, not to mention vellus-to-terminal
hair follicle reconversion in the balding scalp, has as
yet few, if any hard data to show in its support.
Instead, we suspect that inherent epithelial-mesenchymal signaling exchanges within the hair follicle itself, for better or worse, largely dictate subsequent changes in the hair follicle vasculture. Therefore,
targeting the hair follicle vasculature as a tool for therapeutically manipulating human hair growth may well
465
PAUS
turn-out to be a frustrating enterprise, since the hair follicle appears to be “in the driver seat”, not the perifollicular vasculature. In contrast, we propose that it
holds much greater promise to target intrafollicular
signalling events that drive hair follicle cycling - as a
powerful tool for manipulating skin vascularization
and perfusion by unleashing the hair follicle’s inherent power to control the skin vasculature, whenever this
is clinically desired.
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2. Creamer D, Sullivan D, Bicknell R, Barker J. Angiogenesis in psoriasis. Angiogenesis 2002;5:231-6.
3. Dadras SS, Detmar M.Angiogenesis and lymphangiogenesis of skin
cancers. Hematol Oncol Clin North Am 2004;18:1059-70.
4. Hirakawa S, Fujii S, Kajiya K, Yano K, Detmar M. Vascular endothelial growth factor promotes sensitivity to ultraviolet B-induced cutaneous photodamage. Blood 2005;105:2392-9.
5. Yano K, Kadoya K, Kajiya K, Hong YK, Detmar M. Ultraviolet B irradiation of human skin induces an angiogenic switch that is mediated
by upregulation of vascular endothelial growth factor and by downregulation of thrombospondin-1. Br J Dermatol 2005;152:115-21.
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7. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27-31.
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follicles. In: Montagna W, Ellis RA editors. The Biology of Hair
Growth. New York: Academic Press; 1958. p. 189-218.
9. Ellis R, Moretti G. Vascular patterns associated with catagen hair follicles in the human scalp. Ann NY Acad Sci 1959;83:448-57.
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York: Academic Press; 1974
11. Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med
1999;341:491-7.
12. Sholley MM, Cotran RS. Endothelial DNA synthesis in the microvasculature of rat skin during the hair growth cycle. Am J Anat
1976;147:243-54.
13. Stenn MD, Fernandez LA, Tirell SJ. The angiogenic properties of
the rat vibrissa hair follicle associate with the bulb. J Invest Dermatol 1988;90:409-11.
14. Goldman CK, Tsai JC, Soroceanu L, Gillespie GY. Loss of vascular
endothelial growth factor in human alopecia hair follicles. J Invest
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15. Lachgar S, Moukadiri H, Jonca F, Charveron M, Bouhaddioui N,
Gall Y et al. Vascular endothelial growth factor is an autocrine growth
factor for hair dermal papilla cells. J Invest Dermatol 1996;106:17-23.
16. Lachgar S, Charveron M, Aries MF, Gall Y, Bonafe JL. Hair follicles
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Mecklenburg L, Tobin D, Müller-Röver S, Handjiski B, Wendt G,
Peters EMJ et al. Active hair growth (anagen) is associated with angiogenesis. J Invest Dermatol 2000;114:909-16.
Yano K, Brown FL, Detmar M. Control of hair growth and follicle size
by VEGF mediated angiogenesis. J Clin Invest 2001;107:409-17.
Yano K, Brown FL, Lawler J, Miyakawa T, Detmar M. Thrombospondin-1 plays a critical role in the induction of hair follicle involution and vascular regression during the catagen phase. J Invest Dermatol 2003;120:14-9.
Mecklenburg L, Tobin D, Rossiter H, Ellerbrok H, Nakamura M,
Barresi C et al. Hair cycle-dependent remodelling of the skin vasculature is mediated by changes in the expression of key regulators of
blood vessel remodelling and their receptors. J Deut Dermatol Ges
2004; 2:492 (S8.3).
Wang D, Huang HJ, Kazlauskas A, Cavenee WK. Induction of vascular
endothelial growth factor expression in endothelial cells by plateletderived growth factor through the activation of phosphatidylinositol
3-kinase. Cancer Res 1999;59:1464-72.
Seghezzi G, Patel S, Ren CJ, Gualandris A, Pintucci G, Robbins ES
et al. Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis.
J Cell Biol 1998;141:1659-73.
Gille J, Khalik M, Konig V, Kaufmann R. Hepatocyte growth factor/scatter factor (HGF/SF) induces vascular permeability factor
(VPF/VEGF) expression by cultured keratinocytes. J Invest Dermatol 1998;111:1160-5.
Wojta J, Kaun C, Breuss JM, Koshelnick Y, Beckmann R, Hattey E et
al. Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human
keratinocytes and the vascular endothelial growth factor receptor flk1 in human endothelial cells. Lab Invest 1999;79:427-38.
Oike Y, Yasunaga K, Suda T. Angiopoietin-related/angiopoietin-like
proteins regulate angiogenesis. Int J Hematol 2004;80:21-8.
O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses
M et al. Angiostatin: a novel angiogenesis inhibitor that mediates the
suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:
315-28.
Dhanabal M, Ramchandran R, Volk R, Stillman IE, Lombardo M,
Iruela-Arispe L et al. Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma. Cancer Res 1999;59:189-97.
Lindner G, Menrad A, Gherardi E, Merlino G, Welker P, Handjiski B
et al. Involvement of hepatocyte growth factor/scatter factor and Met
receptor signaling in hair follicle morphogenesis and cycling. FASEB
J 2000;14:319-32.
Larcher F, Franco M, Bolontrade M, Rodriguez-Puebla M, Casanova L, Navarro M et al. Modulation of the angiogenesis response
through Ha-ras control, placenta growth factor, and angiopoietin
expression in mouse skin carcinogenesis. Mol Carcinog 2003;37:
83-90.
Kiba A, Sagara H, Hara T, Shibuya M.VEGFR-2-specific ligand
VEGF-E induces non-edematous hyper-vascularization in mice.
Biochem Biophys Res Commun 2003;301:371-7.
Ottobre 2005
The dermatologic implications of novel delivery systems
Z. D. DRAELOS
D
elivery systems are the key to dermatology. Traditional products applied to the skin have taken the
form of lotions, creams, ointments, gels, and solutions, however Santoianni focuses on the new realm in
dermatology, which is the intradermal delivery of
actives? Why is this intradermal technology so important? Simply because the cells that direct the growth
and maturation of the skin lie in the dermis. Deposition
of actives in the dermis is crucial to disease states,
such as atopic dermatitis, psoriasis, discoid Lupus erythematosus, and Lichen planus. In all of these complex
medical conditions, the abnormal pathology lies in
the dermis and must be modified by the medication
reaching this target site. Dermal penetration is also
key to cosmeceuticals where the regeneration of collagen and elastin requires a dermal effect. Many of
the now popular anti-aging ingredients make claims
that can only result from the active reaching the dermis, yet traditional cosmetic creams are not currently
designed with intradermal delivery principles in mind.
Santoianni nicely reviews many different methods
of reaching the dermis from the Stratum corneum
application of actives to include transdermal patches,
sonophoresis, iontophoresis, electroporation, crioelectrophoresis, hydroeectrophoresis, and laser. However, the mechanism of dermal delivery that he discussed which I find most fascinating is the area of folIN THIS ISSUE SEE PAGE 539
Address reprint requests to: Z. D. Draelos, MD, 2444 North Main Street,
High Point, NC 27262. E-mail: [email protected]
Vol. 140 - N. 5
Dermatology Consulting Services
High Point, NC, USA
licular delivery. This interests me because it does not
require the use of a special device to enhance penetration and thus has direct applicability in the current prescription and cosmeceutical worlds as products are
presently dispensed. One of the best examples I can
think of to illustrate my point is the recent reintroduction of an azelaic acid cream for the treatment of rosacea.
Azelaic acid is a particulate that can be ground into a fine
white powder for suspension in a cream or gel.
Azelaic acid has traditionally been formulated as a
cream, but has recently been reformulated as an opaque
water-soluble gel. The gel is the most effective formulation because 25% of the 15% azelaic acid concentration can be solubilized in a gel while only 3% of
the higher 20% azelaic acid concentration can be dissolved in a cream vehicle. This alters the amount of
medication that is available to reach the dermis. Another critical point is the size of the particle. Particles that
are too big cannot lodge in the follicular orifice, while
particles that are too small are easily rinsed away as
sebum is produced. Azelaic acid micronized to a particle size of 1-10 µm can easily lodge within the follicular ostia. Most dermatologics are preferentially
absorbed through the skin, which provides a much
greater surface area for absorption, but particles that are
lodged in the follicular ostia can be time released into
467
DRAELOS
THE DERMATOLOGIC IMPLICATIONS OF NOVEL DELIVERY SYSTEMS
the skin through the appendageal route. This is one of
the current examples of intradermal delivery in the
prescription realm.
One of the biggest hurdles to the development in intradermal delivery systems is accurate measurement of the
amount of active that reaches the dermis. The test typically performed for dermal absorption is the Franz flowthrough diffusion cell study. In this study, human or
mouse skin is placed in a frame. The formulation is
placed on the top of the skin and the amount of active that
passes through the skin is collected below in the receptor fluid. Sometimes the active ingredient is radiolabeled with carbon-14 and the amount of radioactivity
in the collection chamber is felt to equate with the passage of the active through the skin. Thus, the exact
amount of active ingredient that reaches and remains in
the dermis can be determined by looking at the amount
of carbon-14 labeled active ingredient present.
This Franz cell technique was used to aid in the
development of the new azelaic acid delivery system
mentioned previously. What is the difference in the
dermal amount of azelaic acid when comparing 25%
of the active in the gel versus 3% of the active in the
cream? Using the Franz cell flow-through diffusion
cell study, the analysis shows that when azelaic acid
20% cream is applied to the skin surface, 68.4%
remains on the skin, 0.8% penetrates in the Stratum
corneum, 3.4% is present in the dermis, and 16.3%
reaches the receptor fluid. Thus, only 3.4% of the
applied azelaic acid is available to induce a clinical
effect, such as improvement in rosacea. This example illustrates the importance of intradermal delivery.
Compare these findings with azelaic acid 15% gel,
in which the concentration of the active ingredient
applied to the skin is actually lower. The same Franz
cell study shows that 56.7% remains on the skin surface, 3.7% penetrates the Stratum corneum, 25.3% is
468
present in the dermis, and 5.8% is found in the receptor fluid. In this case, nearly 8 times as much medication (25.3%) is available for a clinical effect in rosacea
with the gel vs the cream. The difference between the
amount of drug reaching and remaining in the viable
skin with the gel formulation compared with the cream
is due to the difference in the vehicle and the intradermal appendageal delivery mechanism. And, as
might be expected, the clinical findings are better with
the gel formulation than the cream formulation in the
treatment of rosacea. This is due solely to the ability
of the vehicle to more effectively deliver the active
ingredient to the dermis.
In summary, an understanding of mechanisms of
intradermal derlivery is key to achieving efficacy with
dermatologic medications. In the future, sonophoresis,
iontophoresis, electroporation, crioelectrophoresis,
and hydroeectrophoresis may assume greater importance in dermatologic medication delivery. Transdermal patches have already become a popular delivery
system for medications such as estrogen, both for hormone replacement and contraceptive purposes, and
for nitroglycerin, as vasodilator to prevent myocardial infarction. Lasers are being used as part of photodynamic therapy to initiate drug activation and further developments may yield laser as an at home penetration enhancer for a variety of medications. This
editorial has discussed in depth a current use of the
appendageal route for intradermal delivery. Many
times the active ingredient is only part of the true value of a topical dermatologic medication. The vehicle
may account for 50% to 75% of the ability of the topical to achieve efficacy and the ability for the active to
reach the dermis may account for the rest. Intradermal
delivery accompanied by careful formulation will yield
the next generation of pharmaceuticals and cosmeceuticals for dermatology.
Ottobre 2005
New treatments for basal cell carcinoma: how to make the election
F. M. CAMACHO
B
asal cell carcinoma (BCC) is the most common
malignant tumor in Caucasian people, involving
6% of the consultations in dermatology, and 60% of the
cutaneous tumors. The lifetime risk of having a BCC,
for Latin people, ranges between 11% and 28%,
depending upon factors like age, gender and geographical factors.
Regarding the age, the incidence of BCC is clearly
increased in elderly patients, with the highest rates
between 50 and 70 years, and a peak incidence of this
cancer at 60 years. However, an increase in the incidence of BCC in patients younger than 30, has been
observed in recent years, probably as a result of the
excessive sun exposure during their youth. BCC is
also more frequent in men, especially older than 45
years, although there are no gender differences in
younger patients.
The influence of sun exposure on the development
of a BCC leads to the face and the neck to be the most
common anatomic areas involved, with 85% of the
BCCs arising on these areas. Moreover, the periocular
region, as well as the temples, cheeks and the nose, also
called H zone, are the most involved facial region by
BCC, which means that an early and definitive treatment is mandatory to avoid the destruction of anatomic and functional essential structures.
Address reprint requests to: F. M. Camacho, Departamento de Dermatologia de la Facultad de Medicina de Sevilla, Avda Dr.Fedriani s/n, 41009
Sevilla. E-mail: [email protected]
Vol. 140 - N. 5
Department of Medical-Surgical Dermatology,
University of Seville, Spain
The main aims of our therapeutical attitude indeed
are to carry out an early, radical and definitive treatment.
Thus, the first choice must be the complete surgical
removal of the tumor with a cosmetically acceptable
closure in those cases where this is possible. Nowadays, it is accepted that 4% of the BCCs are incompletely removed, which means a recurrence rate between
30% and 40% although 2-4 mm surgical margins were
excised.1 In BCCs arising on the H zone, a recurrence
rate of 3.2% has been reported for BCCs smaller than
6 mm, with 8-9% of recurrence in tumors larger than this
size. Tumors on H zone should be treated with 4 mm
margins 2 or with Mohs micrographic surgery for histologic margin control because their histologic subtype
are mainly infiltrating or nodulocystic.3 Additionally, the
re-excision of these recurrent tumors present a higher
likelihood to recur, with a rate up to 17.4%; even in
those cases of morpheiform and multilobuled infiltratives
BCCs removed through Mohs micrographic surgery
the recurrence rate is of 5.6%.4 The use of the immunoenzymatic technique with antihuman epithelial antibody Ber-EP4 has led to demonstrate tumoral persistence
in the margins of the tumor and thereby to a slight reduction in the recurrence rates.5
BCC is usually resistant to chemotherapy and radio-
469
CAMACHO
NEW TREATMENTS FOR BASAL CELL CARCINOMA: HOW TO MAKE THE ELECTION
therapy, which may be in relation to the expression of
bcl-2 proto-oncogene.
Ancient techniques as cryosurgery, curettage and
electrodessication, and CO2 laser might not currently
be considered since an appropriate histopathological
study is not allowed and higher recurrence rates are also
expected. For instance, shave excision is only useful in
superficial BCC, although it must not be forgotten that
shave excision in several stages, and with histological
control of the margins, is the procedure carried out in
Mohs micrographic surgery.
In some occasions, the slow evolution of the BCC
leads to the presentation at the clinic after years of
development. In these cases of patients with large
tumoral masses, or in those cases of multiple BCCs,
like in the Bazex and Gorlin syndrome, the surgery is
actually a difficult task non-surgical options must be
offered to our patients.
Several years ago, the administration of intralesional
interferon-alfa-2b, 3 million IU doses 3 times a week
for 3 weeks, was an option in those patients who were
not good candidates for surgery; by stimulating the Tcell immunoresponse to the tumor, a regression, and
even a complete resolution of the tumor could be
observed with this approach.6
However, we now have the possibility to treat multiple BCCs with Imiquimod 5% cream. This topical
drug favours the production of local interferon and
other cytokines which leads to the reduction of the
tumoral bulk, and thus making the surgery of theses
tumors easier, if not unnecessary.
A typical case of Gorlin syndrome with multiple
BCCs successfully treated with imiquimod is discussed by Di Landro et al. I strongly agree in that
imiquimod is the most useful treatment for multiple
superficial BCCs as those in Gorlin syndrome.
Before prescribing imiquimod 5% for BCC, 2
premises must be considered. Firstly, tumors closer
than 1 cm to the lids must be avoided because of the
risk of harming the eyes. Secondly, a more or less
470
degree of local irritation is always expected when
using imiquimod; in this case, an antibiotic ointment,
along with the withdrawal of the drug for several days
is usually enough.7, 8 The accepted guideline for the use
of imiquimod recommends the use of the drug in a
daily basis from Monday to Friday for 6 weeks, with
weekend stops which may help to reduce the local
irritation.9
To sum up, we may currently say that the dream of
a cream to treat cancer, and thus avoiding the operating room, has eventually come true. This approach is
specially interesting in those elderly patients with a
poor general condition, as well as in those cases of
multiple BCCs. Safety and an acceptable effectiveness in these particular cases are further reasons that
support this choice.
References
1. Kimyai-Asadi A, Goldberg LH, Jih MH. Accuracy of serial transverse cross-sections in detecting residual basal cell carcinoma at the
surgical margings of an elliptical excision specimen. J Am Acad Dermatol 2005;53:469-74.
2. Kimyai-Asadi A, Alam M, Goldberg LH, Peterson R, Silapunt S, Jih
MH. Efficacy of narrow-margin excision of well-demarcated primary facial basal cell carcinomas. J Am Acad Dermatol 2005;53:464-8.
3. Leibovitch I, Huilgol SC, Selva D, Richards S, Paver R. Basal cell carcinoma treated with Mohs surgery in Australia. I. Experience over
10 years. J Am Acad Dermatol 2005;53:445-51.
4. Camacho F, Sánchez Conejo-Mir J. Márgenes de escisión en los carcinomas basocelulares. La cirugía micrográfica como el método más
seguro de escisión. Monogr Dermatol 1990;3:245-52.
5. Kist D, Perkins W, Christ S, Zachary CH. Anti-human epithelial antigen (Ber-EP4) helps define basal cell carcinoma masked by inflammation. Dermatol Surg 1997;23:1067-70.
6. Sánchez Conejo-Mir J, Camacho F. Interferón alfa-2b en el tratamiento de los carcinomas basocelulares. Experiencia en 17 casos. Monogr Dermatol 1994;7:259-64.
7. Marks R, Owens M, Walters SA. Efficacy and safety of 5% imiquimod
cream in treating patients with multiple superficial basal cell carcinomas. Arch Dermatol 2004;140:1284-5.
8. Naylor M. Imiquimod and superficial skin cancer. J Drugs Dermatol
2005;4:598-606.
9. Geisse J, Caro I, Lindholm J, Golitz L, Stampone P, Owens M.
Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from two phase III, randomized, vehicle-controlled
studies. J Am Acad Dermatol 2004;50:722-33.
Ottobre 2005
Treatment of psoriasis: the challenge of the side effects
L. DUBERTRET
T
he 3 main progresses in psoriasis treatment has
been 1) a better adjustment of the therapeutic strategy to the patient needs (the so-called “patient based
medicine”); 2) new drugs; 3) better strategy of use of
classical drugs in order to increase the efficacy/toxicity
ratio.
The patient based medicine
We have to solve every day 2 questions: the first
one is how to evaluate the severity of diseases affecting
mainly the quality of life (like psoriasis, vitiligo, atopic dermatitis …). This severity (the main information to choose the more appropriate treatments) is first evaluated by the patient and the patient is not educated to do this. The second one is to find a method to
adjust the knowledge coming from the evidence based
medicine obtained by studying population through
statistical approaches, to an individual patient, always
different and specific.
The so called “patient based medicine” is the association of clinical techniques making possible to solve these 2 questions.
Patient based medicine unfolds in 4 phases: questions, explanations, negotiation (the most important
step) and prescription. The goal of these successive
Address reprint requests to: Prof. L. Dubertret, Institut de Recherche sur
la Peau, Service de Dermatologie, Hôpital Saint Louis, Paris, 1 Av. Cl. Vellefaux, 75475 Paris Cedex 10, France. E-mail [email protected]
Vol. 140 - N. 5
Department of Dermatology
Saint Louis Hospital, Paris, France
phases is to take into account in the therapeutic choice
all the aspects of the patient character and way of life,
co-morbidities and associated treatments, previous experiences of the patient with different treatments, personal evaluation by the patient of the quality of life and of
the impact of the disease and of the treatments on his
quality of life. At the end of the negotiation the patient
must participate to the therapeutic choice. Patient based
medicine is necessary to treat efficiently chronic diseases. Patient based medicine take time (around 45 min by
consultation) but it is a therapeutic revolution with a
strong impact on patient/doctor relationships, medical
education, development of new drugs and on the awareness of the rights and the duty of the patients.
New and classical systemic treatments
The development of new drugs, and mainly today of
the biologicals, opens a great hope for our patients.
For economic reasons they are reserved for high need
patients. These patients do not respond or suffer from
contraindication or side effects to classical systemic
treatments. Thus it is impossible to efficiently adjust
the prescription of biologicals without a previous optimal use of classical treatments and this optimal use
471
DUBERTRET
TREATMENT OF PSORIASIS
is the result of a very specific and important research
on the therapeutic strategies making possible to find the
best efficacy/toxicity ratio for each patient.
The challenge of side effects
In a chronic non life threatening disease like psoriasis, the control of any possible side effect is of major
importance. The side effects of systemic psoriatic
treatments can be classified in 4 categories: the acute
side effects, the chronic side effects associated with the
continuous use of the same treatment but reversible
if the treatment is stopped, the cumulative side effects
not reversible and leading to the concept of a maximal
cumulative dose, the side effects due to co-morbidities,
age, fertility and/or drugs interactions making possible to identify patients at risk for each systemic treatment.
The strategies to control acute side effects are quickly
defined, usually during the phase III of drug development, at the latest.
The control of UVB dose increase according to the
skin type, the adjustment of 8 MOP doses according
to the body weight and of the UVA doses according to
the skin type, the replacement of 8 MOP by 5 MOP in
case of nausea, the use of balneo- PUVA therapy in case
of cataract or of hepatic disease are good examples of
the management of acute side effects during the photo or photo-chemo therapies.
The acute side effects of retinoids can be avoided by
a very progressive increase of the daily dose beginning at low doses (10 mg a day or every other day)
and increasing the average daily dose by 5 mg every 2
weeks or every month. This make possible to find, for
each patient, the highest well tolerated dose (usually
associated with a mild cheilitis). Interestingly the
highest well tolerated dose is the most efficient (PASI
90 for 40% of the patients). Highest doses may induce a Köebner phenomenon due to epidermal fragility.
The respect of the contraindication of retinoids in case
of hypertriglyceridemia prevents retinoid induced pancreatitis. We are looking for short half life retinoids for
easier prescription in fertile ladies. The acute side
effects of methotrexate are usually avoided if the first injection is at low dose (5 mg), if methotrexate in not
prescribed in case of liver disease, in case of macrocytose or in case of decreased kidney functions. High
doses of aspirin or NSAI must be avoided as well as
anti folic drugs like antibiotic containing trimétoprime.
472
Methotrexate is photodestroid by UVA in a very strong
phototoxic compound ([diamino-2,4 pteridinyl]-6 carboxaldehyde) and it is important to avoid UVA exposure after methotrexate intake. The tolerance of methotrexate is clearly increased by folic acid intake (5 mg
a day, but not the methotrexate day).
The frequency of acute side effects of cyclosporine
is decreased if this drug is not prescribed in the elderly,
if the blood pressure is low, if the creatininemia is low,
if the dose is calculated in relation not with the real
body weight but with the theoretical optimal weight,
if the patient do not suffer from chronic infection (look
for the teeth, the gums, look for HPV infection of the
cervix in ladies). The acute side effects of cyclosporine are also very dependent from drug interactions.
The most usual chronic side effect of systemic treatment is the progressive lost of efficacy. In this case it
is useless and dangerous to increase the doses. The
best is to use a different systemic treatment for some
months and the previous one will become efficient
again. This was at the origin of the rotating strategies.
For example we alternate methotrexate (-6 months)
and cyclosporine (6 months) for patients escaping to
these treatments. In case of liver enzymes increase
during retinoids or methotrexate treatments one must
suspect a fatty liver. Decrease of the body weight, suppression of alcohol and decrease of sweet and fatty
food intake may restore a good tolerance.
Cumulative toxicity is the most serious problem for
systemic psoriasis treatment and during many years the
strategy was to use a treatment as long as possible and
the consequence was the progressive increase in the
number of patients without any further therapeutic
possibility. When the cumulative toxic doses of PUVA
are reached this treatment cannot be used again or
with a very severe risk of skin carcinoma. The protection of specific body areas like the genital area or the
eyes (from taking the psoralens until night) may decrease but not suppress the risk of carcinoma or of cataract.
Intermittent PUVA treatment seems less dangerous
than the continuous one but remains at risk. When a
liver fibrosis appears due to continuous treatment by
methotrexate it seems not reasonable to use the drug
again. This was the justification to perform liver biopsy. These biopsies, associated with morbidity and
mortality, are the main limitation of the use of methotrexate. Fortunately thanks to the impulse of rheumatologists who never performe liver biopsy and to the
hepatologists who consider methotrexate not so dan-
Ottobre 2005
TREATMENT OF PSORIASIS
DUBERTRET
gerous for the liver, the need for liver biopsies strongly decrease (after a cumulative dose of 3 g, in not at
risk patients, we ask the advice of hepatologist) and the
American guidelines must not be followed. Most interestingly new non invasive approaches open the hope
to suppress the need of liver biopsies: normal dosage
of procollagen III every 3 months is associated with a
very low risk of liver cirrhosis;1 dynamic hepatic scintigraphy (portal contribution >52% associated with a
95% chance of normal liver biopsy) could also decrease the need in liver biopsy;2 finely new biological tests
predictive for liver fibrosis are coming. It is time now
to organize a consensus meeting on the use of methotrexate in psoriasis.
During 12 years Cyclosporine was used on a chronic way, like the last possible treatment for patient
resistant to the others systemic treatments. Using this
strategy cyclosporine must be definitively stopped
after 2 or 3 years when chronic kidney ischemia began
to impair kidney functions and increases blood pressure. Recently the idea came from Italy to use cyclosporine as a first line systemic treatment but only for
intermittent courses of four months. This was validated by a controlled study.3 Now the ideal patient for
cyclosporine is a slim young lady with low blood pressure and willing to forget her psoriasis for the summer
time and taking cyclosporine 4 months a year. The
Vol. 140 - N. 5
hope is to avoid cumulative kidney toxicity and thus to
preserve the possibility to use cyclosporine for life.
With the same hope short intermittent courses of
methotrexate began to be used.
The study of G. A. Vena et al. published in this issue
goes in the same line. The idea was to give cyclosporine 4 days a week with the hope to increase again the
tolerance without losing efficacy. A 6 month comparison of this discontinuous strategy with the continuous one has been performed on 203 patients. The
results suggest that the efficacy remains good and that
the tolerance is increased. The authors propose confirming these preliminary stimulating data by a controlled study and one can strongly support this project.
References
1. Zachariae H. Liver biopsies and methotrexate: a time for reconsideration? J Am Acad Dermatol 2000;42:531-4.
2. van Dooren-Greebe R, Kuijpers AL, Buijs WC, Kniest PH, Corstens
FH, Nagengast FM et al. The value of dynamic hepatic scintigraphy
and serum aminoterminal propeptide of type III procollagen for early
detection of methotrexate-induced hepatic damage in psoriasis patients.
Br J Dermatol 1996;134:481-7.
3. Ho VC, Griffiths CE, Albrecht G, Vanaclocha F, Leon-Dorantes G,
Atakan N et al. Intermittent short courses of cyclosporin (Neoral(R))
for psoriasis unresponsive to topical therapy: a 1-year multicentre,
randomized study. The PISCES Study Group. Br J Dermatol
1999;141:283-91.
473
ORIGINAL ARTICLES
Gene expression profile in keratinocytes
from atopic dermatitis patients
S. PASTORE 1, L. ROGGE 2, F. MARIOTTI 1, F. MASCIA 1, R. LA SCALEIA 1, C. DATTILO 1, F. SINIGAGLIA 3
G. GIROLOMONI 4
Aim. Keratinocytes of atopic dermatitis (AD) patients show
enhanced production of cytokines and chemokines, a phenomenon that could be relevant in promoting and maintaining
inflammation and hence pivotal in localizing the atopic diathesis to the skin. We performed an oligonucleotide microarrray
analysis to investigate the gene expression profile in keratinocyte
cultures from 6 AD patients in order to search differentially
expressed genes.
Methods. Six well informed volunteer patients with moderateto-severe chronic AD (age range 19-45 years) participated in this
study. Skin involvement ranged from 20% to 60% of the body
surface area. Six well informed volunteer healthy individuals
(age range: 25-50 years) were used as controls.
Results. The microarrray analysis allowed to identify 201 differentially expressed transcripts, including transcriptional regulators, signal transducers, cell cycle regulators and enzymes
involved in inflammation. Ten transcripts were confirmed by
real-time reverse transcription polymerase chain reaction (RTPCR), with some diverged results from gene chip analysis. In
particular, metalloproteinase-2 (MMP-2) and leupaxin were
not confirmed, whereas we found heterogeneous levels for c-fos,
SAP1b, acidic sphingomyelinase (SMPD1), protein phosphatase
2A (PP2A) and GTP cyclohydrolase I regulatory protein among
the 6 AD patients. Still, we confirmed that cathepsin O mRNA
was homogeneously up-regulated in AD keratinocytes. Finally, specific transcripts of cdc2-like PISSLRE and 15-Lipooxigenase (15-LO) were undetectable as compared to controls.
Conclusion. These results indicate that the low transcript levels typical of unstimulated culture conditions may critically
impair the usefulness of microarray technology in the definition
of transcription profile. Furthermore, the disparate pattern
of gene transcript levels in AD donors suggests that multiple and
Address reprint requests to: Dr. S. Pastore, Istituto Dermopatico dell'Immacolata, IRCCS, Via dei Monti di Creta 104, 00167 Roma. E-mail:
[email protected]
Vol. 140 - N. 5
1Laboratory of Biochemistry
Istituto Dermopatico dell'Immacolata, Roma, Italy
2Laboratory of Immunoregulation
Department of Immunology, Institut Pasteur, Paris, France
3BioXell SpA, Milano, Italy
4Unit of Dermatology, Department of Biomedical
and Surgical Sciences, University of Verona, Verona, Italy
distinct molecular changes may concur to establish skin predisposition to a dysregulated response to inflammatory stimuli.
KEY WORDS: Atopic dermatitis - Keratinocytes - Gene expression
profiling.
A
topic dermatitis (AD) is a chronic inflammatory
disease resulting from complex interactions
between genetic and environmental factors.1 Genetic
factors strongly affect susceptibility to atopic diseases
and also influence disease-related quantitative traits.
Twin studies have indicated the presence of strong
genetic factors in the development of AD with heritability of approximately 60%, and segregation analyses suggest that this heritability is due to a few genes
of moderate effect rather than many genes of small
effect. Of note, an altered lipid composition of the
stratum corneum appears responsible of the xerotic
aspect of the skin, and may determine a higher permeability to allergens and irritants. Specific immune
responses against a variety of environmental allergens
are implicated in AD pathogenesis, with a bias towards
Th2 immune responses. Epithelial cells, including epi-
475
PASTORE
dermal keratinocytes, are the outermost components of
skin and mucous membranes, and they can be activated by diverse factors to produce mediators involved
in the initiation and amplification of inflammatory
responses. Of note, keratinocytes of AD patients exhibit a propensity to an exaggerated production of several cytokines and chemokines, a phenomenon that can
be relevant in promoting and maintaining inflammation, and may have a major role in localizing the atopic
diathesis to the skin.2
Both genetic and genomic methodologies are currently being applied to track down the complex molecular basis that may define the predisposition to AD.3
Identifying the relevant genes has been difficult so far,
in part because each causal gene only makes a small
contribution to overall heritability. Genetic association studies offer a potentially powerful approach for
mapping causal genes with modest effects, but are
nowadays limited because only a small number of
genes can be studied at a time. By contrast, genomics
rests on the knowledge of the entire genetic sequence
of an organism to study all of its genes at the same
time. Genomic expression profiling, usually performed
by hybridization of complemetary RNA to oligonucleotide sequences, the so-called oligonucleotide
microarrays, on silicon chips, can be used to gain information about all the processes going on in a particular cell or tissue and their modification in disease.4
Microarray techniques permit the analysis of the
expression levels of thousands of genes simultaneously, both in health and disease. They are thus expected to generate a vast amount of gene expression data
that may lead to a better understanding of the regulatory events involved in pathophysiological processes.
In addition, these techniques offer a systematic
approach for searching for effective targets for drug discovery and diagnostic markers.
Using oligonucleotide microarrays, we have investigated the differential gene expression profile in
unstimulated keratinocytes obtained from non-lesional skin of 6 atopic dermatitis patients when compared
to cells originated from healthy controls. Differentially expressed transcripts could be clustered in functional groups, including transcription factors, signal
transducers, cell cycle regulators and a heterogenous
set of enzymes. On a panel of 10 genes, we then looked
for verification of our gene chip data by quantitative
real-time reverse transcription polymerase chain reaction (RT-PCR).
476
Materials and methods
Subjects
Six well informed volunteer patients with moderateto-severe chronic AD (age range 19-45 years) participated in this study. Skin involvement ranged from
20% to 60% of the body surface area. Serum IgE levels were elevated in 3 patients (120-2 000 kU/mL)
who concomitantly presented associated bronchial
asthma and/or rhino-conjunctivitis. None of the AD
patients received oral corticosteroids within 1 month
of skin biopsy and topical corticosteroids were not
administered for a period of at least one-week prior to
enrollment. Six well informed volunteer healthy individuals (age range 25-50 years) were used as controls.
They had no personal or family history of atopic diseases, and serum IgE levels were within normal limits. Epidermal sheets for keratinocyte cultures were
obtained from the roof of suction blisters raised on
normal-looking skin of all patients and control subjects.5
Keratinocyte cultures
Primary keratinocyte cultures were established by
seeding epidermal cells on a feeder layer of irradiated
3T3/J2 fibroblasts, as previously described.5 Thirdpassage keratinocytes were used in all experiments,
with cells cultured in the serum-free medium keratinocyte growth medium (KGM, Clonetics, San
Diego, CA), prepared from an essential nutrient solution supplemented with optimal concentrations of epidermal growth factor, hydrocortisone, insulin, bovine
pituitary extract, and antibiotics. In the 24 h prior to
RNA extraction, keratinocyte cultures were incubated
in a hydrocorticone-deprived KGM.
Analysis of gene expression using oligonucleotide
microarrays
We carried out parallel analysis of gene expression
with commercial human gene probe arrays with the
capacity to display transcript levels of 12 500 human
genes (U95A array, Affymetrix, Santa Clara, CA).
Sample labeling and processing were performed
according to supplier’s instructions, except that
hybridization was performed in 1xMES buffer (0.1 M
2-(N-Morpholino) ethanesulfonic acid (MES), pH 6.7,
1 M NaCl, 0.01% Triton X-100) and chips were
washed with 0.1xMES buffer, as previously described.6
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100
10
1
The mean of the expression level of each gene has
been calculated from the 6 replicates in each experimental group (AD patients and healthy subjects). A
pair-wise comparison was performed, and transcripts
with a ratio of normalized expression levels >2 or
<0.5, and a confidence level of at least 95% (P<0.05)
were regarded as differentially expressed/modulated.
According to this criterion, 111 genes were significantly down-regulated and 90 up-regulated in AD keratinocytes when compared to healthy controls.
Atopic dermatitis
Statistical analysis of the gene expression data set
1 000
10 000
We collected data by laser scanning and analyzed pixel levels with commercial software (Affymetrix).
Quantification of gene expression by quantitative realtime RT-PCR
Total RNA was prepared using TRIzol reagent (Invitrogen Life Technologies, Milano, Italy), treated with
DNase I (Qiagen, Milano, Italy) to totally eliminate
DNA and reverse transcribed (1 µg) for 15 min at 42°C
using an oligo(dT)12-18 primer (Applera, Monza,
Italy). Quantitative real-time RT-PCR was performed
using the SYBR Green PCR core reagents mix
(Applera). MWG Biotech (Milano, Italy) synthesized
the primers used. They were designed using the ABI
PRISM Primer Express 2.0 software (Applied Biosystems, Foster City, CA), and at least 2 primer pairs were
used for the quantitative detection of each transcript.
Gene-specific RT-PCR products were continuously
measured by incorporation of SYBR Green fluorescent
dye (Applera) into the double stranded DNA, using
the ABI PRISM 5700 sequence detection system
(Applied Biosystems). The transcript level of each
gene was normalized to the transcript level of β-actin.
Five replicates for each experimental point were performed, and differences were assessed with the twotailed Student’s t-test. Results are expressed as the relative fold increase or decrease of the specific transcripts in AD donors over one of the control group,
which was used as a calibrator.
Results
We analyzed differential gene array expression profile in unstimulated cultures of AD keratinocytes as
compared to healthy controls. Raw signal intensity
Vol. 140 - N. 5
1
10
100
1 000
10 000
Controls (arbitrary units)
Figure 1.—Differential gene expression in AD keratinocytes. Total mRNA
was prepared and gene expression profiles in unstimulated keratinocytes
from healthy controls (abscissa) and AD patients (ordinate) were analyzed
by oligonucleotide arrays. Data are represented as log/log scatter plots of
the raw expression values provided by GENECHIP software (Affymetrix).
Transcripts equally expressed in both arrays fell along the central diagonal. Outer diagonals represent differences of two- and five-fold magnitude, respectively. The color-code reflects the confidence level of differential
expression: red: (P = 1) means that there is no difference in the expression
or that the value has to be considered a negligible outlier. Blue (P = 0)
means that the difference in the expression has to be considered significant.
values are shown in Figure 1, where the mean values
of each transcript expression in healthy controls (in
abscissa) were plotted against the corresponding data
in AD keratinocytes. Transcripts equally expressed in
both arrays fell along the central diagonal. Outer diagonals represent differences of two- and five-fold magnitude, respectively. The color-code reflects the confidence level of differential expression, with red dots
representing transcripts with no significant difference
in the expression, or representing outliers, and blue
dots representing statistically significant values. The
majority of the differentially expressed genes were
detected below 1 000 absorbance arbitrary units, indicating that these genes were expressed at very low
levels both in AD and in control keratinocytes. Further
statistical analysis allowed the selection only of those
genes that were differentially expressed at a confidence level of more than 95% (which means P<0.05),
hence indicating that 201 genes (1.6% of all the genes
examined) were significantly dysregulated in AD keratinocytes. Of these, 90 genes were more than two-
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c-fos V01512
Mitocondrial transcription factor (MTF1) M62810
Protooncogene (c-mer) U08023
SRF accessory protein 1b (SAP1b) M85164
ETS-like gene (tel) U11732
Krüppel-associated box (KRAB) AF022158
Transcription factors
0
500
1000
1500
Leupaxin AF062075
Cbl-b U26710
Phosphotyrosine protein of 36 kDa (pp36) AJ223280
Mitogen-activated protein kinase (MEK5c)U71088
Protein phosphatase 2A (PP2A) M65254
Rab-family gene Rab36 NM004914
Phosphodiestarase 1α (PPD1α) D45421
Mitogen-activated protein kinase 4 (63kDa ERK3)U53442
Mitogen-activated protein kinase p38 β X59727
Mitogen-activated protein kinase phosphatase 4 (MAPKP4) Y08302
2000
2500
3000
Signal transducers
0
1000
2000
0
500
1000
3000
4000
Cdc2-like protein (PISSLRE) X78342
p16 INK4a/MTS1 U26727
MAD2 AJ000186
Cyclin-dependent kinase 2 (Cdc2) Y00272
P1-Cdc21 X74794
Cdk inhibitor p57/kip2 U22398
Checkpoint kinase 1 (CHK1) AF016582
HUS1-like checkpoint protein AF076844
Cell cycle regulators
1500
GTP cyclohydrolase I regulatory protein U78190
Acid sphingomyelinase (SMPD1) M81780
Metalloproteinase 2 /MMP-2) Z48482
Cathepsin O X82153
Cytochrome P450 (CYP2A13) U22028
Tyrosinase-related protein precursor (TYRP1) X51420
15-Lipooxigenase (15-LO) M23892
Dioxin-inducibile cytochrome P450 (CYP1B) U03688
Myeloperoxidase (MPO) M19507
2000
Enzymes
0
250
500
7500 1000
Gene expression (AU)
1250
1500
Figure 2.—Differential gene-expression patterns in human keratinocytes from healthy controls (white bars) and AD patients (black bars). Genes were
selected if differential expression between the 2 groups was determined at a confidence level of 95% based on t-test statistics and if at least a two-fold
change in expression level was observed. Each transcript is identified by the historical name of the corresponding gene and its GenBank accession number.
478
Ottobre 2005
6.0
3.00
c-fos
SAP1b
2.25
6.0
6AD
5AD
4AD
3AD
2AD
1AD
6KC
5KC
4KC
6AD
5AD
4AD
3AD
2AD
1AD
6KC
5KC
4KC
0.00
3KC
0.0
2KC
0.75
1KC
1.5
3KC
1.50
2KC
3.0
1KC
Relative units
4.5
Relative units
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3
Cathepsin O
SIMPD1
Relative units
Relative units
4.5
3.0
2
1
4AD
5AD
4AD
5AD
6AD
3AD
2AD
1AD
6KC
5KC
4KC
3KC
2KC
3AD
1.5
1KC
0
6AD
5AD
4AD
3AD
2AD
1AD
6KC
5KC
4KC
3KC
2KC
0.0
1KC
1.5
4
PP2A
GTP cyclohydrolase I reg. prot.
Relative units
Relative units
3
1.0
0.5
2
6AD
2AD
1AD
6KC
5KC
4KC
3KC
2KC
0
1KC
6AD
5AD
4AD
3AD
2AD
1AD
6KC
5KC
4KC
3KC
2KC
0.0
1KC
1
Figure 3.—Transcript levels in single keratinocyte cultures as obtained by quantitative real-time PCR. The levels of a set of transcripts that were
detected as differentially expressed according to gene chip analysis was verified by SYBR Green-based real-time RT-PCR in keratinocytes from
healthy controls (KC, white bars) and from AD patients (AD, black bars).
Vol. 140 - N. 5
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fold increased, with 20 of these more than five-fold
increased in AD keratinocytes. In contrast, 111 transcripts were significantly down regulated, with 39 of
these more than five-fold decreased in AD keratinocytes. Figure 2 shows the expression level of part
of the differentially expressed genes, clustered according to gene function. A first set of differentially
expressed transcripts encoded proteins that operate as
transcription factors. These include c-fos and serumactivated protein 1b (SAP1b). Among signal transducers, we found that AD keratinocytes presented
down-regulation of the transcript encoding protein
phosphatase 2A (PP2A), a key regulator of mitogen
activated protein kinase phosphorylation state,7 and
up-regulation of leupaxin, a cytoplasmic protein most
homologous to the focal adhesion protein paxillin.8
In the cluster of cell cycle regulators, gene chip analysis suggested that AD keratinocytes over-expressed
the cell cycle inhibitors p16 INK4a/MTS1,9 and
PISSLRE, this last a protein functionally homologous
to cycle-dependent cyclin 2 (cdc2).10 Finally, several
enzymes appeared invariably up-regulated in AD keratinocytes, including lipooxigenase (15-LO), metalloproteinase 2 (MMP-2), cathepsin O, acidic sphingomyelinase (SMPD1), and the regulatory protein of
GTP cyclohydrolase I.11 The microarray data were
verified by SYBR green-based quantitative real-time
RT-PCR of a panel of 10 dysregulated genes. The
accuracy of mRNA quantification depends on the linearity and efficiency of PCR amplification. In preliminary tests, we optimized each primer concentration
and verified absence of primer-dimers and nonspecific amplification of other unrelated gene products carefully analyzing the dissociation curve (data not shown).
Non-retro-transcribed controls were always used to
ascertain the absence of genomic DNA. Results of
quantitative real-time RT-PCR analysis in part diverged
from gene chip analysis. In particular, 2 genes (MMP2 and leupaxin) failed to confirm a differential expression among the 2 groups (data not shown). Furthermore, specific transcripts of 2 genes (PISSLRE and 15LO) were undetectable by SYBR green-based RTPCR. We confirmed the up-regulated expression of
cathepsin O transcript in keratinocytes from AD
patients, whereas important interindividual differences
in the expression of the transcripts corresponding to cfos, SAP1b, SMPD1, PP2A and GTP cyclohydrolase
I regulatory protein could be observed among the 6
AD patients (Figure 3). Finally, in contrast to the mean
480
expression data provided by gene chips, real-time RTPCR data of SAP1b transcript appeared down regulated
rather than up-regulated in AD keratinocytes.
Discussion and conclusions
In this study we used microarray analysis to compare
the patterns of gene expression in unstimulated keratinocytes from AD patients versus healthy controls,
in an effort to identify molecular pathways involved in
the localization of the inflammatory atopic disorder
to the skin. A major confounding factor in the interpretation of gene expression profiles in whole (crude)
tissue biopsies derives from the fact that a multitude of
cell populations, distributed throughout disparate levels of functional activation, contribute to the final picture, hence complicating the appreciation of diseaseand tissue- specific gene expression.12-14 To rule out this
problem, we used purified keratinocyte cultures from
healthy controls and AD patients and preliminarily
checked the existence of possible differential gene
expression profiles in unstimulated culture conditions.
Statistical analysis of raw microarray data indicated that
a considerable number of genes were dysregulated in
AD keratinocytes, and allowed their clustering into
relevant functional sets. However, the analysis of 10 differentially expressed transcripts performed by quantitative real-time RT-PCR did not always validate the
microarray results. In particular, the expression of leupaxin and MMP-2 did not display any significant variation between healthy and AD donors. Furthermore,
although we used distinct independent primers, the
cdc2-like PISSLRE and 15-LO could not be detected
by real-time RT-PCR. These observations suggest that
the low levels of specific gene transcripts characteristic of unstimulated culture conditions could critically
impair the reliability of the microarray technique in the
definition of gene transcription profile.
For other transcripts, e.g. c-fos and SAP1b, PP2A
phosphatase, cathepsin O, SMPD1 and GTP cyclohydrolase I regulatory protein, we could observe relevant interindividual differences in their expression
levels. GTP cyclohydrolase I is the rate-limiting
enzyme involved in the biosynthesis of tetrahydrobiopterin, which operates as an indispensable cofactor
of nitric oxide synthase.11 Generation of nitric oxide
plays a major anti-inflammatory effect in chronic
inflammatory skin disorders, down-regulating the
expression of crucial chemokines including IP-10,
Ottobre 2005
MCP-1 and RANTES by epidermal keratinocytes.15
Hence, enhanced expression of the regulatory protein
of GTP cyclohydrolase I in a proportion of AD keratinocytes could implicate a reduction in the release of
nitric oxide also in vivo, and consequently a less efficient control of skin inflammation. We also found that
the transcript encoding for acid sphingomyelinase
(SMPD1) was significantly up-regulated in 3 out of 6
AD keratinocytes. This observation apparently contrasts with the data reported in a recent report, which
indicate that AD skin is characterized by a reduced
sphingomyelinase expression.16 Indeed, acid and neutral sphingomyelinases are involved in the regulation
of ceramides, which possess structural and signal transduction functions in epidermal proliferation and differentiation. A defective permeability barrier leads to
dry skin, which is a pro-inflammatory condition by
itself, and also to the enhanced penetration of environmental aeroallergens into the skin. Both these
processes are deeply involved in AD pathogenesis. Of
note, IL-4, a T cell-derived cytokine characteristically found in AD lesions, has been recognized as a major
factor in the down-regulation of SMPD1 expression in
these lesions.17
Noteworthy, c-fos transcript was significantly up-regulated in 4 out of the 6 patients examined, in keeping
with our previous data which associated enhanced
granulocyte/macrophage colony-stimulating factor
(GM-CSF) expression to higher levels of c-fos and
stronger AP-1 binding activity in a relevant proportion of keratinocytes from AD patients.18, 19 Strong
staining for GM-CSF can be observed in the lesional
skin of AD patients, and can thus contribute to explain
the nature and intensity of the dendritic cell infiltrate
found in the tissues affected by AD inflammation.
Keratinocytes from AD patients were also previously
shown to produce increased amounts of
RANTES/CCL5, a chemokine with a broad spectrum
of target cells, including monocytes, dendritic cells
and T lymphocytes.20 Quite interestingly, abnormal
expression of GM-CSF and RANTES was similarly
found in epithelial cells isolated from the respiratory
tract of asthmatic atopic patients in response to air
pollutants, including diesel exhaust particles, ozone
and nitrogen dioxide.21, 22
SAP1b, which was expected to be expressed at higher levels in AD keratinocytes according to microarray data, appeared rather down regulated when examined by quantitative real-time RT-PCR. In contrast to
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SAP1a, SAP1b lacks a critical portion of its C-terminus. Consequently, SAP1b can not participate to ternary
complex formation on c-fos promoter and opposes
SAP1a-driven promoter trans-activation.23 Hence it
appears highly presumable that its expression levels
could be inversely correlated to c-fos levels in AD keratinocytes, suggesting its contribution to the regulation
of c-fos expression in human keratinocytes.
In 3 out of the 6 AD cases examined, the PP2A transcript was found expressed at least four-fold less than
the levels found in normal controls. PP2A activity was
shown to play a critical role in the regulation of AP-1
transcriptional activity in different leukocyte types,
due to its rapid de-phosphorylation of mitogen activated
protein kinases.7, 24 It is thus reasonable to hypothesize
that the reduced expression of this phosphatase may
increase the persistence of AP-1 activity in response to
pro-inflammatory stimuli.
Uniformed high expression levels of cathepsin O
(also identified as cathepsin K) were observed in AD
keratinocytes. This is a lysosomal cysteine proteinase
of the papain family with high sequence homology to
cathepsins S and L, expressed predominantly in osteoclasts.25 Cathepsin O is the cysteine protease with the
highest matrix degradation activity yet measured, suggesting its involvement in the process of tissue destruction and remodeling following secretion into the extracellular space. In the lung, a relevant source is represented by the respiratory (bronchial and alveolar)
epithelial cells.26 However, the regulation of the expression of cathepsin O and its functional significance in
physiopathologic processes remain to be investigated, not only in the lung but also in the skin. A vast
body of evidence over the last decade has shown that
proteases are involved in critical steps of mammalian
skin homeostasis, including the correct formation of the
stratum corneum and consequently a valid epidermal
barrier.27, 28 A characteristic feature of AD skin is its
abnormal epidermal barrier, which leads to the penetration of irritants and allergens, a facilitated inflammatory response and reactive hyperplasia.1 The regulation of proteolytic enzyme activity is a fragile balance
of many factors, including their cognate protease
inhibitors. The importance of regulated proteolysis in
epithelia is well demonstrated by the discovery of the
Kazal-type 5 (SPINK5) serine protease inhibitor as
the defective gene in Netherton syndrome, a congenital ichthyosis associated with erythroderma, a specific hair shaft defect, and atopic features.29 The defec-
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tive inhibitory regulation of the protein product of
SPINK5, LEKT1, results in increased protease activity in the stratum corneum, accelerated degradation
of desmoglein-1 and over-desquamation of corneocytes.30 Whether a similar defect in LEKT1 function
contributes to AD pathogenesis is, however, not confirmed yet. An independent line of research also suggested that an up-regulated activity of the stratum
corneum serine proteases, in particular of the stratum
corneum chymotryptic enzyme (SCCE), may lead to
a defective epidermal barrier similar to that observed
in chronic AD.31 Indeed, a statistically significant association was recently found between a genetic variation (AACC insertion) in the coding region of SCCE
and AD,32 which is now under further investigation
for confirmation of its functional meaning in the pathophysiology of this disease. In their whole, these data
suggest that the functional characterization of dysregulated proteolytic activity, which could possibly
include cathepsin O, may help identify the complex
molecular mechanisms underlying skin barrier perturbation in AD.
Riassunto
Profilo dell’espressione genica nei cheratinociti dei pazienti affetti da dermatite atopica
Obiettivo. I cheratinociti di pazienti con dermatite atopica presentano una spiccata tendenza a rilasciare livelli elevati di alcune citochine e chemochine, che, pertanto, possono
giocare un ruolo importante nella promozione e nel mantenimento dell’infiammazione. Inoltre, questo meccanismo
molecolare può essere implicato nella localizzazione cutanea
della sindrome atopica. La conoscenza del profilo qualiquantitativo dei trascritti genici attraverso la tecnologia dei
chip oligonucleotidici in una popolazione cellulare pura può
aiutare a individuare quali processi molecolari sono attivi
in vivo. Questa tecnologia è basata sull’ibridazione del RNA
complementare opportunamente marcato con oligonucleotidi specifici per migliaia di trascritti, i quali sono fissati su
un chip di silicone idoneo alla successiva analisi computerizzata del risultato. Partendo dall’RNA totale estratto da colture non stimolate di cheratinociti abbiamo ricavato il profilo dei trascritti differenzialmente espressi tramite analisi con
chip di oligonucleotidi.
Metodi. Nello studio sono stati arruolati 6 pazienti, con
un’età compresa tra i 19 e i 45 anni, affetti da dermatite
atopica da lieve a severa. Sei volontari sani, di età compresa tra i 25 e i 50 anni, sono stati usati come gruppo di controllo.
Risultati. I livelli relativi di espressione della maggior
parte dei 201 trascritti differenzialmente espressi sono risul-
482
tati globalmente piuttosto bassi (inferiori a 5 000 U di assorbanza) sia nei cheratinociti di controllo sia in quelli provenienti da soggetti atopici, a sottolineare il fatto che le colture
sono state esaminate in condizioni basali, ovvero in assenza
di stimolazione. Tra questi, si sono individuati trascritti codificanti per fattori di trascrizione, secondi messaggeri, regolatori del ciclo cellulare ed enzimi coinvolti nella risposta infiammatoria dei cheratinociti. Al fine di verificare i dati ottenuti dai chip di oligonucleotidi, si è impiegata una tecnica
indipendente di analisi quantitativa dei livelli di trascritto,
ovvero la real-time reverse transcription-polymerase chain
reaction (real-time RT-PCR) basata sull’impiego dell’intercalante SYBR Green. A questo scopo, si sono impiegate
almeno 2 coppie di primer per la quantizzazione accurata
di ogni singolo trascritto. Quest’analisi è stata applicata a
10 diversi trascritti appartenenti ai diversi gruppi funzionali
individuati, alcuni dei quali erano risultati sovra-espressi o
sotto-espressi nei cheratinociti di pazienti con dermatite
atopica in base all’analisi dei chip di oligonucleotidi. I dati
forniti dalla real-time RT-PCR sono risultati in parte non
sovrapponibili a quelli ottenuti con i chip di oligonucleotidi. In particolare, l’espressione dei trascritti per la metalloprotease (MMP)-2 e per la leupaxina, proteina associata alle
focal adhesion kinases, non è parsa differenzialmente espressa tra i 2 gruppi di donatori. Inoltre, il fattore di trascrizione
SAP1b non è risultato sovra-espresso, ma piuttosto sottoespresso nei cheratinociti da pazienti con dermatite atopica.
Trascritti per l’oncosoppressore PISSLRE (proteina cdc2-simile) e per l’enzima 15-lipoossigenasi (15-LO), implicato nella generazione dei leucotrieni, sono risultati talmente poco
rappresentati da essere sotto il limite di sensibilità della tecnica in tutti i soggetti esaminati. Infine, è risultata importante la variabilità interindividuale dell’espressione dei
trascritti specifici per il fattore di trascrizione c-fos, per la sfingomielinase acida (SMPD1), per la protein fosfatasi 2A
(PP2A) e per la proteina regolatoria della GTP cicloidrolasi. Solo il trascritto per la cistein proteasi catepsina O è
risultato omogeneamente più espresso in tutti i cheratinociti di pazienti con dermatite atopica.
Conclusioni. Globalmente, i dati raccolti indicano che l’esiguo numero dei donatori e i bassi livelli di espressione genica caratteristici della condizione di coltura basale possono
seriamente inficiare l’utilità di una tecnologia fine come quella dei chip di oligonucleotidi nella definizione del profilo di
espressione genica. Inoltre, l’esistenza di importanti differenze
interindividuali nell’espressione di alcuni geni (c-fos, PP2A)
indica la complessità dell’identificazione delle basi molecolari della predisposizione alle patologie infiammatorie, anche
tramite un approccio semplificato monocellulare. Tuttavia, il
dato dell’aumentata espressione di catepsina O, se confermato a livello proteico nelle colture cellulari e nella cute del
paziente atopico, può fornire un ulteriore elemento significativo a riprova dell’evidenza che un disturbo dell’equilibrio
proteasi-antiproteasi sta alla base del difetto di barriera epidermica caratteristico della dermatite atopica.
PAROLE CHIAVE: Dermatite atopica - Cheratinociti - Espressione genica.
Ottobre 2005
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Dermatol 2005;152:489-504.
13. Nomura I, Goleva E, Howell MD, Hamid QA, Ong PY, Hall CF et al.
Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin
prevents induction of innate immune response genes. J Immunol
2003;171:3262-9.
14. Nomura I, Gao B, Boguniewicz M, Darst MA, Travers JB, Leung
DYM. Distinct patterns of gene expression in the skin lesions of atopic
dermatitis and psoriasis: a gene microarray analysis. J Allergy Clin
Immunol 2003;112:1195-202.
15. Giustizieri ML, Albanesi C, Scarponi C, De Pita O, Girolomoni G.
Nitric oxide donors suppress chemokine production by keratinocytes
in vitro and in vivo. Am J Pathol 2002;161:1409-18.
16. Jensen J-M, Förstel-Host R, Baranowsky A, Schunck M, WinotoMorbach S, Neumann C et al. Impaired sphingomyelina activity and
epidermal differentiation in atopic dermatitis. J Invest Dermatol
2004;122:1423-31.
17. Hatano Y, Terashi H, Arakawa S, Katagiri K. Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier functions induced by tumor necrosis factor-α and interferon-γ in human epidermis. J Invest Dermatol 2005;124:786-92.
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18. Pastore S, Corinti S, La Placa M, Didona B, Girolomoni G. Interferon-γ promotes exaggerated cytokine production in keratinocytes cultured from patients with atopic dermatitis. J Allergy Clin Immunol
1998;101:538-44.
19. Pastore S, Giustizieri M, Mascia F, Giannetti A, Kaushansky K,
Girolomoni G. Dysregulated activation of activator protein 1 in keratinocytes of atopic dermatitis patients with enhanced expression of
granulocyte/macrophage-colony stimulating factor. J Invest Dermatol 2000;115:1134-43.
20. Giustizieri ML, Mascia F, Frezzolini A, De Pità O, Chinni ML,
Giannetti A et al. Keratinocytes from patients with atopic dermatitis and psoriasis show a different chemokine production profile in
response to T cell-derived cytokines. J Allergy Clin Immunol
2001;107:871-7.
21. Bayram H, Devalia JL, Khair OA, Abdelaziz MM, Sapsford RJ, Sagai
M et al. Comparison of ciliary activity and inflammatory mediator
release from bronchial epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients and the effect of diesel exhaust
particles in vitro. J Allergy Clin Immunol 1998;102:771-82.
22. Bayram H, Sapsford RJ, Abdelaziz MM, Khair OA. Effect of ozone
and nitrogen dioxide on the release of proinflammatory mediators
from bronchial epithelial cells of nonatopic nonasthmatic subjects
and atopic asthmatic patients in vitro. J Allergy Clin Immunol
2001;107:287-94.
23. Dalton S, Treisman R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element.
Cell 1992;68:597-612.
24. Avdi NJ, Malcolm KC, Nick JA, Worthen GS. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils. J Biol Chem 2002;277:40687-96.
25. Drake FH, Dodds RA, James IE, Connor JI, Debouk C, Richardson
S et al. Cathepsin K, but not cathepsins B, L or S, is abundantly
expressed in human osteoclasts. J Biol Chem 1996;271:12511-6.
26. Bühling F, Gerber A, Häckel C, Krüger S, Köhnlein T, Brömme D et
al. Expression of cathepsin K in lung epithelial cells. Am J Respir
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27. Zeeuwen PLJM. Epidermal differentiation: the role of proteases and
their inhibitors. Eur J Cell Biol 2004;83:761-73.
28. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell
death in the skin. Nar Rev Mol Cell Biol 2005;6:328-40.
29. Bitoun E, Chavanas S, Irvine AD, Lonie L, Bodemer C, Paradisi M et
al. Netherton syndrome: disease expression and spectrum of SPINK5
mutations in 21 families. J Invest Dermatol 2002;118:352-61.
30. Descargues P, Deraison C, Bonnart C, Kreft M, Kishibe M, IshidaYamamoto A et al. Spink5-deficient mice mimic Netherton syndrome
through degradation of desmoglein 1 by epidermal protease hyperactivity. Nat Genet 2005;37:56-65.
31. Hansson L, Backman A, Ny A, Edlund M, Ekholm E, Ekstrand Hammarstrom B et al. Epidermal overexpression of stratum corneum chymotryptic enzyme in mice: a model for chronic itchy dermatitis. J
Invest Dermatol 2002;118:444-9.
32. Vasilopoulos Y, Cork MJ, Murphy R, Williams HC, Robinson DA, Duff
GW et al. Genetic association between an AACC insertion in the
3’UTR of the stratum corneum chymotryptic enzyme gene and atopic
dermatitis. J Invest Dermatol 2004;123:62-6.
483
Thyroid diseases and skin disorders in pediatric patients
V. BRAZZELLI, 1 D. LARIZZA, 2 F. PRESTINARI, 1 T. BARBAGALLO, 1 M. M. LAURIOLA, 1 V. CALCATERRA, 2
A. DE SILVESTRI, 2 G. BORRONI 1
Aim. Thyroid disorders have a high prevalence in medical practice; they are associated with a wide range of diseases, which
they may or may not share etiological factors with. One of the
organs showing pathological manifestations is the skin, therefore, it is not surprising that patients with thyroid diseases
show a large variety of skin changes; the majority of them are
not the only association, however, they may provide valuable
clues in diagnosing thyroid diseases. Cutaneous changes, in
fact, are frequently the first manifestation to be noted. Furthermore, the link between skin disorders and thyroid diseases
in pediatric patients is very subtle. This study focuses on specific cutaneous/thyroid lesions and non specific cutaneous alterations of the hyperthyroid and hypothyroid states, as well as frequent associations of thyroid diseases with other cutaneous
and/or systemic disorders in pediatric patients.
Methods. An epidemiological study was performed on 123 consecutive pediatric patients (100 female, 23 male; F/M: 4.34/1;
mean age: 15.57+6.95 years) with a thyroid disease, treated in
the Endocrinology Unit of the Pediatric Department, Policlinico San Matteo, University of Pavia, Pavia, Italy.
Results. Non-specific manifestations secondary to hyperthyroid and hypothyroid states were found to be xerosis, keratosis pilaris, hyperhidrosis, hypertrichosis, striae cutis distensae.
Conclusion. The well known clinical aspects of hypo- and hyperthyroidism were not found in our study, but the close relationship between skin and thyroid diseases in young patients have
to be stressed.
KEY WORDS: Thyroideal diseases - Skin - Child.
C
utaneous manifestations of thyroid diseases are
frequent even though, in most cases, they are not
Address reprint requests to: V. Brazzelli, MD, Clinica Dermatologica,
Università degli Studi di Pavia, IRCCS-Policlinico S. Matteo, Piazza C.
Golgi 2, 27100 Pavia, Italia. E-mail: [email protected]
Vol. 140 - N. 5
1Department of Human and Hereditary Pathology
Institute of Dermatology, IRCCS Policlinico S. Matteo
University of Pavia, Pavia, Italy
2Department of Pediatric Sciences
IRCCS Policlinico S. Matteo
University of Pavia, Pavia, Italy
the only association. Some cutaneous signs may aid to
disclose a thyroid disease.
Dermatologic manifestations of thyroid diseases
include specific lesions (thyroglossal duct cysts and
cutaneous metastases from thyroid malignancies), nonspecific signs secondary to hyperthyroid and hypothyroid states, and the association of thyroid diseases with
other dermatologic and systemic disorders.1-6
Few reports in the literature concern cutaneous disorders associated with thyroid diseases in childhood.7
This study focuses on the occurrence of cutaneous
disorders in a cohort of 123 pediatric patients with
thyroid diseases.
An epidemiological study was performed on 123
consecutive pediatric patients (100 female; 23 male;
F/M: 4.34/1; mean age: 15.57+6.95 years) with a thyroid disease, treated in the Endocrinology Unit of the
Pediatric Department, Policlinico San Matteo, University of Pavia, Pavia, Italy.
485
BRAZZELLI
THYROID DISEASES AND SKIN DISORDERS IN PEDIATRIC PATIENTS
Among the 123 patients, 71 (13 M/58 F) had
Hashimoto’s thyroiditis, 25 (7 M/18 F) had congenital hypothyroidism, 18 (2 M /16 F) had
Graves’disease and 9 (2 M/7 F) developed iatrogenic
thyroid disease owing to radiotherapy for a neoplasia (1 patient had rabdomyosarcoma, 3 patients had
neuroblastoma e 5 patients had acute lymphatic
leukaemia).
The 15 patients with hyperthyroidism were treated with metimazole; 1 patient with hyperthyroidism
underwent a thyroidectomy; 77 patients (25 with
congenital hypothyroidism, 48 with Hashimoto’s
thyroiditis and 4 with iatrogenic thyroid disease)
were treated with levothyroxine; 30 patients were
not treated because they were in a state of euthyroidism (2 patients with a past hyperthyroidism, 23
with autoimmune thyroiditis and 5 oncological
patients).
The current skin status was carefully examined with
particular attention to cutaneous adnexa, temperature
and perspiration, to cutaneous pigmentation, to the
presence of congenital lesions (melanocytic nevi, cafèau-lait spots, angiomas), to the presence of specific
dermatologic diseases or systemic disorders and to
the body mass index (BMI) (body weight in kilograms
divided by the height in meters squared used as a practical marker to assess obesity).
We also considered a control group composed by
100 healthy subjects, homogeneous for age and origin
to the group of patients with thyroid diseases (65 F/35
M; F/M: 1.85/1; mean age: 14.36±4.71 years).
Comparisons between thyroiditis patients and control subjects were performed with 2 or Fisher exact
test, as appropriate.
Furthermore Bonferroni correction was used for
multiple comparisons. Due to the small number of
patients we excluded iatrogenic thyroid diseases in
oncological patients (N=9).
Results
Results are summarized in Table I.
Non-specific manifestations secondary to hyperthyroid and hypothyroid states were found to be
xerosis (9.75% of patients with thyroid diseases and
8% of those belonging to the control group); keratosis pilaris (16.26% of the total and, particularly,
22.22% of patients with hyperthyroidism, 16.9% of
patients with Hashimoto’s thyroiditis and 16% of
486
the ones with congenital hypothyroidism versus 10%
of control subjects); hyperhidrosis (16.26% of
patients and, particularly, 21.12% of patients with
Hashimoto’s thyroiditis versus 4% of control subjects: P=0.0004; Pc=0.012); hypertrichosis (8.94%
of the total and, particularly, 11.26% of patients with
Hashimoto’s thyroiditis, 11.11% of the ones with
iatrogenic thyroid disease and 5.55% of those with
hyperthyroidism, versus 2% of control subjects);
striae cutis distensae (20.32% of the total and, particularly, 27.77% of hyperthyroid patients [P=0.007;
Pc=ns], 22.53% of the ones with Hashimoto’s thyroiditis [P=0.0005; Pc=0.015] and 12% of those with
congenital hypothyroidism versus 5% of control
subjects).
BMI was performed in the 25 patients with striae and
it was 25.72±5.00; BMI of the 98 patients without
striae was 19.98±3.67 (P<0.0001).
The dermatologic disorders related to thyroid diseases were found to be café-au-lait spots (25.2% of
the total and, particularly, 44.44% of patients with
hyperthyroidism [P=0.0001; Pc=0.003], 25.35% of
patients with Hashimoto’s thyroiditis [P=0.007; Pc=ns],
22.22% of the ones with iatrogenic thyroid disease
and 12% of those with congenital hypothyroidism versus 10% of control subjects); halo nevi (3.25% of the
total and, in particular, 4.22% of patients with
Hashimoto’s thyroiditis and 4% with congenital
hypothyroidism versus 2% of control subjects); congenital nevi (13% of the total and, in particular, 15.49%
of patients with Hashimoto’s thyroiditis, 12% of the
ones with congenital hypothyroidism, 11.11% of those
with iatrogenic thyroid disease and 5.55% of patients
with hyperthyroidism versus 20% of control subjects);
capillary hemangiomas (8.94% of the total and, particularly, 11.11% of both those patients with iatrogenic thyroid disease or with hyperthyroidism, 8.45%
of the ones with Hashimoto’s thyroiditis and 8% of
those with congenital hypothyroidism versus 12% of
control subjects).
With regard to autoimmune or immunomediate dermatologic diseases we report: alopecia areata (1.62%
of the total and, particularly, 5.55% of hyperthyroid
patients and 1.4% of patients with Hashimoto’s thyroiditis versus 2% of control subjects); vitiligo (2.81%
of patients with Hashimoto’s thyroiditis and no one
of the control group); atopic dermatitis (7.31% of the
total and, particularly, 11.11% of patients with hyperthyroidism, 8.45% of patients with Hashimoto’s thy-
Ottobre 2005
BRAZZELLI
TABLE I.—. Dermatologic disorders associated with thyroid diseases in 123 pediatric patients. Data have been compared with a control group
homogeneous for age and origin.
Iatrogenic
thyroid diseases
in oncological
patients
Total patients
with thyropathy
Control group
71
18
(25.35%)
P=0.007
Pc=0.049
16
(22.53%)
P=0.0005
Pc=0.0035
12
(16.9%)
6
(8.45%)
9
2
(22.22%)
123
31 (25.2%)
M=4 F=27
100
10 (10%)
M=4 F=6
1
(11.11%)
25
(20.32%)
M=5 F=20
5 (5%)
M=1 F=4
0
10 (10%)
M=4 F=6
8 (8%)
M=4 F=4
15
(21.12%)
P=0.0004
Pc=0.049
8
(11.26%)
P=0.01
Pc=ns
11
(15.49%)
0
20
(16.26%)
M=2 F=18
12
(9.75%)
M=2 F=10
20
(16.26%)
M=4 F=16
1
(11.11%)
11
(8.94%)
M=0 F=11
2 (2%)
M=0 F=2
1
(11.11%)
20 (20%)
M=9 F=11
1 (4%)
3
(4.22%)
0
2 (11.11%)
2 (8%)
6
(8.45%)
1
(11.11%)
16
(13%)
M=2 F=14
4
(3.25%)
M=1 F=3
11
(8.94%)
M=1 F=10
2 (11.11%)
1 (4%)
6
(8.45%)
0
10 (10%)
M=3 F=7
Psoriasis
1 (5.55%)
0
1
(1.4%)
0
Alopecia areata
1 (5.55%)
0
1
(1.4%)
0
0
0
2
(2.81%)
0
9
(7.31%)
M=2 F=7
2
(1.62%)
M=0 F=2
2
(1.62%)
M=0 F=2
2
(1.62%)
M=0 F=2
Hyperthyroidism
(Graves’ disease)
Congenital
hypothyroidism
Autoimmune
thyroiditis
Number of patients
Cafe-au-lait spots
18
8 (44.44%)
P=0.0001
Pc=0.0007
25
3 (12%)
Striae distensae
5 (27.77%)
P=0.007
Pc=0.049
3 (12%)
Keratosis pilaris
4 (22.22%)
4 (16%)
Xerosis
2 (11.11%)
3 (12%)
Hyperhidrosis
2 (11.11%)
3 (12%)
Hypertrichosis
1 (5.55%)
1 (4%)
Congenital nevi
1 (5.55%)
3 (12%)
Halo nevi
0
Angiomas
Dermatologic-immunomediate
diseases
Atopic dermatitis
Vitiligo
roiditis and 4% of the ones with congenital hypothyroidism versus 10% of control subjects); psoriasis
(1.62% of the total and, particularly, 5.55% of hyperthyroid patients and 1.4% of patient with Hashimoto’s thyroiditis versus 1% of control subjects); autoimmune polyendocrinopathy (one patient with Hashimoto’s thyroiditis and no one of the control group);
antiphospholipid antibodies syndrome (one patient
Vol. 140 - N. 5
1
(11.11%)
4 (4%)
M=3 F=1
2 (2%)
M=0 F=2
12 (12%)
M=3 F=9
1 (1%)
M=0 F=1
2 (2%)
M=0 F=2
0
with Hashimoto’s thyroiditis and no one belonging to
the control group).
Other disorders as 3-β-hydroxysteroid-dehydrogenase (3-beta-HSD) deficiency (1 patient), growth hormone (GH) deficiency, polymalformative syndrome,
psycho-motor retardation, mental retardation, hydrocephalus, hypertrophic cardiomyopathy, pyelonephritis, congenital cataract, strabismus have been found.
487
BRAZZELLI
The relationship between the thyroid gland and the
skin is dynamic, complex and difficult to recognize,
particularly during childhood.
In general, dermatologic manifestations of thyroid
diseases are directly proportional to the age of the
patients and inversely proportional to the timeliness of
thyropathy diagnosis and to the hormonal compensation obtained with therapy.
The dermatologist, therefore, has an important role
in the precocious diagnosis of thyroid disease through
physical/skin examination.
In our study the most frequent specific dermatologic diseases in patients with a thyroid disorder are
café-au-lait spots present in 25.2% of total patients
and, particularly, in 44.44% of those with hyperthyroidism versus 10% of control subjects (P=0.0001;
Pc=0.0007).
Keratosis pilaris, consisting in keratinous plugs in the
follicular orifices surrounded by a variable degree of
erythema over the face, arms and tights,7-9 has been
found in 16.26% of total patients with a thyropathy
versus 10% of control subjects.
Furthermore, 17.97% of patients with an autoimmune thyroid disease showed keratosis pilaris, probably also related to the presence of atopic dermatitis.
Striae cutis distensae were observed in 20.32% of
total patients versus 5% the of control subjects. This can
be explained by rapid weight changes reported by
patients and influenced both by the hyperthyroid state
and by the therapy (metimazole, ablative). The BMI
showed a mean value significantly higher in the 25
patients with striae than in those without striae (BMI
25.72 versus 19.98; P < 0.0001).
Kirkeby et al.10 demostrated that cortisol disappears
more rapidly from the serum of hyperthyroid patients,
resulting in increased levels of ACTH in response to
increased cortisol degradation caused by elevated thyroid hormone activity.
Many authors 11-14 have suggested that ACTH
induces striae in those tissues that are mechanically
heavily strained, particularly in subjects with a constitutional predisposition. The catabolic effect of ACTH
mainly affects the activity of fibroblasts and leads to
a decreased deposition of mucopolysaccharides in the
basic substance.
Hypertrichosis was present in 11.26% of patients
with Hashimoto’s thyroiditis (versus 2% of control
subjects) and, particularly, in 14.58% of the ones in
488
a state of hypothyroidism treated with levothyroxine.
This is a distinctive pediatric cutaneous sign characterized by coarse dark terminal hairs appearing
on the back and upper arms. This is not hirsutism,
because the hair is not a secondary sexual distribution.2, 15
Hyperhidrosis is present in 16.26% of total patients
(versus 4% of control subjects), but the percentage
increased up to 21.12% in patients with Hashimoto’s
thyroiditis (P=0.0004; Pc=0.049) and up to 26% in
the ones with thyroiditis not treated. No pathogenetic
mechanism, however, can be supposed because every
patient was in a hyperthyroid state at the time of our
observation. A hypothetical link with transient hyperthyroid states, sometimes possible in Hashimoto’s thyroiditis, is presumable.16
Atopic dermatitis was more frequent in patients
with autoimmune thyropathies, that is to say Graves’
disease and Hashimoto’s thyroiditis. No increased
occurrence of alopecia areata and vitiligo was found
in comparison with the control group, contrary to the
association of such diseases with thyropathies in adults
reported in literature.17-22 Considering the pediatric
age of the recruited subjects, we supposed that these
diseases had not been revealed yet, but could subsequently appear.
No dermatological manifestations were found, neither in the group of 9 oncologic patients with iatrogenic thyroid disease nor in those with an acquired
hypothyroid state. The absence of cutaneous manifestations in these patients, without an autoimmune
trend and in a state of hormonal compensation (euthyroidism), is a further element proving the existence
of non-casual correlation among dermatologic diseases found in the different groups of patients and
their thyropathies.
In conclusion, the well known clinical aspects of
hypo- and hyperthyroidism were not found in our
study, but the close relationship between skin and
thyroid diseases in young patients have to be
stressed.
Riassunto
Patologie tiroidee e cutanee nei pazienti pediatrici
Obiettivo. Numerose sono le alterazioni della cute e degli
annessi cutanei descritti in pazienti con patologie tiroidee.
Sebbene, nella maggior parte dei casi, non si tratti di associazioni esclusive, alcune lesioni possono fornire un valido
Ottobre 2005
aiuto per la diagnosi di una patologia tiroidea sottostante.
Gli aspetti dermatologici correlabili alle patologie della tiroide possono essere divisi in lesioni specifiche (cisti del dotto tireoglosso e metastasi cutanee da neoplasie tiroidee),
alterazioni non specifiche (legate allo stato di ipertiroidismo o di ipotiroidismo) e patologie dermatologiche o sistemiche associate. In generale, le alterazioni di cute e annessi sono direttamente proporzionali all’età dei pazienti e inversamente proporzionali alla tempestività della diagnosi endocrinologica e al compenso terapeutico raggiunto. In Letteratura, pochi sono gli studi riguardanti le alterazioni cutanee
in corso di patologie tiroidee in età pediatrica. Scopo di questo lavoro è stato lo studio di pazienti pediatrici distiroidei,
al momento dell’osservazione, in compenso ormonale, per
evidenziare possibili correlazioni con la patologia cutanea e
degli annessi.
Metodi. È stato condotto uno studio epidemiologico su
123 pazienti pediatrici consecutivi (100 di sesso femminile,
23 di sesso maschile, F/M: 4,34/1; età media 15,57±6,95
anni) affetti da patologie tiroidee, trattati nell’Unità di Endocrinologia del Dipartimento di Pediatria del Policlinico S.
Matteo di Pavia.
Risultati. Nell’ambito dello studio, le lesioni dermatologiche di più frequente riscontro sono risultate le chiazze
caffè-latte, lo spinulosismo, le striae cutis distensae, l’ipertricosi e l’iperidrosi. Le patologie dermatologiche, di natura autoimmune o immunomediate come la dermatite atopica, sono state maggiormente riscontrate nei pazienti con
tireopatie autoimmuni, cioè con malattia di Graves oppure con
tiroidite di Hashimoto. Non è stato, invece, riscontrato un
aumento di alopecia areata e vitiligine, spesso descritto nei
pazienti adulti. Nel gruppo dei 9 pazienti oncologici, non
sono state rilevate alterazioni cutanee di rilievo. Tale dato
avvalora una correlazione non casuale tra le alterazioni dermatologiche riscontrate negli altri gruppi di pazienti e le
patologie tiroidee di base.
Conclusioni. Questo lavoro mette in evidenza uno stretto
legame tra cute e patologia tiroidea anche nei pazienti pediatrici ormai in compenso ormonale. Le alterazioni dermatologiche sono fini ma peculiari, spesso di ausilio alla diagnosi precoce di distiroidismo.
PAROLE CHIAVE: Tiroide, patologie - Cute - Età pediatrica.
Vol. 140 - N. 5
BRAZZELLI
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489
Magnetic resonance imaging application
in infantile hemangiomas and vascular malformations
C. GELMETTI, I. MOGLIA, L. RESTANO
Aim. The aim of this paper was to review the role of modern
diagnostic imaging in the evaluation of patients with vascular
anomalies: hemangiomas and vascular malformations.
Methods. We have analyzed 20 pediatric patients using the
magnetic resonance imaging (MRI) and the magnetic resonance angiography. For each pathology, we have compared
the literature findings with our clinical cases.
Results. MRI leaves some doubts in the diagnosis of 2 types of
lesions. The first type concerns the high-flow lesions: in this
case the differential diagnosis among a small arteriovenous
fistula, a hemangioma in the proliferation phase, a venous malformation with high feeding arteries and an arteriovenous malformation in the preclinical phase can be impossible. The second type concerns the low-flow lesions, in which the diagnosis
of combined vascular malformations such as venolymphatic
malformations can be very difficult.
Conclusion. Nowadays, MRI is the best technology in order to
provide information about type, location and extension of this
type of lesions. It is not invasive neither dangerous, does not
require the use of ionizing radiations, and presents minimum
disadvantages as sedation. Nevertheless, MRI alone is not sufficient to provide the necessary informations for the diagnosis
and treatment of all vascular anomalies.
KEY WORDS: Child - Hemangioma - Arteriovenous malformations - Magnetic resonance imaging.
V
ascular lesions are frequent, and up to 1/3 of
livebirths presents some kind of vascular marks,
that fortunately are unconsequential in the great majority of cases. Large vascular lesions or lesions located
Address reprint requests to: Prof. C. Gelmetti, Clinica Dermatologica,
Via Pace 9, 20122 Milano. E-mail: [email protected]
Vol. 140 - N. 5
Pediatric Dermatology Unit
Institute of Dermatological Sciences
University of Milan,
IRCCS Foudation, Ospedale Maggiore Policlinico
Mangiagalli, Regina Elena, Milan, Italy
in unfavourable sites, that may cause cosmetic problems, functional disability or even life-threatening
complications, are far less frequent, but not exceedingly rare. Unfortunately, at the present time, the vascular abnormalities are relatively poorly known among
non-experts, and a patient with this kind of lesions
will often receive different diagnoses and treatment
options from different phisicians. The need for homogeneous standards of diagnosis and treatment for these
lesions is, therefore, a significant issue. The nomenclature and classification for superficial vascular
anomalies have been discussed in international workshops for over 20 years since the first publication of
John Mulliken.1 The classification used in this article
is that approved by the International Society for the
Study of Vascular Anomalies (ISSVA) initiated in
1992. This classification is based on clinical, radiological, histopathological and hemodynamic characteristics; it was further completed by the work of
Requena et al. in 1997.2 According to this system,
there are 2 major categories of superficial vascular
anomalies found in infancy and chilhood: infantile
hemangiomas (IH) and vascular malformations
491
GELMETTI
MAGNETIC RESONANCE IMAGING APPLICATION IN INFANTILE HEMANGIOMAS AND VASCULAR MALFORMATIONS
TABLE I.—Summary of the clinical findings in our patients.
Name
Age
Sex
No. of lesions
A. T.
V. Y.
C. S.
C. M.
M. M.
M. C.
B. G.
R. S. A.
C. A.
D. S.
C. V.
L. M.
S. A.
P. E.
R. R.
T. A.
F. A.
L. L.
O. S.
L. C.
5 years
8 and 10 years
4 years
4 years
6 years
6 years
11 months
3 months
1 year
10 months
4 years
1 year
2 years
1 and 3 months
6 years
4 years
4 months
1 year
3 months
4 years
F
M
F
F
M
F
F
F
F
F
F
M
F
F
F
F
F
M
F
M
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
(VM).3, 4 IH exhibit cellular proliferation; they grow
during infancy, involute in childhood and never appear
in an adolescent or an adult. In contrast, VM are
anatomical abnormalities and consist of dysplastic
vessels. These lesions do not exhibit endothelial cell
proliferation and they never regress. Some VM are
stable, whereas others expand; VM can be further
divided into 2 categories, based on rheology and channel morphology. Fast-flow vascular malformations
may be either arterial malformation (aVM), arteriovenous fistula (avF) or arterial venous malformation (avVM). Slow-flow vascular malformations may
be venous malformations (vVM), lymphatic malformations (lVM) and capillary malformations (cVM).
There are also complex-combined vascular malformations as capillary-venous malformations (cvVM),
lymphatic-venous malformations (lvVM) and so on.
Many eponymous syndromes fall into these categories.
Twenty patients suffering from IM or VM followed
in our Department during their infancy or childhood
period were submitted to magnetic resonance imaging (MRI). The characteristics of the patients are summarized in Table I. All patients, but one (C.V.) who had
2 lesions, had a single lesion. Two patients (P. E. and
492
Localization
Right parotid area
Left upper limb
Head
Head
Right lower limb
Right lower limb
Sternal area
Left thorax
Trunk
Left parotid area
Right upper limb
Right cheek
Glabella
Head
Left cheek
Chin
Thorax, abdomen, lower limbs
Right parotid area
Head
Left foot
Diagnosis
MRI
IH
lvVM
IH
cVM
IH
vVM
IH
lVM
lvVM
IH
lVM
vVM
IH
PHACE
lvVM
IH
lv VM
IH
cVM
vVM
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
SE GE MRA
V. Y.) had 2 exams, all the others had one MRI. One
patients suffered from PHACE syndrome.5
Results
Infantile haemangiomas
IH in the proliferation phase are well-circumscribed
soft-tissue densely lobulate lesions. They have no capsules and show diffuse enhancement. Dilated feeding
arteries and draining veins are seen within and around
the lesion. The spin-echo (SE) shows T1-weighted
iso-hypointense image, similar to the muscle tissue
and T2-weighted hyperintense image, similar to the fat
(Figures 1, 2).
Both show flow voids within and around the lesion.
They represent high flow vessels, typical of hemangiomas. After an intravenous gadolinium administration there is a diffuse enhancement with contrast
between the lesion (bright appearance) and the tissues
around (dark appearance). This technique with contrast
is used only in ambiguous cases. Gradient-recalled
echo sequences (gradient echo, GE) show fast flow
vessels within and around the mass with a high intensity signal opposed to the low intensity signal of the tissues around, hemangioma mass included. IH in the
involution phase, present a higher quantity of fat tissue,
which shows a more or less homogenous T1- and T2-
Ottobre 2005
Figure 1.—Hemangioma in a 4-year-old little girl on the lower lip and on
the chin area.
weighted hyperintense signal in SE, depending on the
involution degree.
Vascular malformations
In avVM, dilated high flow vessels could be seen in
SE as flow void areas (black tubular structures), and in
GE as enhancement signal areas (white tubular structures). Depending on vessel dimensions, the avVM
could be more or less detectable. Instead of hemangiomas, avVM could not present an associated parenchy-
GELMETTI
ma mass but could present some abnormal signals linked
to a fibroadipose mass, an edema or a hemorrhage/
thrombosis. In these cases, SE shows several high-intensity small pointed diffuse areas. The MRI shows the
avVM location and the soft tissue overgrowth degree.
vVMs represent the most common lower extremity
vascular malformations. They do not display high flow
component. In these lesions, SE often shows a mass
enhanced diffusely with intravenous contrast. Rounded T1- and T2-weighted hypointense areas represent
phleboliths and thrombi; vVM could present a variable
rapport S/R in T1-and T2-weighted signals depending
on a hemorrhage and/or a thrombosis. Normally they
present more often T1-weighted intermediate intensity (isointense) signals (higher than muscle signal) and
T2-weighted high intensity (hyperintense) signals
(higher than subcutaneous fat). GE shows non-enlarged
arteries, but dilatations and anomalies of the axial
venous systems may be seen. Phleboliths, when present, are seen as flow void areas.
lVM show 2 components in SE: a lymphatic tissue
component with T1-weighted isointense signal and a
cystic component with T2-weighted lightly hyperintense homogenous signal. The lymphatic cyst components are not enhanced with gadolinium contrast
but the edges and septum do. Gradient-recalled echo
sequences do not show flow voids neither enhanced
flow. Otherwise, nearby veins could be dilated or
abnormal.
Figure 2.—A) Sagittal T1-weighted MR imaging on the lower lip and on the chin. The hemangioma is evident as a uniform soft tissue mass with hypointense
signal similar to surrounding muscle. Flow voids, indicating dilated feeding draining vessels, seen within and adjacent to the mass. B) Sagittal T2-weighted sequence also shows the uniform signal enhancement of the hemangioma with internal and surrouding flow voids, similar to the fat. Some flow voids
depending on the presence of high flow vessels are seen. C) Gradient recalled echo technique shows in the axial section a hypointense lesion with high
flow vessels within, detectable by a hyperintense signal as carotid and jugular vessel signal.
Vol. 140 - N. 5
493
GELMETTI
Figure 3.—A 4-year-old female infant newborn with a predominant capillary malformation on the trunk, abdominal area and lower extremities.
Figure 4.—Axial proton density image: this lesion is so superficial that normally it involves just the cutaneous tissue. The soft parts are lightly thickened with a not homogenous signal.
In most of cases our study confirms the data of the
literature; however, we found a number of discrepancy between the data of the literature and MRI imaging
in some of our cases. In the complex malformations,
in particular, the MRI did not provide enough elements for a precise diagnosis, as for instance in our case
of lvVM in a 4-month-old female infant newborn (FA)
presented with a predominant capillary malformation
on the thorax, abdominal area and lower extremities
(Figures 3, 4).
MRI evaluation only confirms a lesion maintaining
a superficial location, i.e. a capillary lesion. Only the
clinical diagnosis permitted us to identify the lymphatic and venous component of the lesion (an axilla
swelling the former, an angiokeratoma the latter).
Also the diagnosis of vVM gave us some problem:
in a child (LC) with a venous malformation confirmed
by angiography, we expected to find a T1-weighted
isointense signal, a T2-weighted hyperintense homogenous signal and only dilated veins in GE. In this case,
however, T2-weighted flow voids and brilliant tubular
structures in GE were also visible, suggesting the presence of a high-flow component that was not confirmed
by angiography.
Discussion
The diagnosis of VM located on the skin could be
simply performed looking at their history and clinical
aspect. Problems may arouse when the clinical diag-
494
nosis is not reliable, or when a possible extension of the
lesion in underlying tissues or the presence of visceral lesions must be evaluated. In these cases imaging is
needed for diagnosis. Values and limits of the imaging
techniques more commonly employed for the diagnosis of vascular lesions are briefly summarized below.
Ecography represents a low cost, non invasive technique, but its value is limited by its inadequate capacity to differentiate the vascular lesion from the surrounding tissue. It is mainly employed to visualize
liquid-filled lesions, such as the cystic component of
a cystic hygroma. It is also used to look for hepatic
lesions in patients with diffuse hemangiomatosis.
Eco-color-Doppler represents a chief diagnostic tool
in the diagnosis of aVM and avVM and permits to
easily differentiate vVM and lVM. Rapidly expanding
hemangiomas, however, can simulate the presence of
several small arteriovenous fistulas. The main limit
of eco-color-Doppler consists in the fact that this exam
relies mainly on the ability of the executor, and does
not permit to estabilish precisely the extension of the
lesion and its relationship with the surrounding structures.
Angiography also represents an important diagnostic tool in the study of aVM and avVM, and it can
be used to differentiate between a rapidly expanding
IH and a avVM. Being that this diagnostic issue can
arouse only in infants, it must be remembered that in
this age group angiography is a relatively high-risk
Ottobre 2005
GELMETTI
Gradient recalled echo
High flow?
No
Yes
T1, T2-weighted
T1-weighted + gadolinium
Discrete soft tissue mass?
No
Yes
Arteriovenous
malformation
Infantile
hemangioma
Diffuse enhancement?
Septal enhancement?
Arteriovenous
malformation
Lymphatic
malformation
Figure 5.—Flow chart for the diagnosis of vascular pathologies using MRI evaluation.
technique, due to the small size of the vessels and their
tendency to spasm. Angiography is always required
before an embolization procedure.
Phlebography with direct injection of the contrast
medium in the lesion represents a useful tool in vVM;
lymphography and lymphoscintigraphy with 99mTc
are used for the diagnosis of lVM.
CT scan with contrast medium does not furnish a
high-quality imaging of vascular lesions, and is mainly used to detect subtle lesions of the cerebral tissue in
patients with Sturge-Weber syndrome. This technique
is effective to evaluate the relationship between the
soft tissues involved in the vascular lesions and the
bone in cases, for instance, of spine dysraphism.
At the present time, MRI represents one of the best
technologies in order to provide information about
vascular lesions; the limit of this technique is represented by its inadequate ability to visualize bone and
calcifications. MRI is not invasive or dangerous,
because it is based on the use of magnetic fields and
radio waves and not of ionizing radiations, and it presents the minimum disadvantage of requiring sedation. MRI imaging for the study of vascular lesions
relies on different techniques. Briefly, for the imaging
of vascular lesions the following techniques are used:
SE T1- and T2 weighted images; saturation recovery,
partial recovery and inversion recovery images; SE
images; in selected cases the intravenous contrast
medium gadolinium can be used.6-16
Magnetic resonance angiography (MRA) provides
a good imaging of large vessels without the use of
Vol. 140 - N. 5
contrast medium, and for this reason it is particularly
useful in pediatric patients; it does not allow to visualize the small vessels. The techniques of time of flight
(TOF) angiography and phase contrast (PC) angiography are the most used. In contrast enhanced
MRA 9, 10 an intravenous contrast medium is added.
Conclusions
At the end of our study we thus can affirm that MRI
might present some doubts in 2 cases:
— in high-flow lesions, in the differential diagnosis
among a small avF, an IH in the proliferation phase, a
vVM with a high feeding arteries and an avVM in the
preclinical phase;
— in low flow lesions, in the diagnosis of combined
vascular malformations (venolymphatic).
Figure 5 shows the flow chart recommended for the
diagnosis of vascular lesions using MRI techniques.
MRI allow to differentiate amongst 4 main different
types of lesions: IH, avF, vVM and lVM. The first step
consists in acquiring GE imaging, that assess the flowspeed of the lesions and thus permits to differentiate
between high-flow lesions and low-flow lesions. Then
the GE technique is used: T1 and T2-weighted MR
imaging can separate high-flow lesions in IH (that
show a discrete soft tissue mass with diffuse enhancement) and avF, wich lakcs a soft tissue mass and show
a non-homogenous enhancement.
495
GELMETTI
As regards low-flow lesions, after gadolinium contrast T1-weighted imaging of these lesions allows to differentiate between vVM (a mass diffusely or homogenously enhanced by the contrast medium) and lVM (a
“septate image”, i.e. lymphatic cyst components that are
not enhanced with gadolinium contrast and edges and
septa of the lesions that are enhanced by it).
Aknowledgements.—The authors wish to thank Dr V. Branca, head of
the Nuclear Magnetic Resonance Unit of our Hospital and Dr L. Balzarini, head of the Nuclear Magnetic Resonance Unit of the Humanitas Institute of Milan, for their kind cooperation in reading this paper.
Riassunto
Obiettivo. In questo lavoro viene valutato il ruolo della risonanza magnetica nucleare (RMN) nella diagnosi delle anomalie vascolari che sono suddivisibili in 2 principali categorie:
gli emangiomi capillari immaturi (ECI) e le malformazioni
vascolari (MV).
Metodi. Venti pazienti in età pediatrica sono stati studiati tramite RMN o angiorisonanza. Tali metodiche rappresentano la tecnica non invasiva che permette una diagnosi più
precisa della natura e dell’estensione della lesione senza
esporre il paziente a radiazioni ionizzanti. Mediante la RMN
è possibile differenziare le patologie vascolari in 4 patologie
di base: ECI, fistole artero-venose, MV venose e MV linfatiche. La tecnica gradient echo (GE) permette di differenziare
lesioni ad alto e basso flusso. La tecnica spin-echo (SE),
mediante le proiezioni pesate T2 e T1, differenzia le lesioni
ad alto flusso: quelle dotate di una discreta massa di tessuto molle con segnale omogeneo sono ECI, quelle prive di tale
massa e aventi segnale disomogeneo sono fistole arterovenose. Mediante la proiezione in T1 pesata e l’iniezione di
gadolinio, le lesioni a basso flusso si differenziano in malformazioni venose, se il segnale proveniente dal mezzo di contrasto è diffuso o uniforme, o in malformazioni linfatiche, se
il segnale è settato, ossia solo le pareti delle cisti linfatiche
hanno captato il gadolinio, divenendo brillanti.
Risultati. Nella casistica presentata, in molti casi le lesioni hanno mostrato un quadro RMN conforme alla letteratura; tuttavia, sono stati rilevate alcune discrepanze. Per quanto riguarda le malformazioni complesse, in particolare, la
RMN, spesso, non fornisce elementi sufficienti, come si è evidenziato in una paziente affetta da MV capillaro-veno-linfatica prevalentemente capillare localizzata al tronco, addome e arti inferiori. In questo caso, la RMN confermava una
lesione a estensione del tutto superficiale, quindi una lesione capillare, mentre la clinica evidenziava la componente
linfatica e venosa della lesione (caratterizzate la prima da
un rigonfiamento a livello ascellare, la seconda da un angiocheratoma). In un altra paziente che presentava una malformazione venosa, la RMN avrebbe dovuto riscontrare un
segnale isointenso in T1, uno iperintenso in T2 e solo una dilatazione del sistema venoso alla GE. Invece, è comparsa anche
496
una componente ad alto flusso che risulta in T2 con un vuoto di flusso e brillante alla GE. Questi dati non permettono
di affermare con certezza che si tratti di una lesione venosa
pura; tale diagnosi è stata confermata dall’esecuzione di
un’angiografia.
Conclusioni. Sebbene la RMN debba essere considerata
la tecnica di prima scelta per lo studio delle anomalie vascolari, soprattutto in pediatria, dove l’impiego delle radiazioni
ionizzanti o di altre tecniche invasive appare particolarmente rischioso, essa non è, tuttavia, ancora capace di fornire, da
sola, tutte le informazioni necessarie. In particolare, in questa casistica, la RMN ha presentato risultati dubbi in 2 casi: nelle lesioni ad alta portata, nella diagnosi differenziale tra una
piccola fistola arterio-venosa, un angioma capillare infantile
in fase proliferativa, una malformazione venosa con arterie
afferenti ad alta portata e una malformazione arterio-venosa
in fase preclinica; nelle lesioni a bassa portata, nella diagnosi delle malformazioni vascolari combinate veno-linfatiche.
Parole chiave: Età pediatrica - Emangiomi - Malformazioni
artero-venose - Risonanza magnetica nucleare.
References
1. Mulliken JB, Glowacki J. Hemangiomas and vascular malformations
in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg 1982;69:412.
2. Requena L, Sangueza OM. Cutaneous vascular anomalies. Part I.
Hamartomas, malformations, and dilatation of preexisting vessels. J
Am Acad Dermatol 1997;37:523-49.
3. Powell J. Update on hemangiomas and vascular malformations. Curr
Opin Pediatr 1999;11:457-63.
4. Drolet BA, Esterly NB, Frieden I. Hemangiomas in Children. N Engl
J Med 1999;341:173-81.
5. Metry DW, Dowd CF, Barkovich AJ, Frieden IJ. The many faces of
PHACE syndrome. J Pediatr 2001;139:117-23.
6. Bernard AM, De Certaines JD, Le Jeune JJ. Risonanza Magnetica
Nucleare, applicazioni biomediche. Milano: Masson; 1989.
7. Del Maschio A, Uslenghi CM, Musumeci R. Risonanza Magnetica in
Oncologia. Milano: Masson; 1993.
8. Guida all’imaging vascolare con risonanza magnetica. Milano: General Electric; 1997.
9. Korosec FR, Mistretta CA. MR Angiography. MRI Clin North Am
1998;6:223-56.
10. Laub G. Principles of Contrast – Enhanced MR Angiography. MRI Clin
North Am 1999;7:783-95.
11. Chaft J, Blei F. Prenatal diagnosis of vascular anomalies: update and
review of the literature. Lymphat Res Biol 2003;1:309-12.
12. Laor T, Burrows PE. Congenital anomalies and vascular birthmarks
of the lower extremities. MRI Clin North Am 1998;6:497-519.
13. Burrows PE, Laor T, Paltiel H, Robertson RL. Diagnostic imaging in
the evaluation of vascular birthmarks. Pediatr Dermatol 1998;16:
455-88.
14. De Franco A, Monteforte MG., Maresca G, De Gaetano AM., Manfredi R, Marano P. Diagnostica integrata dell’angioma epatico: Confronto tra eco color Doppler, Tomografia Computerizzata, Risonza
Magnetica. Radiol Med 1997;93:87-94.
15. Wenger DE, Wold LE. Benign vascular lesions of bone: radiologic and
pathologic features. Skeletal Radiol 2000;29:63-74.
16. Dubois J, Garel L. Imaging and therapeutic approach of hemangiomas
and vascular malformations in the pediatric age group. Pediatr Radiol 1999;29:879-93.
Ottobre 2005
Expression of placenta growth factor in mouse hair follicle cycle
F. CIANFARANI, M. L. ZACCARIA, T. ODORISIO, G. ZAMBRUNO
Aim. The hair follicle cycle comprises an anagen growth phase
followed by a catagen regression phase and a telogen resting
phase. During anagen neoangiogenesis events occur leading to
a marked increase in the extension of the perifollicular vascular network. The aim of the present study was to evaluate the
expression of the angiogenic cytokine placenta growth factor
(PlGF) in hair follicle and its relationship with changes in perifollicular vascularization during hair cycle.
Methods. A murine model of synchronized hair follicle cycling
induced by depilation was employed to obtain skin samples at different time points of the hair cycle. The evaluation of PlGF
expression and perifollicular vessels was performed by immunohistochemical analysis. A computer assisted morphometric analysis was used to quantify perifollicular vessel size and density.
Results. Immunoreactivity for PlGF was detected during anagen in follicular keratinocytes of the outer root sheath, with
maximum signal 8 days after depilation (middle anagen), while
no PlGF staining was observed in catagen and telogen phases.
Computer assisted morphometric analysis in perifollicular
dermis showed a significant increase in average vessel size and
in percentage of dermal area occupied by blood vessels during
late anagen.
Conclusion. The present study shows that PlGF protein is
upregulated in anagen mouse hair follicles, immediately before
the maximum peak of perifollicular angiogenesis. These results
suggest a role for PlGF in modulation of hair follicle-associated angiogenesis events. PlGF might act in synergism with the
closely related vascular endothelial growth factor (VEGF)
which has recently been shown to promote anagen angiogenesis and hair growth.
KEY WORDS: Hair cycle- Anagen - Hair follicle keratinocytes Angiogenesis - Angiogenic growth factors.
Address reprint requests to: F. Cianfarani, Laboratorio di Biologia
Molecolare e Cellulare , Istituto Dermopatico dell'Immacolata, IDI-IRCCS,
Via Monti di Creta 104, 00167 Roma, Italy. E-mail: [email protected]
Vol. 140 - N. 5
Laboratory of Molecular and Cell Biology,
Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy
D
uring all postnatal life hair growth takes place
through a cyclic regeneration phenomenon, in
which the hair follicle undergoes repeated cycles. Each
cycle comprises an active growth phase (anagen) with
proliferation of follicular keratinocytes and elongation and thickening of the hair shafts, followed by a
regression phase (catagen), that leads to involution of
the hair follicle itself, and, finally, by a resting phase
(telogen).1] Like the interfollicular epidermis, hair follicles are avascular. However, neoangiogenesis skin
events are associated with the anagen phase of cell
proliferation of the growing hair to support the
increased nutritional needs.2, 3 In the past years, dye
injection studies in rat and rabbit skin revealed changes
in the arrangement of perifollicular blood vessels during synchronized switches of hair follicles from anagen to telogen, suggesting that hair development is
linked to vessel proliferation.4 Furthermore, degeneration of the capillary loops within the dermal papilla
was observed during human hair follicle catagen.5
More recently, studies on the murine hair cycle have
revealed the crucial role played by both pro-angiogenic and anti-angiogenic growth factors in regulating
angiogenic events occurring during hair growth. Mice
overexpressing the angiogenesis inhibitor Thrombospondin-1 (TSP-1) are characterized by a delayed
hair follicle growth and reduced perifollicular angio-
497
CIANFARANI
EXPRESSION OF PLACENTA GROWTH FACTOR IN MOUSE HAIR FOLLICLE CYCLE
genesis, while in TSP-1 deficient mice the anagen follicle growth phase is significantly prolonged and associated with increased perifollicular vascularization.6
Vascular endothelial growth factor (VEGF) is an
homodimeric glycoprotein that exerts a strong angiogenic and permeabilizing activity by binding 2 tyrosine
kinase receptors on endothelial cells, VEGFR-1 and
VEGFR-2.7 In the skin, VEGF is constitutively
expressed at very low levels by keratinocytes and
strongly upregulated during the wound healing process
and in inflammatory and neoplastic diseases associated
with angiogenesis.8-11 VEGF mRNA has also been
shown to be strongly up-regulated in mouse follicular
keratinocytes of the outer root sheath during anagen,
in association with a pronounced increase in perifollicular vascularization.3 Placenta growth factor (PlGF)
is a dimeric glycoprotein structurally related to VEGF,
that binds with high affinity to the VEGFR-1.12 In the
last few years, PlGF has been demonstrated to represent a key mediator of adult angiogenesis in pathological conditions.13 In addition, the analysis of a transgenic mouse model overexpressing PlGF in basal keratinocytes has pointed out a role for this cytokine in
modulating angiogenesis in the cutaneous system.14, 15
In order to evaluate a potential involvement of PlGF
in the control of hair growth associated angiogenesis,
we have analyzed the expression of this factor in hair
follicles and its relationship with vascular development in perifollicular dermis using a mouse model of
highly synchronized hair follicle cycling.
Animals
C57Bl/6 6-8 weeks old female mice (Charles River,
Calco, Italy) were selected for their hair follicle telogen
state on the basis of pink color of dorsal skin.16 Mice
were anesthetized by intraperitoneal injection of 15
µL/g of 2.5% 2.2.2-tribromoethyl alcohol (Sigma
Aldrich, Milwaukee, WI, USA) and stripped of hair
using a wax hot mixture which was painted over their
dorsal region. After hardening, the wax was peeled-off,
inducing the resting follicles to enter anagen.17
Skin specimen collection
6-mm-diameter punch biopsies were performed on
mouse dorsal skin at 0, 1, 5, 6, 8, 12, 19 and 25 days
498
after depilation (3 mice per time point). Animals were
then sacrificed by cervical dislocation under anesthesia. Skin biopsies were immediately fixed in freshly
prepared 4% paraformaldehyde (Sigma Aldrich) in
phosphate-buffered saline (PBS) for 24 h, then ethanoldehydrated and paraffin embedded.
Immunohistochemical analysis
The antibodies used for immunohistochemistry
were: anti-mouse polyclonal PECAM/CD31 (M-20,
Santa Cruz Biotechnology, Santa Cruz, CA), at a concentration of 1 µg/mL overnight at 4°C, and anti mouse
polyclonal PlGF (M-18, Santa Cruz) at a concentration
of 4 µg/mL for 2 h at room temperature. Immunohistochemistry was performed on 4 µm thick paraffinembedded skin sections. Specimens were deparaffinized in xylene (Sigma-Aldrich), then ethanol rehydrated (Carlo Erba Reagenti, Rodano, Italy) and treated for 20 min with 0.3% H2O2 in PBS. Heat-induced
epitope retrieval treatment was performed in Chemate
citrate buffer pH 6.0 (Dako Cytomation, Glostrup,
Denmark) for 5 min in microwave oven at a power of
650 W for PlGF, or for 2 rounds of 2 and 5 min at 650
W for PECAM/CD31. Specimens were then preincubated with 3% bovine serum albumin (BSA, SigmaAldrich) in PBS for 1 h, and incubated with primary
antibodies at the indicated concentrations. Sections
were subsequently treated for 45 min with specific
biotinilated IgG (Vector Laboratories, Burlingame,
CA, USA) diluted 1:150 in 3% BSA in PBS and for 1
h with avidin-biotin peroxidase complex (Vectastain
Elite ABC kit, Vector Laboratories). Immunoreactivity was visualized with peroxidase reaction using
diaminobenzidine tetrahydrochloride (DAB) or 3amino-9-ethylcarbazole (AEC) in H2O2 and specimens counterstained with hematoxylin (Dako Cytomation). Negative controls were done by omitting the
primary antibody.
Computer assisted morphometric analysis of perifollicular blood vessels
Six different microscopic fields of PECAM/CD31
stained skin sections were examined at 100x magnification for each time point after depilation. The fields
analyzed were localized within 30 µm distance from
individual hair follicles. Vessel areas were calculated
by computer assisted image analysis, using a Zeiss
KS300, Version 3.0 program. Data were expressed as
Ottobre 2005
CIANFARANI
Figure 1.—Immunohistochemical localization of PlGF expression in mouse hair follicle. (A, B) Longitudinal section of hair follicles from a mouse
skin specimen taken 8 days after depilation (middle anagen). PlGF is expressed in the outer root sheath keratinocytes of the lower portion of the anagen hair follicle (arrows). (C, D) PlGF is not detectable in follicular telogen (C) and catagen (D) keratinocytes. Bars: A: 100 µm; B: 50 µm; C, D: 25
µm. Original magnifications: A: 25x; B: 50x; C and D: 100x.
Vol. 140 - N. 5
499
CIANFARANI
Mean vessel area
400
Vessel area (%)
4.5
*
*
4.0
3.5
300
%
µm2
3.0
200
2.5
2.0
1.5
100
1.0
0.5
0
1
6
A
8
12
0
19
1
6
B
Day after depilation
8
12
19
Vessel density
600
Vessels/mm2
500
400
300
200
100
0
C
1
6
8
12
19
means ± standard error. Student’s two-tailed t-test was
performed and a P < 0.05 was considered statistically
significant.
Results
PlGF is expressed in anagen hair follicles
To localize PlGF in mouse hair follicle, an immunohistochemical staining with a polyclonal antibody
against murine PlGF was performed on sections of
dorsal skin biopsies taken at 0, 1, 5, 6, 8, 12 19 and 25
days after depilation. These time points are known to
cover the 3 phases of hair follicle development: telogen (day 0 and 25), early (day 1 and 5), middle (day 6
and 8) and late anagen (day 12) and catagen (day 19).18,
19 At these time points, the hair cycle stage was also
confirmed by visual analysis of mice skin color, as
the development of mature anagen is associated with
500
Figure 2.—Analysis of perifollicular vascularization during hair cycle.
Computer assisted image analysis reveals significant changes of average
vessels size (A) and of relative areas covered by vessels (B), with a threefold increase of both parameters during anagen. Vessel densities was not
significant modified during the hair cycle (C). *P<0.01.
progressive skin pigmentation.19 No PlGF staining
could be visualized in early anagen (days 1 and 5 after
depilation). A faint cytoplasmic reactivity was observed
at day 6 (middle anagen) in the outer root sheath keratinocytes and maximal cytoplasmic PlGF staining
was detected 8 days post depilation. The staining was
present in all cell layers of outer root sheath and it
appeared limited to the lower portion of the hair follicle (Figure 1A, 1B). In late anagen (12 days after
depilation), a faint residual PlGF immunostaining was
still visible in approximately one half of hair follicles,
while no immunoreactivity was detected during catagen and telogen phases (Figure 1C, 1D).
PlGF expression in hair cycle is associated with perifollicular angiogenesis
In order to correlate PlGF expression during anagen
with perifollicular angiogenesis phenomena, a com-
Ottobre 2005
puter-assisted morphometric analysis was performed
on back skin sections taken at different time points
during hair cycle, and immunostained for the endothelial cell marker PECAM/CD31.20 During late anagen
vessels appeared larger and more elongated as compared to early anagen and also to catagen and telogen
phases. Indeed, a three-fold increase in perifollicular
vessel size was observed during late anagen (312±65.4
µm2, Figure 2A), as compared with early and middle
anagen. During the catagen phase of hair cycle perifollicular vessel size appeared markedly reduced
(75.8±25.8 µm2). In contrast vessel density, defined as
the number of vessels for area unit, showed a slight not
significant increase during anagen, if compared with
catagen phase (Figure 2C). Overall, the percentage of
perifollicular dermis occupied by blood vessels (Figure 2B) was significantly increased during late anagen
(3.3%(0.45 at day 12), as compared with early anagen
and catagen phases (0.92%(0.33 at day 1; 1.27%(0.22
at day 5; 1.02%(0.22 at day 19).
In the present study we investigated the involvement of PlGF in the modulation of angiogenic events
that are associated with hair follicle cycling. To this purpose, a well-established murine model of synchronized hair follicle cycling induced by depilation was
used. Our results show that PlGF is expressed in keratinocytes of the outer root sheath during the anagen
follicular active growth phase. PlGF protein was upregulated during the middle anagen, followed by a downregulation starting from late anagen, and its expression
was no more detectable in catagen involution phase and
in telogen resting phase. These results indicated a correlation between PlGF expression in hair follicles and
angiogenic events that were described to sustain the
anagen phase of cell proliferation.2 To define the temporal relationship between PlGF expression and angiogenesis events, we have then quantified the perifollicular dermal vascolature during hair cycle in the
same skin specimens. In keeping with previous studies 3 our results show a significant increase in both
perifollicular vessel size and percentage of perifollicular dermal surface occupied by blood vessels during
anagen. Maximum PlGF protein expression immediately preceded the peak of these angiogenic events.
Conversely, this growth factor was no more detectable
during catagen, when dermal vascolature has regressed.
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CIANFARANI
These results suggest a role for PlGF in angiogenic
events occurring during active anagen follicular growth
phase. Indeed, timely expression of PlGF by follicular keratinocytes could contribute to increased perifollicular anagen vascularization. During the last years
several evidences pointed out a role for PlGF in skin
angiogenesis. The expression of this growth factor is
normally extremely low in the skin and upregulation
has been reported in events associated with neoangiogenesis in the adult. In particular, PlGF upregulation occurs during wound healing keratinocytes 14 and
overlaps in time that of keratinocyte-released VEGF.
Together with the delayed wound closure observed in
PlGF knockout mice,13 these findings support a role for
PlGF in promoting angiogenesis during tissue repair.
Moreover, PlGF expression by melanoma cells appears
linked to tumor growth.21 Finally, the generation of
mice overexpressing PlGF in the epidermis 15 has contributed to define the angiogenic properties of this factor in the skin. Starting from fetal life and continuing
throughout lifetime, these mice show a striking increase
in the size of dermal blood vessels, with only a moderate increase in their density. In agreement with these
data, PlGF expression by anagen outer root keratinocytes is followed by a marked enhancement in
size and only a modest not significant increase in density of perifollicular vessels.
The presence of PlGF in the follicular keratinocytes
during anagen is also concomitant with VEGF expression in the same follicular compartment.3, 22 Using the
murine model of synchronized anagen hair follicles
induction, VEGF mRNA has been shown to be
expressed in outer root sheath keratinocytes during
middle anagen. In addition, transgenic overexpression
of VEGF in outer root sheath keratinocytes of hair follicles strongly induced perifollicular vascularization,
resulting in accelerated hair regrowth after depilation
and increased size of hair follicles and hair shafts.
Conversely, systemic treatment with a neutralizing
anti-VEGF antibody led to hair growth retardation
and reduced hair follicle size.3, 22 Together with our
results, these data suggest a potential cooperation
between PlGF and VEGF in modulating angiogenic
anagen events. Several experimental evidences have
shown that angiogenic activity of PlGF is mainly mediated through synergy with VEGF.13 A suggested explanation is that the binding of PlGF to VEGFR-1 could
displace VEGF from this receptor making it available
for binding to VEGFR-2, the main receptor mediating
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CIANFARANI
VEGF angiogenic activities. Another explanation for
the observed PlGF-VEGF synergism has come from
the demonstration that activation of VEGFR-1 by PlGF
transphosphorylates the VEGFR-2 receptor, amplifying VEGF activity. Moreover, PlGF/VEGF heterodimers could stimulate angiogenesis through the
formation of VEGFR-1/VEGFR-2 heterodimeric
receptors.23 Further studies are, however, required to
determine the role of PlGF-VEGF synergism in modulating hair cycle associated angiogenesis.
Impaired vascularization of the hair follicle has been
previously suggested to play a role in the pathogenesis of disorders characterized by hair loss, including
androgenic alopecia.24, 25 The evidence that PlGF
expression is finely modulated in hair follicles during
hair cycle suggests that this growth factor might represent a potential tool for therapeutic approaches aimed
at increasing the vascular support of hair follicles is
some forms of human alopecia.
Acknowledgements.—The authors wish to thank N. De Luca
and D. Carlone for skillful technical assistance.
Riassunto
Espressione del placenta growth factor nel follicolo pilifero murino
Obiettivo. Nel corso della vita adulta, i follicoli piliferi
sono caratterizzati da un’attività ciclica in cui a una fase di
crescita attiva (anagen), caratterizzata da un intenso processo di proliferazione cellulare e dall’accrescimento del fusto
del pelo, seguono una fase di involuzione (catagen) e una
fase di quiescenza (telogen). Numerose evidenze sperimentali hanno dimostrato che, nel corso dell’anagen, si verificano
fenomeni di neoangiogenesi che determinano l’estensione
della rete vascolare perifollicolare, necessaria per il corretto apporto di ossigeno e nutrienti nella fase di crescita attiva del follicolo. Il placenta growth factor (PlGF) è un fattore di crescita appartenente alla famiglia del vascular endothelial growth factor (VEGF), coinvolto nella modulazione dei
fenomeni angiogenici che si verificano durante la vita adulta. In particolare, negli ultimi anni si sono accumulati numerosi dati che indicano un ruolo del PlGF nel controllo dell’angiogenesi cutanea. In questo lavoro sono state analizzate l’espressione di questo fattore di crescita e la sua relazione con i cambiamenti della vascolatura perifollicolare durante le varie fasi del ciclo del pelo.
Metodi. Un modello murino in cui la sincronizzazione
del ciclo dei follicoli piliferi viene indotta attraverso depilazione a strappo della regione dorsale è stato utilizzato per
ottenere campioni istologici. Il prelievo di biopsie cutanee a
giorni successivi dalla depilazione (0, 1, 5, 6, 8, 12, 19 e 25
502
giorni) ha consentito di ottenere campioni istologici rappresentativi di tutte le fasi del ciclo del pelo.
Risultati. Attraverso un’analisi immunoistochimica è stato dimostrato che il PlGF viene espresso dai cheratinociti
della guaina follicolare esterna del segmento inferiore del follicolo pilifero già nella fase centrale dell’anagen, con un’intensità massima del segnale immunoistochimico rilevabile 8
giorni dopo la depilazione. Una debole immunopositività
per il PlGF è ancora rilevabile nell’anagen tardivo (12 giorni dopo la depilazione), ma non più durante il catagen o il telogen. Parallelamente, è stata condotto uno studio della vascolatura presente nella regione perifollicolare durante le varie
fasi del ciclo del pelo, attraverso un’analisi immunoistochimica con un anticorpo diretto contro l’antigene endoteliale
PECAM/CD31, seguita da un esame morfometrico computerizzato delle sezioni di cute immunomarcate. I risultati
ottenuti dimostrano che, nella fase finale dell’anagen, i vasi
presenti nel derma perifollicolare possiedono delle dimensioni
medie sensibilmente aumentate (aumento di circa tre volte)
rispetto a quelle misurabili sia nel catagen che nel telogen.
Un incremento analogo è stato osservato per quanto riguarda l’area percentuale del derma perifollicolare occupata dalla vascolatura. La densità vascolare, intesa come numero di
vasi per unità di area, è risultata, invece, aumentata in anagen in maniera non significativa rispetto alle altre fasi del ciclo
del pelo. I risultati ottenuti indicano, quindi, che il picco di
aumento della vascolatura perifollicolare è immediatamente successivo alla massima espressione del PlGF da parte
dei cheratinociti della guaina follicolare esterna. Un’analoga induzione è stata recentemente descritta anche per il
VEGF, la cui espressione, presente nei cheratinociti follicolari durante l’anagen, promuove l’estensione della rete
vascolare perifollicolare e la crescita del pelo. È noto che
gli effetti angiogenici del PlGF sono mediati, per una parte
importante, da un sinergismo con il VEGF. Un’azione sinergica del PlGF e del VEGF potrebbe, quindi, essere implicata nella modulazione dell’angiogenesi associata al ciclo del
follicolo pilifero.
Conclusioni. In alcune forme di alopecia, tra cui l’alopecia androgenetica, la vascolatura perifollicolare appare alterata. La modulazione dell’espressione del PlGF potrebbe,
dunque, rappresentare un potenziale strumento terapeutico
per aumentare l’apporto vascolare ai follicoli piliferi in specifiche forma di alopecia.
PAROLE CHIAVE: Ciclo cellulare - Capelli - Cheratinociti Angiogenesi - Fattori di crescita.
References
1. Paus R, Cotsarelis G. The biology of hair follicles. New Engl J Med
1999;341:491-7.
2. Mecklenburg L, Desmond JT, Muller-Rover S, Handijski B, Wendt G,
Peters EMJ. Active hair growth (anagen) is associated with angiogenesis. J Invest Dermatol 2000;114:909-16.
3. Yano K, Brown FL, Detmar M. Control of hair growth and follicle size
by VEGF mediated angiogenesis. J Clin Invest 2001;107:409-17.
4. Durward A, Rudall KM. The vascularity and patterns of growth of hair
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follicles. In: Montagna W, Ellis RA editors. The biology of hair
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Yano K, Brown FL, Lawler J, Miyakawa T, Detmar M. Thrombospondin-1 plays a critical role in the induction of hair follicle involution and vascular regression during the catagen phase. J Invest Dermatol 2003;120:14-9.
Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:1029-39.
Velasco P, Lange-Asschenfeldt B. Dermatological aspects of angiogenesis. Br J Dermatol 2002;147:841-52.
Brown L, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF et al.
Expression of vascular permeability factor (vascular endothelial
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Brown LF, Harrist TJ, Yeo KT. Stahle-Backdahl M, Jackman RW,
Berse B et al. Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis
herpetiformis and erythema multiforme. J Invest Dermatol
1995;104:744-9.
Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW
et al. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med
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Park J, Chen HH, Winer J, Houck KA, Ferrara N. Placenta growth factor. J Biol Chem 1994;269:25646-54.
Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol
M et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nature Med 2001;7:575-83.
Failla C, Odorisio T, Cianfarani F, Schietroma C, Puddu P, Zambruno
G. Placenta Growth Factor is induced in human keratinocytes during
wound healing. J Invest Dermatol 2000;115:388-95.
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15. Odorisio T, Schietroma C, Zaccaria ML, Cianfarani F, Tiveron C,
Tatangelo L et al. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J Cell Sci
2002;115:2559-67.
16. Paus R, Stenn KS, Link RE. Telogen skin contains an inhibitor of
hair growth. Br J Dermatol 1990;122:777-84.
17. Silver AF, Chase HB. An in vivo method for studying the hair cycle.
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18. Paus R, Van der Veen C, Eicmuller S, Kopp T, Hagen E, Muller-Rover
S. Generation and cycling remodeling of the hair follicle immnune system in mice. J Invest Dermatol 1998;111:7-18.
19. Slominski A, Paus R, Plonka P, Chakrabotry A, Maurer M, Pruski D.
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503
REVIEWS
Oxidative stress in the human epidermis
K. U. SCHALLREUTER 1, 2
The skin represents with 1.8 m2 the largest organ of the human
body protecting against water loss and physical, chemical and
biological threats. Besides the important barrier function, the
outer epidermal layer relies on a complicated well-tuned signalling network. One major role of the epidermal compartment is the maintenance of the redox balance, due to constant
exposure of reactive oxygen species (ROS). In this article the
basic biochemistry for ROS generation and their effects on
epidermal integrity and function is reviewed. Special emphasis is put forward to demonstrate the relevance of epidermal
hydrogen peroxide as contributor to oxidative stress and cell signalling/transcription depending on the concentration of this
ROS using the depigmentation disorder vitiligo as a model disease.
KEY WORDS: Epidermis - Hydrogen peroxide - Enzyme deactivation - Transcriptional activation - Antioxidants.
T
he human skin represents with 1.8 m2 the largest
organ of the human body. It is this organ which
has constantly to combat with endogenous and exogenous reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radicals and singlet oxygen. Hence their generation implies powerful
defence mechanisms for fine control of the redox staThe research was supported by the NIH, Stiefel International, German
Vitiligo Society (Deutscher Vitiligo Verein e.V. Hamburg/Germany) and
by private donations.
Address reprint requests to: Professor K. U. Schallreuter, Clinical and
Experimental Dermatology/Department of Biomedical Sciences, University of Bradford, Bradford/West Yorkshire, BD7 1DP, UK.
Vol. 140 - N. 5
1Unit
of Clinical and Experimental Dermatology
Department of Biomedical Sciences
University of Bradford, Bradford, UK
2Institute for Pigmentary Disorders in Association
with the Ernst Moritz Arndt University Greifswald, Germany
and University of Bradford, UK
tus to guarantee cellular homeostasis. Nowadays, there
is increasing knowledge that low concentrations of
hydrogen peroxide are essential in cell signalling.1-3
Unfortunately we are still far away from understanding the exact mechanisms.4-6 Defence strategies include
enzymes, such as superoxide dismutases, catalase,
glutathione reductase/glutathione, glutathione peroxidase, thioredoxin reductase/thioredoxin and the thioredoxin peroxidases.7-10 Moreover, small trapping molecules such as thiols (glutathione and thioredoxin),
vitamins C and E, the amino acids methionine and
tryptophan, 6 and 7 tetrahydrobiopterins and lipoic
acid play an important role. Here it is also noteworthy
that besides the absorbing effects of the skin pigment
the actual biopolymer melanin contains free radical
traps.11, 12 Oxidative stress occurs only when the rate
of ROS generation exceeds the capacity of the cell for
their removal. In this context it should be noted that a
correlation exists between skin colour and individual
free radical defence capacity because darker skin phototypes (Fitzpatrick classification) express significantly more effective mechanisms against UV induced
damage.13-15
505
SCHALLREUTER
OXIDATIVE STRESS INTHE HUMAN EPIDERMIS
TABLE I.—Exogenous and endogenous reactive oxygen species (ROS)
in the human skin.
—
—
—
—
—
—
—
—
Singlet oxygen
Superoxide anion
Hydroxyl radical
Hydroperoxyl radical
Hydrogen peroxide
Organic peroxides and hydroperoxides
Peroxynitrite anion
Nitric oxide
Structure and function of the human epidermis
The epidermis with its outer stratum corneum
forms the upper layer of the skin. The major cellular components are the keratinocytes, besides
melanocytes, Langerhans cells and Merkel cells.
The melanocyte is surrounded by approximately 36
keratinocytes forming the epidermal unit. 16 Both
cells have the full capacity for autocrine adrenergic
and cholinergic signal transduction.17, 18 A plethora
of mechanisms controls proliferation and differentiation of the epidermal compartment where calcium
is a major player.19
Generation of ROS from molecular oxygen
Despite oxygen (dioxygen, O2) is absolutely necessary for human life, it can be also toxic. This paradox is based on its electronic structure yielding a biradical nature. The reduction of O2 to water (H2O) requires
the transfer of 4 electrons. Although both O2 and H2O
are essential for life, intermediates produced by oneelectron reduction of O2 along this reduction pathway,
lead to the formation of ROS, which in turn can severely affect the homeostasis of all cells.20 The human epidermis is especially vulnerable due to its constant
exposure to high levels of ROS produced by physical, chemical and biological reactions in the milieu
exterior and interior. Table I summarises the individual ROS species generated in the human epidermis. In
order to avoid oxidative stress neutralising mechanisms must be in place to maintain homeostasis in this
compartment.
The most potent ROS is the hydroxyl radical (OH.)
because this radical can react with nucleotide bases
resulting in irreversible damage of DNA. Furthermore
it reacts with fatty acids and amino acids leading to lipid
Fenton reaction
Fe3+ + OH -+OH
Fe2+ + H 2O2
.
e-
.
A
O2 - + H 2O2
O2 + OH -+OH
e-
.-
Molecular oxygen
Superoxide anion
CIOe.g. by hypochlorite
D
506
H 2O2
B
.
HO
-
HO
-
O2
O2
C
.
+
.-
O2
O2
Haber-Weiss reaction
Hydroxyl radical
.
O2
H 2O2
Hydrogen peroxide
Cl- + H2O
1
O2
Figure 1.—Mechanisms
of ROS generation. A) Formation of. hydroxyl
.
radicals (OH ). B) Formation of superoxide anion radicals (O2 -). C) 2 ereduction of oxygen to hydrogen peroxide. D) Formation of singlet oxygen (1O2).
Ottobre 2005
SCHALLREUTER
7
875 cm-1
900 890 880 870 860 850
3500
A
3000
2500
2000
1500
1000
500
0
Wavenumber (cm-1)
Figure 2.—Idendification of H2O2 using Fourier-Transform-Raman spectroscopy. A) The H2O2 peak (8.8 mM) could be assigned to the O-O stretch
at 875 cm-1.21. B) Dose dependent in vivo epidermal H2O2 generation
using Fourier-Transform-Raman spectroscopy. Each measurement was
taken directly after the application of 1 MED UVB (311 nm) exposure on
skin phototype V (Fitzpatrick classification).21
peroxidation and to distorted membrane and protein
function, enzyme deactivation and disruption of transcription.20
OH. is produced directly from hydrogen peroxide (H2O2) by the Fenton reaction and by the HaberWeiss reaction (Figure 1A). In the Fenton reaction
electrons from free ferrous ions (Fe2+) produce OH.
and the hydroxyl ion (OH-), meanwhile the superoxide anion (O2-), produced from many single electron donors (Figure 1B), reacts with H2O2 yielding
O2, OH. and OH- by the Haber-Weiss reaction (Figure 1A). The two-electron reduction of O2 yields
H2O2 by sequential single electron transfer reactions
(Figure 1C). Moreover, the most damaging form of
O2 is singlet oxygen (1O2) which can be produced by
photosensitisers such as haem proteins (e.g.
cytochrome P 450, cytochrome c and haemoglobin).
Hypochlorite produced from myeloperoxidase or
from exogenous sources reacts with H2O2 to produce singlet oxygen (1O2) (Figure 1D). One clinical example for the damaging effect of this ROS is
porphyria cutanea tarda. Finally the generation of
nitric oxide (NO) by the NO synthases leads to the
peroxynitrite (OONO-) anion due to the rapid reaction between NO and O 2-. Peroxynitrite anion is
more reactive then H2O2.
Vol. 140 - N. 5
Raman intensity at 875 cm-1 (arbitrary units)
Raman intensity (arbitrary units)
6
5
4
3
2
1
0
0
B
0.15
0.3
0.45
0.6
0.75
0.9
UVB dose at 311 nm (Jules/cm2)
Evidence for exogenous
and endogenous H2O2 generation
in the human epidermis
In vivo evidence for epidermal H2O2 formation by
UV- irradiation
To date there is compelling evidence that UV–light
can generate ROS.21, 22 Recently it was shown in vivo
using FT-Raman spectroscopy that both UVA and
UVB produce H2O2.21 H2O2 production depends on
the dose. However, the values reach levels in the mM
range.21, 23 Figure 2A-B demonstrates this result by
following the O-O stretch at 875 cm-1.21
Catecholamine degradation in the human epidermis
leads to H2O2 formation
Both epidermal melanocytes and keratinocytes hold
the capacity for autocrine catecholamine synthesis and
degradation.24 Monoamine oxidase A (MAO-A), the
degrading enzyme for noradrenaline, produces H2O2 as
a reaction product from the oxidation of this catecholamine (Figure 3).25 For example it has been shown
that epidermal MAO-A enzyme activities are significantly increased in patients with vitiligo contributing to
H2O2 concentrations in the mM range.21, 23, 25
507
SCHALLREUTER
NH3
CHO
H
C
H
H
C
OH
MAO-A
+
O2
+
H
C
OH
H 2O
+
NH 4
+
H 2O2
OH
OH
OH
OH
3,4 DIHYDROXYPHENYLGLYCOALDEHYDE
NOREPINEPHRINE
Figure 3.—H2O2 production from norepinephrine by MAO-A.
NADPH
O2
2+
Fe
personal communication). After disproportionation
this concentration produces a 10 fold increase in H2O2.
Hence, this reaction can significantly contribute to
oxidative stress in loco.
3+
Fe
NADPH
oxidase
NADPH +
O2
OH
H 2O2
SOD
.
Fenton
reaction
or spontaneous reaction
Figure 4.—The oxygen burst initiated by NADPH-oxidase. NADPH-oxidase produces O2- which is degraded spontaneously or by SOD into H2O2.
This. ROS is converted by the Fenton reaction to the hydroxyl radical
(OH ).
Oxygen burst via NADPH–oxidase
Disruption of the epidermal homeostasis can induce
inflammation in association with infiltrating cells such
as activated leucocytes and macrophages yielding the
biological oxygen burst via the enzyme NADPH-oxidase.26 This membrane integrated haem protein uses
NADPH and arachidonic acid to produce superoxide
anion (O2-) from O2 which is spontaneously or enzymatically converted to H2O2 followed by the Fenton
reaction to produce the hydroxyl radical (OH.) (Figure
4). Its activity is regulated by calcium.26 In a normal
inflammatory response this ROS concentration can
increase up to 20 fold after only 15 min 20 (Wood JM,
508
Photo-oxidation of pterins produces H2O2 and pterin
–6-carboxylic acid
Earlier it has been shown that both epidermal keratinocytes and melanocytes hold the capacity for the
de novo synthesis/recycling and regulation of the essential cofactor (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin
(6BH4).27 In this context, H2O2 production was demonstrated after perturbed recycling of the cofactor due
to deactivation of the enzymes 4a-pterin carbinolamine
dehydratase (PCD) and dihydropteridine reductase
(DHPR).28, 29 Moreover, it was also shown that 6BH4
is readily photo-oxidised to 6-biopterin due to H2O2
generation by UV light 30, 31 and it was shown that 6biopterin can be cytotoxic to melanocytes under in
vitro conditions.32 Later it was recognised that 6biopterin as well as sepiapterin are further photo-oxidised to pterin-6-carboxylic acid producing stoichiometric amounts of H2O2.33
Superoxide dismutase and its induction by TNFα leads
to H2O2 generation in the mitochondrion
Nowadays it is well established that any physical or
chemical stress to the human skin causes the rapid
release of TNFα.15, 34 More than 20 years ago Wong
Ottobre 2005
et al. were the first to show that mitochondrial superoxide dismutase (MnSOD) was under transcriptional control of TNFα.35 It has also been shown that this
enzyme increases H 2O 2 concentrations in this
organelle by disproportionation of O2- leaking from
coenzyme Q in the respiratory chain.20 Here it is noteworthy that high concentrations of H2O2 actually lead
to the release of cytochrome c from the mitochondria
followed by initiation of the caspase mediated apoptosis cascade.36
The production of H2O2 from nitric oxide synthases
and xanthine oxidase
Both the nitric oxide synthases (NOS) and xanthine
oxidase are highly expressed in epidermal keratinocytes
and melanocytes. Tetrahydrobiopterin (6BH4) is the
critical cofactor for all NOS. In the absence of the
cofactor/substrate the enzyme can become uncoupled
producing ROS instead of NO.37 Under those conditions NOS produces O2- from O2. O2- can couple with
NO to produce the highly reactive and damaging peroxynitrite anion or it can spontaneously disproportionate to H2O2. However, xanthine oxidase produces
H2O2 from the degradation products of purine bases
released due to DNA damage for example after UVR
exposure. In this context it is noteworthy that both
enzymes require the essential pteridine cofactors, 6BH4
or molybdopterin respectively.38, 39
Cytochrome P450 produces H2O2 by the oxidation of
oestrogens and androgens
Cytochrome P450 enzymes are a superfamily of
structurally related monoxygenases which hydroxylate
a number of physiological compounds including
steroids, fatty acids and various xenobiotics.40 Recently it was recognised that oestrogens produce H2O2 in
blood lymphocytes and in human spermatozoa leading
to DNA damage shown by the COMET assay.41 Since
the human epidermis produces oestrogens and androgens it could be possible that these hormones could
contribute to oxidative stress under certain conditions
and this has been suggested in the case of vitiligo.42
Oxidation of hydroquinones produces H2O2
The oxidation of hydroquinone in the epidermal
compartment involves first the formation of a semiquinone radical followed by its oxidation to 1,4 ben-
Vol. 140 - N. 5
SCHALLREUTER
zoquinone. From this 2 electron oxidation reaction 2
O2- are produced. After disproportionation by SOD 1
O2 and 1 H2O2 are formed. This reaction can be blocked
by the glucuronide of hydroquinone or by the so called
Michael addition reaction.43, 44 Both of these reactions
occur in epidermal keratinocytes and melanocytes due
to the presence of thiol and selenohydryl antioxidant
enzymes.43-45 These conjugation reactions represent
the first line of defence preventing the generation of
ROS. When the concentrations of hydroquinone exceed
the capacity of the thiol containing anti-oxidant
enzymes these mechanisms can be directly deactivated by the compound.44 In this context it has been shown
that quinones are suicide inhibitors of thiol and selenohydryl based enzymes.43, 45
Melanin and H2O2 production - a widely underestimated paradox
Already at the turn of the century it was observed
and reported that low concentrations of H2O2 promote tyrosinase activity, meanwhile high concentrations of this ROS had the opposite effect by bleaching human skin.46-49 However, the accurate mechanism has been only now elucidated and it was shown
that H2O2 in concentrations of 10-6 M activates the
enzyme, whereas concentrations in the 10-3 M range
yield deactivation.50 While the black eumelanin works
as a UV filter, it also binds strongly transition metals
and calcium.51 Consequently this polymer can interfere with the Fenton reaction and adjusts homeostasis. Hence, it can be concluded that eumelanin not
only traps ROS, it also acts as a redox buffer due to
the removal of calcium.51 The red pigment pheomelanin has no photo-protection capacity because it
generates high concentrations of ROS such as O2-,
H2O2 and OH..12
ROS and cellular damage
Proteins, membrane lipids, carbohydrates and nucleic acids - all are subjects to cellular damage by oxygen
radicals. In this context it has been shown that the
amino acids histidine, proline, arginine, cysteine,
methionine and tryptophan are especially susceptible
to the attack by the hydroxyl radical leading to oxidation and consequently to protein fragmentation, crosslinking and aggregation and possible proteolytic digestion 20 (Figure 5).
509
SCHALLREUTER
Protein damage (oxidation of amino acid
residues, cysteine/SH groups and S-S bridges)
Mitochondrial DNA damage
RER
SER
Membrane damage
OH
H2O2
.
Mitochondrial DNA
Nucleus
DNA
_
O2
Nuclear DNA damage
_
O2
H2O2
Plasma membrane
Lipid peroxidation and massive calcium influx
Figure 5.—The effects of ROS on the cellular integrity.
Lipid peroxidation is a free radical chain reaction
resulting in the formation of the lipid radical which
is further propagated by O2 forming the lipid peroxy
radical and lipid peroxide. One compound formed is
the soluble malondialdehyde 20 (Figure 5).
Peroxidation of the lipid molecules leads to irreversible structural changes and disruption in the lipid
bilayer of membranes fostering a massive influx of
extra cellular calcium giving even more rise for cellular
damage (Figure 5).
ROS are also a major source for nuclear and mitochondrial DNA strand breaks/damage (Figure 5).
Moreover, ROS have also been invoked directly in
mutation and carcinogenesis.
Effects of H2O2 on the epidermal redox status a 2 edged sword
It is beyond any doubt that the human epidermis is
a major target for exogenous as well a endogenous
ROS formation, but oxidative stress is only the result
510
TABLE II.—Antioxidant enzymes in the human skin.
—
—
—
—
—
—
Catalase
Glutathione reductase
Thioredoxin reductase
Thioredoxin peroxidases
Glutathione peroxidases
Superoxide dismutases
TABLE III.—Low molecular weight antioxidants in the human skin.
—
—
—
—
—
—
—
—
Vitamins C and E
Reduced glutathione
Thioredoxin
Glutaredoxin
6 and 7-Tetrahydrobiopterin
Lipoic acid
Methionine, tryptophan
Selenium
of ROS generation exceeding the removal capacity of
the cellular defence mechanisms. H2O2 production is
under physiological conditions counteracted by
enzymes such as catalase, glutathione reductase, glu-
Ottobre 2005
SCHALLREUTER
Catalase
PCD/DCoH
Control
+H2O2 (10-3 M)
+H2O2 (10-6 M)
Figure 6.—Effects of H2O2 on epidermal catalase and 4a-pterin-carbinolanine dehydratase (PCD). Catalase (H2O2 degradation) and PCD (6BH4-recycling) protein expression are significantly reduced in the presence of 10-3 M H2O2, whereas both proteins are upregulated in the presence of 10-6 M H2O2.
tathione peroxidase, thioredoxin reductase and the
thioredoxin peroxidases (Table II) and small trapping molecules such as reduced glutathione, glutaredoxin, thioredoxin, lipoic acid, amino acids methionine and tryptophan, thiols and disulfide bridges,
tetrahydrobiopterins, vitamins E and C and selenium
(Table III).10, 20 However, under certain conditions, for
example in vitiligo skin, H2O2 can accumulate up to
mM levels consequently destroying the prosthetic
group of catalase.21, 52 Low epidermal catalase levels
are indeed observed in this depigmentation disorder
and after UVB exposure in association with mM concentrations of H2O2.53, 54 Recently deactivation of
several other important enzymes was documented in
vitiligo skin due to oxidation of methionine and/or
tryptophan residues in the active site of the proteins
Vol. 140 - N. 5
by concentrations of H2O2 in the mM range.28, 29, 55 In
this context it was shown that the recycling of the
cofactor 6BH4 is severely interrupted by deactivation of 2 enzymes i.e. PCD and DHPR.28, 29 Only
very recently it was recognised that acetylcholinesterase, the acetylcholine degrading enzyme,
is also deactivated by this ROS.55 After reduction of
the epidermal H2O2 concentrations with a pseudocatalase PC-KUS in patients with vitiligo it was
observed that catalase, PCD, DHPR as well as acetylcholinesterase enzyme activities and levels either
return to normal or are even upregulated. Figure 6
demonstrates the influence of H2O2 on epidermal
catalase and PCD protein expression in the presence
of mM H2O2 and after reduction of this ROS by a
pseudocatalase PC-KUS complex. Moreover, after
511
SCHALLREUTER
0.6
***
Conclusions
*
mU DHPR/mg Hb
0.5
0.4
0.3
0.2
0.1
n=10
n=27
n=10
Controls
10-3 M
10-6 M
0
Epidermal H2O2 concentrations
Figure 7.—Influence of epidermal H2O2 on dihydropteridine reductase
(DHPR) enzyme activities in whole blood of patients with vitiligo Epidermal H2O2 (10-3 M) decreases DHPR significantly, whereas concentration of 10-6 M upregulate significantly enzyme activities in whole blood.
This result confirmed H2O2 transfer from the epidermis to the vascular system.29
careful enzyme kinetics using recombinant enzymes,
it was observed that enzyme activities are under fine
control by H2O2 in a concentration dependent manner. In fact it was shown that low levels of H2O2 had
a very beneficial effect on all above enzyme activities
and on up-regulation of enzyme protein expression.28,
29, 55 The reduction of epidermal H O by a topical
2 2
applied pseudocatalase PC-KUS complex affected
surprisingly even the recovery of systemic blood
DHPR activities in patients with vitiligo (Figure 7),
confirming that epidermal generated ROS can clearly be transferred from the epidermis to the vascular
system.29
To continue, it has been shown that micromolar concentrations of H2O2 induce functioning wild-type p53
in the epidermis of patients with vitiligo.56, 57 In this
context it is of interest that the up-regulated p53 is
neither associated with increased apoptosis nor with an
increased prevalence of skin cancer in this patient
group.57, 58 Whether this up-regulated p53 points to a
permanent increased epidermal H2O2 level in vitiligo
needs to be shown. Moreover, H2O2 activates the transcription of important downstream signalling pathways for example NFκ B, AP1, heat shock proteins, Jun
and ERK.59 H2O2 also increases the phosphorylation
by inhibition of phosphatases.60 Furthermore this ROS
has been invoked in the activation of lymphocytes and
dendritic cells connecting this signal to the cellular
immune response.3, 61, 62
512
Taken together, nowadays it is evident that H2O2
has 2 faces which seem to be comparable with its
mother molecule dioxygen. Too much is deleterious
and toxic while little is of great benefit. Clearly this
ROS is of major importance in cell homeostasis. What
is exciting to this author is that the clinical observation
is such a wonderful teacher for basic science. Working
with patients suffering from vitiligo provides a great
model to study the influence of H2O2 on epidermal
homeostasis in great detail.
Future work must aim to get a fully balanced understanding of the fascinating ROS which can indeed be
a 2 edged sword.
Acknowledgement.—The author wishes to acknowledge her husband Professor J.M. Wood for stimulating discussions on the subject throughout the years. Moreover, without the help of the numerous MD and PhD students who joined her during many years little
progress would have been made.
Riassunto
Stress ossidativo nell’epidermide umana
La cute rappresenta, con i suoi 1,8 m2, l’organo più grande
del corpo umano ed essa lo difende dalla perdita di acqua e
da danni fisici, chimici e biologici. Lo strato più superficiale dell’epidermide, accanto all’importante funzione di
barriera, dipende da un complicato sistema ben modulato
di segnali. Uno dei ruoli maggiori del compartimento epidermico è rappresentato dal mantenimento del bilancio redox,
dovuto alla costante esposizione a specie reattive all’ossigeno (ROS).
In questo articolo viene rivista la biochimica di base per
la generazione di ROS e i loro effetti sull’integrità e funzionalità dell’epidermide. Una speciale enfasi è stata posta
nel dimostrare l’importanza del perossido di idrogeno epidermico quale contributo allo stress ossidativo e al segnale/trascrizione cellulare dipendente dalla concentrazione
di questi ROS, utilizzando il disturbo depigmentativo vitiligine quale modello di patologia.
PAROLE CHIAVE: Epidrmide - Idrogenioni perossidati - Deattivazione enzimatica - Attivazione trascrizionale - Antiossidanti.
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Ottobre 2005
Cosmetic dermatology: a review of skin care
Z. D. DRAELOS 1, 2
Dermatologic treatment involves 2 phases: therapeutic and
maintenance. The therapeutic phase of dermatologic treatment includes the diagnosis of the condition and the development of a plan to alleviate the signs and symptoms of disease.
The maintenance phase of dermatologic treatment involves
the recommendation of a skin care routine that prevents the
recurrence of the disease state, since failure to identify and
eliminate the causative factors will result in disease recurrence.
This review article deals with the maintenance phase of dermatologic treatment by discussing the use of cleansers, moisturizers, sunscreens, and facial cosmetics to maintain the integrity of the skin barrier and the ultimate health of the skin.
KEY WORDS: Dermatology - Cosmesis - Skin.
C
omplete skin care from the dermatologic standpoint involves the treatment of disease and the
maintenance of healthy skin. The difference between
healthy skin and diseased skin is proper functioning of
the skin barrier, the production of the correct amount
of melanin, and an intact immune system. The goal
of skin care maintenance is to keep these cutaneous systems functioning optimally. This means that skin
hygiene delivered through the use of cleansers to
remove sebum, environmental dirt, bacteria, fungus,
and yeast must prevent infection while leaving the
intercellular lipids untouched and the skin barrier
intact. Moisturizers can supplement the skin barrier
by temporarily restoring transepidermal water loss to
normal levels and creating an environment for barrier
Address reprint requests to: Z. D. Draelos, MD, 2444 North Main Street,
High Point, North Carolina 27262, USA. E-mail: [email protected]
Vol. 140 - N. 5
Wake Forest University School of Medicine
Winston-Salem, North Carolina, USA
2Dermatology Consulting Services
High Point, NC, USA
repair. Pigmentation problems and cutaneous immunosuppresion can be minimized with protective sunscreens that prevent UVB and UVA radiation from
striking the skin. Lastly, facial cosmetics can be used
to provide adornment, as well as moisturization and sun
protection. This review article will discuss the use of
these categories of skin care products including basic
formulation, variety within the marketplace, and skin
benefits.
Cleansing
Skin cleansing involves the use of a variety of surfactants that can be applied with many different implements. The ultimate result of the cleansing depends
on the ability of the cleanser to remove sebum and
desquamating corneocytes from the skin surface.
Aggressive removal of sebum and intercellular lipids
accompanied by the excessive loss of desquamating
corneocytes can lead to skin disease. Thus, cleansing
must maintain skin hygiene without producing barrier damage.
515
DRAELOS
COSMETIC DERMATOLOGY
TABLE I.—Types of surfactants.
Cleanser
type
Composition
Soap
Syndet
Combar
Synthetic detergents
Combination of soap
and syndet
PH
Attributes
9-11
Excellent thorough sebum
removal
Mild sebum removal
Moderate sebum removal
5.5-7
7-9
Types of soaps
There are many different methods for skin cleansing
(Table I). The dermatologist should understand all of
these techniques and pick the one for the patient that
provides the best combination of skin hygiene and
barrier maintenance. The basic cleanser is bar soap,
however, not all bar soaps are the same. In the basic
chemical sense, bar soap is a reaction between a fat and
an alkali resulting in a fatty acid salt with detergent
properties.1 These true soaps are composed of long
chain fatty acid alkali salts with a pH between 9-10.2
The high pH of these cleansers is excellent at sebum
removal, but can also damage the intercellular lipids in
diseased or sensitive skin. A newer class of cleansers
is known as a synthetic detergent, or syndet, and contains less than 10% real soap with a pH adjusted to
5.5-7. These are the cleansers that form the bulk of
the products found in a dermatologist’s sample closet and are less likely to damage the intercellular lipids,
but also may not remove all of the sebum from extremely oily skin. A third type of cleanser known as a combar, combines alkaline soaps with syndets to create a
bar with greater cleansing abilities, but less intercellular
lipid damage.3
Selecting the proper type of cleanser may be confusing at first, but once the 3 categories of cleansers are
identified, the task becomes much easier. In general,
all beauty bars, mild cleansing bars, and sensitive skin
bars are of the syndet variety (Oil of Olay, Dove,
Cetaphil). Most deodorant bars or highly fragranced
bars are of the combar variety (Dial, Coast, Irish
Spring) and very few true soaps are currently on the
market (Ivory).
to dry or moistened skin, rubbed to produce a lather,
and rinsed or wiped away (Cetaphil cleanser, Aquanil).
These products may contain water, glycerin, cetyl
alcohol, stearyl alcohol, sodium laurel sulfate and
occasionally propylene glycol. They leave behind a
thin moisturizing film and can be used effectively in
persons with excessively dry, sensitive, or dermatitic
skin. They do not have strong antibacterial properties,
however, and may not remove odor from the armpit or
groin. They also are not good at removing excessive
environmental dirt or sebum. Lipid-free cleansers are
best used where minimal cleansing is desired.
Cleansing creams
Cleansing creams offer gentle cleansing and moisturization in one step. Cleansing creams are composed
of water, mineral oil, petrolatum, and waxes.4 The
most common variant of cleansing cream, known as
cold cream, is created by adding borax to mineral oil
and beeswax (Pond’s Cold Cream).5 These products are
popular to remove cosmetics and provide cleansing
for patients with dry skin in one step.
Abrasive scrub
The recognition that exfoliation of desquamating
corneocytes was desirable to produce smooth skin in
maturing patients, led to the concept of an abrasive
scrub. Abrasive scrubs incorporate polyethylene beads,
aluminum oxide, ground fruit pits, or sodium tetraborate decahydrate granules to induce various degrees
of exfoliation.6 The most abrasive scrub is produced by
aluminum oxide particles and ground fruit pits. In general, products containing these rough edged particulates
are not appropriate for sensitive skin patients. More
mild facial scrubbing is produced by polyethylene
beads which are smooth and round (Clinique 7th Day
Scrub). The least aggressive abrasion of the skin is
found in products that contain sodium tetraborate decahydrate granules, which soften and dissolve during
use. The main problem with abrasive scrub products for
epidermabrasion is the firm nature of the scrubbing
granules that do not deform when pressed too hard
against the skin.
Lipid-free cleansers
Lipid-free cleansers are liquid products that clean
without fats, which distinguishes them from the soap
type cleansers previously discussed. They are applied
516
Woven meshes
Woven mesh products were introduced about the
same time as abrasive scrubs to also induce exfoliation,
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DRAELOS
but with an implement instead of a particulate. The
most popular product to enter the marketplace was
composed of a nonwoven polyester fiber web sponge
(Buf Puf).7 Originally, this product was designed to
remove open comedones, but later the web stiffness
was decreased and the sponge impregnated with a
mild cleanser to produce products designed for various
skin types.
Face clothes
The desire for thorough, but less abrasive cleansing led to the development of the disposable cleansing
cloth. These cloths are composed of a combination of
polyester, rayon, cotton, and cellulose fibers held
together via heat through a technique known as thermobonding. Additional strength is imparted to the
wipe by hydroentangling the fibers. This is achieved
by entwining the individual rayon, polyester, and wood
pulp fibers with high pressure jets of water, which
eliminates the need for adhesive binders thereby creating a soft, strong cloth. These clothes are packaged
dry and impregnated with a cleanser that foams modestly when the cloth is moistened. The type of cleanser
in the cloth depends whether strong sebum removal is
required by oily skin or modest sebum removal is
required by dry skin. Humectants and emollients can
also be added to the cloth to decrease barrier damage
with cleansing or to smooth the skin scale present in
xerosis.
In addition to the composition of the ingredients
preapplied to the dry cloth, the weave of the cloth will
also determine its cutaneous effect. There are 2 types
of fiber weaves used in facial products: open weave and
closed weave. Open weave cloths are so named because
of the 2-3 mm windows in the cloth between the adjacent fiber bundles. Closed weave cloths, on the hand,
are designed with a much tighter weave and provide a
more aggressive exfoliation. Ultimately, the degree of
exfoliation achieved is dependent on the cloth weave,
the pressure with which the cloth is stroked over the
skin surface, and the length of time the cloth is applied.
In general, open weave clothes are used in persons
with dry and/or sensitive skin to increase the softness
of the cloth and decrease the surface area contact
between the cloth and the skin yielding a milder exfoliant effect. Closed weave cloths are used in by persons
with oily skin or for the delivery of additional substances to the skin surface for treatment purposes. One
side of the closed weave cloth is textured and impreg-
Vol. 140 - N. 5
nated with a synthetic detergent cleanser designed to
optimize the removal of sebum, cosmetics, and environmental dirt while providing an exfoliant effect. The
opposite side of the cloth is smooth and designed for
rinsing the face and possibly applying skin conditioning agents, such as petrolatum, silicone, etc. The
closed weave cleansing cloth provides mechanical
exfoliation on the skin surface and around the follicular ostia due to the ability of the textured cloth to traverse the irregular topography of the skin more effectively than the hands or a washcloth.
Cleansing pouches
The cleansing pouch represents a variation on the
fibered cloths, however it can also be used as a metered
delivery system for skin cleansing and conditioning
agents. It is possible to place a plastic membrane
between 2 fibered cloths containing holes of various
diameters. The size of the hole determines how quickly the contents of the pouch are released onto the skin
surface. Typically, the cleansing pouch does not produce as much exfoliation as a plain cleansing cloth, but
can deliver a variety of botanical agents to modify the
skin surface, as well as for aromatherapy purposes.
Moisturizing
Many terms are used to describe the effects of creams
and lotions: lubricants, moisturizers, repair or replenishing products, emollients etc. These terms do not
have scientific meaning since the mechanisms for
rehydrating dry skin or rejuvenating damaged skin
remain to be elucidated. In basic terms, lubricants
refer to those products that increase skin slip in dry skin
that is rough and flaky; moisturizers impart moisture
to the skin, increasing skin flexibility; and repair or
replenishing products are intended to reverse the
appearance of aging skin. All 3 classes of products
are based on emollients. An understanding of the function of moisturizers and their formulation is essential
to the dermatologist who must maintain the health of
xerotic skin once the dermatitis has resolved.8
Physiology of xerosis
Xerosis is a result of decreased water content of the
stratum corneum which leads to abnormal desquamation of corneocytes. For the skin to appear and feel
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DRAELOS
TABLE II.—Steps for skin remoisturization.
1. Initiation of barrier repair
2. Alteration of surface cutaneous moisture partition coefficient
3. Onset of dermal-epidermal moisture diffusion
4. Synthesis of intercellular lipids
TABLE III.—Occlusive moisturizers.
1. Hydrocarbon oils and waxes: petrolatum, mineral oil, paraffin, squalene
2. Silicone oils: dimethicone, cyclomethicone
3. Vegetable and animal fats
4. Fatty acids: lanolin acid, stearic acid
5. Fatty alcohol: lanolin alcohol, cetyl alcohol
6. Polyhydric alcohols: propylene glycol
7. Wax esters: lanolin, beeswax, stearyl stearate
8. Vegetable waxes: carnauba, candelilla
9. Phospholipids: lecithin
10. Sterols: cholesterol
normal, the water content of this layer must be above
10%.9 Water is lost through evaporation to the environment under low humidity conditions and must be
replenished by water from the lower epidermal and
dermal layers.10 The stratum corneum must have the
ability to maintain this moisture or the skin will feel
rough, scaly and dry. However, this is indeed a simplistic view as there are minimal differences between
the amount of water present in the stratum corneum of
dry and normal skin.11 Xerotic skin is due to more
than simply low water content.12 Electron micrographic
studies of dry skin demonstrate a stratum corneum
that is thicker, fissured and disorganized.
There are 3 intercellular lipids implicated in epidermal barrier function: sphingolipids, free sterols and
free fatty acids.13 In addition, it is thought that the
lamellar bodies (Odland bodies, membrane-coating
granules, cementsomes), containing sphingolipids,
free sterols and phospholipids, play a key role in barrier function and are essential to trap water and prevent
excessive water loss.14, 15 The lipids are necessary for
barrier function since solvent extraction of these chemicals leads to xerosis, directly proportional to the
amount of lipid removed.16 The major lipid by weight
found in the stratum corneum is ceramide, which
becomes sphingolipid if glycosylated via the primary
alcohol of sphingosine.17 Ceramides possess the majority of the long-chain fatty acids and linoleic acid in
the skin. Perturbations within the barrier result in rapid
518
lamellar body secretion and a cascade of cytokine
changes associated with adhesion molecule expression and growth factor production.18 If skin with barrier perturbations is occluded with a vapor-impermeable wrap, the expected burst in lipid synthesis is
blocked. However, occlusion with a vapor-permeable
wrap does not prevent barrier recovery.19 Therefore,
transepidermal water loss is necessary to initiate synthesis of lipids to allow barrier repair.20, 21
Remoisturization of the skin must then occur in 4
steps listed in Table II.22 It is generally thought in the
cosmetics industry that a stratum corneum containing between 20% and 35% water will exhibit the softness and pliability of normal stratum corneum.23 Thus,
the goal of all moisturizer formulas is to restore water
levels in xerotic skin.
Functional classification of moisturizers
There are 3 functional classifications of moisturizers: occlusives, hydrophilic matrices, and humectants.24 All moisturizers function through one or a
combination of these mechanisms provided by the
various ingredients present in the formulation. It is
important to remember that the skin is moisturized
from the inside of the body, not from the outside.
Dietary water intake and externally applied water do
not remoisturize dry skin. Moisturizers only function
to retard water loss or enhance the water holding capacity of the skin. Occlusives and hydrophilic matrices
function to retard water loss while humectants increase
the water holding capacity of the skin.
Occlusives
Skin that is dehydrated and dry has a damaged barrier such that excessive water is lost to environment.
One method of decreasing the water loss and promoting barrier repair is to put a water impermeable coating over the skin surface. As mentioned previously,
the coating must allow at least 1% water loss to occur
in order to signal barrier damage and the need for the
body to synthesize intercellular lipids. Oily substances
are used to decrease transepidermal water loss and
occlude the skin surface. There are 10 major classes of
substances that can function as occlusive moisturizers
listed in Table III.25
The most commonly employed occlusive moisturizer is petrolatum.26 It appears, however, that total
occlusion of the stratum corneum is undesirable. While
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the transepidermal water loss can be completely halt- TABLE IV.—Humectant moisturizers.
ed, once the occlusive is removed, water loss resumes 1. Glycerin
at its preapplication level. Thus, the occlusive mois- 2. Honey
turizer has not allowed the stratum corneum to repair 3. Sodium lactate
its barrier function.27 Petrolatum is the ideal occlu- 4. Urea
sive moisturizer because it is not an impermeable bar- 5. Propylene glycol
Sorbitol
rier, but rather permeates throughout the interstices 6.
7. Pyrrolidone carboxylic acid
of the stratum corneum allowing barrier function to 8. Gelatin
be reestablished.28 The only drawback to petrolatum is 9. Hyaluronic acid
that it is greasy and has an undesirable smell. For this 10. Vitamins
reason, mineral oil was developed and placed in mois- 11. Hydrolyzed proteins
turizers, but it is not nearly as effective at occluding the
stratum corneum as petrolatum. Concerns about the
oiliness of mineral oil led to the development of sili- maintain their required water content. Humectants are
cone moisturizer additives, such as cyclomethicone also applied to the skin to attract water from the derand dimethicone. These occlusives are the basis for mis and viable epidermis to the stratum corneum. Subthe oil free moisturizers currently popular in the mar- stances that function as humectants are listed in Table
IV.25, 29
ketplace.
Cosmetic chemists have theorized that humectants
could be used to draw water from the environment,
Hydrophilic matrices
under conditions where the ambient humidity exceeds
Another technique of occluding transepidermal 70%, but more commonly humectants attract water
water loss from the skin surface is the use of hydro- from the deeper epidermal and dermal tissues. Water
philic matrices. Hydrophilic matrices are large mole- that is applied to the skin in the absence of a humeccular weight substances that form a physical barrier tant is rapidly lost to the atmosphere.30 Humectants
over the skin surface. The oldest hydrophilic matrix may also allow the skin to feel smoother by filling
moisturizing agent is colloidal oatmeal, that became holes in the stratum corneum through swelling.31 Howpopular for use with atopic dermatitis patients to pre- ever, under low humidity conditions humectants, such
vent water loss through the damaged barrier during as glycerin, will actually draw moisture from the skin
tub bathing. The colloidal oatmeal soothed the skin, but and increase transepidermal water loss.32 Therefore, a
also provide a protective blanket against water evap- good moisturizer must combine a humectant and an
oration.
occlusive. The humectant draws water to the dehyAnother commonly used hydrophilic matrix is drated stratum corneum and the occlusive traps the
hyaluronic acid. It is found in many high-end mois- water preventing evaporation and rehydrating the skin
turizers for its ability to hold water. Hyaluronic acid is temporarily until barrier repair can occur.
also the basis for a number of injectable filler materials that are placed into the dermal to increase the water Moisturizer formulation
holding capacity of the skin and soften the appearMost moisturizers consist of water, lipids, emulsiance of wrinkles on the skin surface.
fiers, preservatives, fragrance, color and specialty additives. Interestingly enough, water accounts for 60Humectants
80% of any moisturizer, however, externally applied
Another concept in rehydrating the stratum corneum water does not remoisturize the skin. In fact, the rate
is the use of humectants. Humectants are substances of water passage through the skin increases with
that attract water and act as a sponge to hold water. increased hydration.33 The water functions as a diluent
Humectants have been used in cosmetics for many and evaporates leaving the active agents behind. The
years to increase shelf life by preventing product evap- water and the oily occlusive agents are mixed togethoration and subsequent thickening due to variations er and stabilized with emulsifiers, which are generalin temperature and humidity. For example, humec- ly soaps, such as triethanolamine, in concentrations
tants are a necessary part of all oil-in-water creams to of 0.5% or less.
Vol. 140 - N. 5
519
DRAELOS
Moisturizer types
A quality moisturizer formulation must fulfill 3 criteria: it must increase the water content of the skin
(moisturization), it must make the skin feel smooth
and soft (emolliency) and it must protect injured or
exposed skin from harmful or annoying stimuli (skin
protectant). Moisturizers can be customized for the
specific needs of a variety of areas including the face,
body, and hands.
Facial moisturizers
Facial moisturizers are the largest segment of the
moisturizer market. There are 2 basic formulations:
oil-in-water emulsions in which water is the dominant
phase, and water-in-oil emulsions in which oil is the
dominant phase. Oil-in-water formulations are used
for the thinner daytime facial moisturizers and waterin-oil formulations are used for night creams or facial
replenishing creams. Oil-in-water emulsions can be
identified by their cool feel and nonglossy appearance
while water-in-oil emulsions can be identified by their
warm feel and glossy appearance.34 Daytime moisturizers are generally composed of mineral oil or dimethicone, propylene glycol, and water in sufficient quantity to form a lotion. Night creams are composed of
petrolatum, mineral oil or dimethicone, lanolin alcohol,
and water to form a cream. The differences between
products thus lie in the addition of fragrances, exotic
oils, vitamins, proteins, and other specialty additives.
The plethora of facial moisturizers has made categorization of the various products difficult; however,
a brief look at the claims and composition of some
key products is valuable. The cosmetic companies
market facial moisturizers based on skin type. Naturally, products designed for oily complexions are oilfree or contain small amounts of light oils. Products for
normal skin contain moderate amounts of light oils, and
products for dry skin contain increased amounts of
heavier oils. The lighter oil used is generally mineral
oil and the heavier oil is petrolatum. Thus, moisturizing products can be developed for all skin types based
on varying water to oil ratios.
Oily complexion products that are oil-free are composed of water and silicone derivatives, such as
cyclomethicone or dimethicone. This combination has
been shown to be noncomedogenic in the rabbit ear
assay. These products are nongreasy since the bulk of
the product evaporates from the face. Many oily com-
520
plexion moisturizers also claim to provide oil control,
which is accomplished with oil-absorbing substances
such as talc, clay, starch or synthetic polymers.
Products designed for normal or combination skin
contain predominantly water, mineral oil and propylene
glycol with very small amounts of petrolatum or lanolin. These products leave a greater oily residue on the
face than oil-free formulations. Moisturizers in this
line are also called anti-wrinkle lotions, protective
creams or sport creams if they contain sunscreen agents.
Dry skin moisturizers contain water, mineral oil,
propylene glycol and larger amounts of petrolatum or
lanolin in addition to low concentrations of numerous additives claiming to rebuild, renew or replenish.
The patient should realize that the perfect skin moisturizer does not exist. Creams and lotions that claim to
restore or rebuild tissue in the dermis do not penetrate
deeply to have any effect. The extremely high cost of
some moisturizers is not justified by the value of the
ingredients. Patients are buying a certain feel, fragrance, or image. If the patient achieves more selfconfidence or an increased sense of well being after
using a certain facial cream, the money has been well
spent. The role of the dermatologist should be to identify which cosmetic claims are unfounded so that the
patient has a medical perspective on the product he
or she chooses to purchase.
Body moisturizers
Body moisturizers come in a variety of preparations
including lotion, cream, mousse, and ointment. Lotions
are the most popular formulation. Creams and ointments can be used on the body, but are more difficult
to spread, especially in hair-bearing areas. Female
patients desire a nongreasy body lotion with a rich
texture; however, a rich texture does not necessarily
identify a superior moisturizer. Richness can be added
to a thin lotion with water-soluble gums that impart a
silky feel to the skin but do not provide improved
moisture retention.
Body lotions are generally oil-in-water emulsions
containing 10-15% oil phase, 5-10% humectants, and
75-85% water phase. More specifically, they are composed of water, mineral oil, propylene glycol, stearic
acid, and petrolatum or lanolin. Most also contain an
emulsifier such as triethanolamine stearate. Humectants
such as glycerin or sorbitol, may also be used. Other
additives include vitamins, such as A, D, and E, and
soothing agents, such as aloe and allantoin.
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Hand moisturizers
The simplest hand moisturizer is petroleum jelly,
but most patients find it too greasy and aesthetically
unacceptable. To improve cosmetic appeal, the petroleum jelly can be whipped with water, color, and fragrance to make a hand cream. Thus, hand creams are
oil-in-water emulsions with 15-40% oil phase, 5-15%
humectants, and 45-80% water phase.35 The addition
of silicone derivatives can also render the hand cream
water-resistant through 4 to 6 washings. Some hand
creams even include a sunscreen agent.
Assessing moisturizer efficacy
The efficacy of moisturizers can be difficult to assess,
however several excellent noninvasive methods have
been developed: regression analysis, profilometry,
squametry, in vivo image analysis, twistometre, impedance measurement, and evaporimetry.36
Regression analysis is a method of evaluating moisturizer efficacy under clinical conditions. Here patients
are selected and treated by an objective observer with
moisturizers at a predetermined test site for 2 weeks.
The test site is evaluated on days 7 and 14. If improvement is noted, moisturizer application is discontinued
and the test site evaluated daily for 2 weeks, or until the
baseline pathology has reappeared.37 This method is
particularly valuable since the efficacy of all moisturizers is excellent immediately following application,
but the true effectiveness can only be assessed with
the passage of time.38
Profilometry involves analysis of silicone rubber
replicas of the skin surface. The replicas are created by
placed unpolymerized silicone in a ring form that is
attached usually to skin just lateral to the eye. The silicone is allowed to polymerize and a negative replica
of the skin surface is created. These silicone replicas
are digitally scanned to obtain a two- or three-dimensional topogram. The topogram gives an indication of
the depth of the wrinkles present on the skin surface.
Unfortunately, this method can be inaccurate since
the silicone application to the skin surface tends to
flatten and disturb the desquamating skin scale.39
Squametry involves analysis of skin corneocytes
harvested by pressing a sticky tape against the skin. The
outmost, loosely adherent skin scale is then removed.
The tape provides a specimen that retains the topographical relationships of the skin surface and the pattern of desquamation. Image processing is then used
Vol. 140 - N. 5
to evaluate the scaliness of the skin.40 The tapes containing the harvested skin scale can also be affixed to
a black background to visually assess the amount of
skin scale removed.
An assessment of skin scale can also be achieved
through in vivo image analysis from video microscope
images. The images can be digitally recorded and analyzed to assess skin surface features.41 Care must be
taken to standardize lighting and camera angles to
insure accurate data for analysis.
Twistometre, impedance measurement and evaporimetry are mechanistic methods of evaluating skin
dryness and moisturizer effectiveness. The twistometre uses torsion to measure in vivo the influence of
stratum corneum hydration on skin extensibility. A
weak torque is applied to a rotating disc that is placed
in contact with the skin. It has been shown that dry
skin is much less extensible than well hydrated skin.42
Skin impedance can also be evaluated. Here a dry electrode consisting of 2 concentric brass cylinders separated by a phenolic insulator operating at 3.5MHz is
applied to the skin.43 The impedance drops as the skin
is better hydrated.44 This technique can evaluate the
efficacy and the duration of effect of moisturizers.45
Lastly, evaporimetry can be used to measure the cutaneous transepidermal water loss.46 More occlusive
substances would be expected to lower water loss
while some humectants, such as glycerin, actually
increase water loss.47, 48
Even though these sophisticated noninvasive methods of cutaneous evaluation sound appealing, there is
no substitute for the opinion of a trained unbiased
observer when evaluating moisturizer effectiveness
or the perceptions of the consumer. Mechanistic evaluation can be easily biased to produce data that serves
the best interest of the manufacturer. Computers cannot yet accurately synthesize all the tactile and visual
information that can be obtained with human evaluation. The noninvasive techniques simply present another tool for assessing moisturizer function, but the opinion of the dermatologist and the patient are still the
most important factors in assessing moisturizer efficacy.49
Sunscreens
Probably the most important skin care product to
prevent premature aging and insure skin health is sunscreen. The skin contains natural protective mecha-
521
DRAELOS
TABLE V.—Natural ultraviolet protective mechanisms.
Cutaneous structure
Compact horny layer
Keratinocyte melanin
Carotenoid pigments
Urocanic acid
Superoxide dismutase
Epidermal DNA excision repair
Sun procedure mechanism
Absorbs and scatters UV
1. UV absorbing filter
2. Free radical scavenger
3. Dissipates UV as heat
4. Undergoes oxidation in 300-360
nm range to produce im-mediate
pigment darkening (IPD)
1. Membrane stabilizers
2. Quench oxygen radicals
Oxidized to stabilize UV-induced
oxygen radicals
1. Oxygen radical scavenger
2. Protects cell membrane from lipoprotein damage
Repairs UV induced DNA damage
nisms in the dermis and stratum corneum that are listed in Table V, however sunscreen use is necessary to
achieve the protection required to insure skin health.
In each layer of the skin, there are a variety of endogenous mechanisms used to reflect ultraviolet radiation,
quench oxygen radicals, and repair the resultant cellular
damage. Beginning at the outermost structure of the
skin with the stratum corneum, ultraviolet radiation
is scattered and reflected by the corneocytes. This is
why the endogenous SPF of the skin is lower through
procedures that induce exfoliation, such as chemical
face peels. The use of topical hydroxy acid facial moisturizers also removes the corneocytes further decreasing the sun protection factor (SPF) of the skin. Most
currently marketed sunscreens operate to enhance the
reflective and scattering properties of the stratum
corneum.
The next natural defense against UV radiation is
the melanin. Melanin performs numerous functions
to function as a UV absorber and dissipate the heat
byproduct. Melanin itself is a free radical scavenger and
undergoes oxidation in the 300-360 nm range. It is
this oxidation of melanin that results in the immediate
pigment darkening phenomenon associated with the
dermatologic use of therapeutic UVA exposure. There
is no sunscreen active that uses the same mechanism
of action as melanin for photoprotection. Even synthetic topical melanin does not provide the protection
of endogenous melanin.
There are a variety of substances in the skin that
function as endogenous antioxidants. These include
the carotinoid pigments, urocanic acid, and superox-
522
ide dismutase which quench oxygen radicals and stabilize cell membranes. No sunscreen can duplicate
the protection of these endogenous substances. There
are oral and topical supplements that claim to enhance
this mechanism of photoprotection, but none have
been proven effective. This then leads to a discussion
of the mechanism of action of currently marketed efficacious sunscreens.50
Sunscreen mechanism of action
Sunscreens cannot duplicate these protective mechanisms, thus they must rely on substances that can
chemically absorb UV radiation or physically reflect
UV radiation. This yields the 2 classes of sunscreens,
chemical sunscreens, and physical sunscreens. Chemical sunscreens function by transforming UV radiation to heat through a process known as resonance
delocalization. This is accomplished through the presence of an electron-releasing group either in the ortho
or para position or both on the benzene ring. The chemical sunscreen is excited to a higher energy state from
ground state by absorbing UV radiation. When the
chemical sunscreen returns to the ground state, the
absorbed energy is emitted in a less energetic form, usually heat.
The mechanism of action for chemical sunscreens
is quite different from the functioning of physical sunscreens. Physical sunscreens are formulated from
ground particulates that either reflect or scatter the
UV radiation. These particulates are primarily chemically inert, however some of the changes described for
sunscreens previously may occur to a lesser degree.
Sunscreen actives
Sunscreens are designed to absorb UVA radiation
(320-360 nm), UVB radiation (290-320 nm), or both.
UVA radiation is primarily responsible for the cutaneous changes associated with aging while UVB radiation causes sunburn. A good sunscreen must provide
protection against both spectrums of UV radiation.
Table VI lists those sunscreen actives that are currently
approved for use by the FDA.51
The primary UVA absorbers on this list are the benzophenones, anthralinates, and avobenzone.52 The primary UVB absorbers are the PABA derivatives, salicylates, and cinnamates, while those substances that
absorb both UVB and UVA are titanium dioxide and
zinc oxide. Most quality sunscreens combine these
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DRAELOS
ingredients to yield a product with excellent photoprotection that is cosmetically elegant. No one ingredient can yield the optimal sunscreen, thus there is
tremendous art to the creation of a quality formulation.
The benzophenones (oxybenzone, dioxybenzone,
sulisobenzone) are popular UVA sunscreening agents,
but they primarily absorb below 330 nm and may be
allergenic.53 For this reason, they are usually found in
low concentration sometimes combined with the
anthralinates (menthyl anthranilate), which have low
allergenicity and high stability. Avobenzone is a common UVA sunscreen, but must be combined with other agents since it is rapidly degraded by UV exposure.
Almost 36% of the avobenzone in a sunscreen formulation becomes chemically inactive on initial exposure.
The UVB sunscreen actives are also commonly used
in combination, with the disadvantages of one active
compensated for by the advantages of another active.
The PABA derivatives (octyl dimethyl PABA,
aminobenzoic acid) are also inactivated by 15.5% on
exposure to UVB and absorb maximally at 296 nm. For
this reason, they are combined with the salicylates
(octyl salicylate, homomenthyl salicylate, trolamine salicylate), which are weak UVB absorbers, but have
their maximal absorption at 300-310 nm complimenting the PABA absorption spectrum. The salicylates are less allergenic than PABA derivatives and
have an excellent safety record. The most popular
UVB sunscreen active is octyl methoxycinnamate. It
has its maximal absorption at 305 nm and is remarkably stable with only 4.5% experiencing degradation
following UV exposure.
The most effective sunscreen actives are the physical particulates, consisting of zinc oxide and titanium
dioxide, since they can reflect both UVB and UVA
radiation. They are white powders that must be used in
combination with some of the chemical actives discussed previously, since they can only be used in low
concentration due to unattractive whitening of the
skin. Micronized particulates have particles of many
sizes while microfine particulates have particles of
even size. The microfine formulations produce less
skin whitening than the micronized formulations. The
particulates are often silicone coated to decrease the
generation of secondary oxygen radicals when struck
by UV radiation. At present, there is a great deal of
research into the development of new particulate sun-
Vol. 140 - N. 5
TABLE VI.—Category 1 monographed sunscreen active ingredients.
Active sunscreen ingredient
Maximum
concentration (%)
Aminobenzoic acid (PABA)
Avobenzone
Cinoxate
Dioxybenzone (benzophenone-8)
Homosalate
Menthyl anthranilate
Octocrylene
Octyl methoxycinnamate
Octyl salicylate
Oxybenzone (benzophenone-3)
Padimate-O (octyl dimethyl PABA)
Phenylbenzimidazole sulfonic acid
Sulisobenzone (benzophenone-4)
Titanium dioxide
Trolamine salicylate
Zinc oxide
15
3
3
3
15
5
10
.57.5
5
6
8
4
10
25
12
25
screens based on pigments adapted from the textile
industry.
Facial cosmetics
Facial cosmetics are also important for the benefits
they provide to the facial skin. Facial cosmetics can provide moisturization, oil control, sun protection, camouflaging, and aesthetic benefits. This final topic of the
review paper will focus on facial foundations, a colored
cosmetic applied to the face for the purpose adding
color, covering blemishes, and blending uneven facial
color. Facial foundations represent the class of cosmetics about which patients most frequently question
their dermatologist. Since a foundation is usually
applied to the entire face, used on a daily basis, and
worn for an extended period; it has a dramatic effect
on the skin. The variety in formulation, type and color of facial foundations is wide and may be bewildering to physicians and patients alike. This discussion
presents basic information on facial foundations for
oily, normal, combination, and dry skin; with a final
discussion on foundation selection in patients with
facial scarring.
Facial foundation formulation
There are 4 basic facial foundation formulations:
oil-based, water-based, oil-free, and water-free forms.
Oil-based foundations are water-in-oil emulsions containing pigments suspended in oil, such as mineral oil
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DRAELOS
or lanolin alcohol. Vegetable oils (coconut, sesame,
safflower) and synthetic esters (isopropyl myristate,
octyl palmitate, isopropyl palmitate) may also be incorporated. The water evaporates from the foundation
following application, leaving the pigment in oil on the
face. This provides facial skin with a moist feeling,
especially desirable in dry complected patients. Oilbased foundations are popular since they undergo no
change in color with wearing, known as color drift.
The pigment is already fully developed in oil, reducing the effect of facial sebum. These foundations are
easy to apply, since the playtime (the time from application to setting) is prolonged, allowing manipulation
of the pigment over the face for up to 5 min.
Water-based facial foundations are oil-in-water
emulsions containing a small amount of oil in which
the pigment is emulsified with a relatively large quantity of water. The primary emulsifier is usually a soap
such as triethanolamine or a nonionic surfactant. The
secondary emulsifier, present in smaller quantity, is
usually glyceryl stearate or propylene glycol stearate.
These popular foundations are appropriate for minimally dry to normal skin. Since the pigment is
already developed in oil, this foundation type is also
not subject to color drift. The playtime is shorter than
with oil-based foundations, however, due to the lower oil content. These products are usually packaged
in a bottle.
Oil-free facial foundations contain no animal, vegetable, or mineral oils. They may, however, contain
other oily substances, such as the silicone derivatives
dimethicone or cyclomethicone. These foundations
are usually designed for oily skin individuals with
acne or oily skin, since they emphasize the absence
of oily substances believed to induce comedogenesis.
The pigment is dissolved in water and other solvents
leaving the skin with a dry feeling resulting from the
absence of oils. However, the color is more prone to
drift with wearing as the pigment mixes with sebum.
Foundation playtime is extremely short as the water and
solvents evaporate quickly. Thus, oil-free foundations
require rapid blending into one facial area at a time
to prevent streaking. They can be packaged in bottles,
jars, tubes, or compacts.
Water-free or anhydrous foundations are waterproof.
Vegetable oil, mineral oil, lanolin alcohol and synthetic esters form the oil phase, which may be mixed
with waxes to form a cream.54 They can be dipped
from a jar, squeezed from a tube, wiped from a com-
524
pact or stroked from a stick. These foundations have a
long playtime, no color drift, extended wear and may
be opaque, making them valuable for patients with
facial scarring.
The coloring agents in all facial foundations are
based on titanium dioxide with iron oxides, occasionally in combination with ultramarine blue. Titanium dioxide acts both as a facial concealing or covering agent and a physical sunscreen.
Facial foundations also contain talc and kaolin to
function as fillers and blotters. A filler gives substance
to the foundation while a blotter functions to absorb
facial secretions. Oil-control foundations contain
increased concentrations of the blotters talc, kaolin,
starch, or polymers to absorb facial sebum, thus preventing the development of facial shine. Oil-control
foundations are not necessarily oil-free, however. The
oil content of a foundation can be assessed by placing
a drop of the product on a sheet of 25% cotton bond
paper. Oil-containing foundations will leave an oil
ring on the paper while oil-free foundations will not.
The size of the oil ring is proportionally related to the
foundation’s oil concentration.55
Facial foundation characteristics
COVERAGE
The ability of a foundation to conceal or cover the
underlying skin is known as coverage. The coverage of
a foundation is directly related to the amount of titanium dioxide, zinc oxide, talc, kaolin, and precipitated chalk it contains. Sheer coverage foundations with
minimal titanium dioxide are almost transparent and
have a SPF around 2, moderate coverage foundations
are translucent and have an approximate SPF of 4 to 5
and anhydrous high-coverage foundations with large
amounts of titanium dioxide are opaque, acting as a
total physical sunblock. Some foundations, known as
sportwear or antiaging foundations, may contain additional chemical sunscreening agents such as PABA
esters or cinnamate derivatives.
TYPES
Facial foundations are available in a variety of
forms: liquid, mousse, water-containing cream, soufflé, anhydrous cream, stick, cake and shake lotion.56
Liquid formulations are most popular because they
are the easiest to apply, provide sheer to moderate
coverage and create a natural appearance. As previ-
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DRAELOS
ously mentioned, they contain mainly water, oils,
and titanium dioxide (TiO 2). If the liquid is
aerosolized, a foam foundation known as a mousse
is produced. A cream foundation has the additional
ingredient of wax, which makes a thicker, occlusive,
more moisturizing formula. Whipping the cream produces a soufflé foundation. An anhydrous cream with
no water in its formulation provides more occlusion
and superior, long-lasting coverage. Addition of
increased amounts of wax to an anhydrous cream
yields a product that can be extruded into a rod and
packaged in a roll-up tube. These are stick foundations. Cake-type foundations, also known as
cream/powder foundations, consist of talc, kaolin,
precipitated chalk, zinc oxide and titanium dioxide
compressed into a cake that is applied to the skin
with a dry sponge (powder) or moistened sponge
(cream). Shake lotions are pigmented talc suspended in water and solvents that evaporate, leaving a
thin layer of powder on the face.
FINISHES
Facial foundations are manufactured in a variety of
finishes: matte, semimatte, moist semimatte and shiny.
The finish is the surface characteristic of a cosmetic.
Matte finish foundations yield a flat look with no shine
and generally are oil-free. They are good for patients
with oily skin who tend to develop some shine after a
foundation has been applied. A semimatte finish has
minimal shine and is generally an oil-free foundation
or water-based foundation with minimal oil content.
This finish performs well on slightly oily to normal
skin. A foundation with more shine is known as a
moist semimatte foundation and is generally waterbased with moderate oil content. Shiny finishes are
found in oil-based foundations and are only appropriate for persons with dry skin. The shinier foundations
with increased oil content also have increased moisturizing ability.
Facial foundation application technique
The foundation selected should match the natural
facial color as closely as possible. This can be difficult,
however, since the nose and cheeks have redder tones
than the forehead and chin. The foundation is matched
to the skin along the jawline, since this is where the color must be carefully blended beneath the chin. Mismatched facial foundations generally leave a line at
Vol. 140 - N. 5
the jawline. A foundation color should also be selected in natural sunlight: the bright, artificial fluorescent
lights used in most stores will distort color perception. This may result in selection of a dark foundation
that will appear unnatural under conventional lighting. The patient should be urged to apply a sample of
foundation to the jawline in the store and then walk outside to examine the color match with a compact mirror.
In general, facial foundation should be applied with
the fingertips. A dab of foundation should be placed on
the forehead, nose, cheeks, and chin and then blended with a light circular motion until it is evenly spread
over all the facial skin, including the lips. Finally, a puff
or sponge should be used, stroking in a downward
direction, to remove any streaks and to flatten vellus
facial hair. Special care should be taken to rub the
foundation into the hairline, over the tragus and beneath
the chin. Foundation should also be blended around the
eyes and may even be applied to the entire upper eyelid if desired. The foundation should be allowed to set
or dry until it can no longer be removed with light
touch. If additional coverage is desired, a second layer of foundation can be applied. Makeup sponges are
used to apply cake foundation, but otherwise are not
recommended since they absorb a tremendous amount
of foundation.
Facial foundation purchase
Facial foundations can be purchased at a mass merchandiser, drug store, department store, or boutique.
Less expensive wide appeal foundations are sold by
mass merchandisers and drug stores. More expensive
upscale foundations are sold by department stores
and very expensive specialty foundations are sold by
elite department stores and boutiques. Dermatologists are frequently asked by patients if they are getting higher quality with an expensive cosmetic compared to a less expensive cosmetic. It is true that part
of the cost of a more expensive cosmetic goes into
attractive packaging and a prestige name or image.
However, a wider range of colors is generally available
with the more expensive product lines, and in some
instances, the increased cost yields superior ingredients that perform better. This especially seems to be
the case with facial foundations. In general, the more
expensive foundations have less color drift, better
coverage, easier application, and more uniform appearance.
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DRAELOS
Facial foundation considerations for various skin
types
ACNE AND/OR OILY SKIN FOR VARIOUS SKIN TYPES
Patients with acne and/or oily skin generally do best
with matte or semimatte finish, oil-free or low oil content facial foundations. While this type of foundation
performs best in the long run, many patients initially
are dissatisfied with such a product because oil-free or
low oil foundations neither cover as well nor wear as
long as oil-containing foundations. They are also more
prone to collect or cake around facial blemishes and
their short playtime makes application more difficult.
The original oil-free foundations were shake lotions
composed of pigmented talc in a water and solvent
vehicle. Shaking was required to suspend the powder
in the liquid prior to application. After the vehicle
evaporated, a thin layer of powder was left on the face.
The coverage of this type product is extremely sheer
and the playtime is extremely short, approximately
30 s, thus requiring skilled application. Coverage can
be improved by repeated application, however, most
patients find this inconvenient. The weartime of shake
lotions on an oily face is very short, approximately 1
to 2 h. Breakdown of the thin foundation layer, known
as separation, occurs commonly over the oily nose
and medial cheeks as the powder foundation preferentially adheres to the pores and is wiped from the
surrounding skin. A similar-appearing foundation can
be made by first applying an oil-free moisturizer and
then a loose transparent powder.
Patients seem to find the newer oil-free liquid foundations more appealing. These products may require
minimal shaking prior to use but do not separate like
a shake lotion. Oil-free foundations differ tremendously by brand in terms of coverage. Extremely sheer
or transparent coverage may be appropriate for the
patient with open comedones and no inflammatory
papules, but the patient with more facial erythema
may need more coverage with a sheer product. Moderate or translucent coverage will be required by the
patient with inflammatory papules. Patients with scars
may need the extremely good, heavy coverage only
afforded by cake foundations with minimal oil. Unfortunately, this look can be quite theatrical.
Acne patients may apply medicated water-based
creams or alcohol-based liquids under an oil-free foundation. Gel-type products that leave a film on the skin,
such as topical antibiotic gels or gel sunscreens, will
526
either change the foundation color or prevent the foundation from adhering. Any product used in this manner must be allowed to dry thoroughly before foundation is applied.
Most oil-free or low oil foundations wear longer if
a loose transparent powder is applied to the face. The
powder increases the weartime of the foundation by
absorbing oil and increasing coverage. The loose powder should be rubbed into the foundation with the fingertips and the excess dusted away with a loose powder brush.
NORMAL SKIN FACIAL FOUNDATIONS
Patients with normal skin and no tendencies toward
acne have numerous facial foundations from which
to choose. These patients usually prefer a semimatte to
moist semimatte finish since their facial oil production
is not excessive and matte finish cosmetics will give a
floured look. Water-based foundations perform best
if a small amount of oil is included in the formulation
to improve coverage, wear and application ease. Sheer
coverage can be useful in the patient who simply needs
facial color. Increased coverage is required by the
patient with facial telangiectasias and lentigines. The
mature patient with normal skin and actinically induced
dyspigmentation needs moderate coverage to deemphasize facial wrinkles.
DRY SKIN FACIAL FOUNDATIONS
Patients with dry skin need a foundation that provides all day moisturization. Water-based foundations
with moderate oil content or oil-based foundations
may be used. The oil content required gives the foundation a moist semimatte or shiny finish. Foundations
with substantial oil content are easy to apply because
of their long playtime, cover extremely well and are
long wearing. In some cases, selection of the appropriate foundation may obviate the need for a daytime
facial moisturizer. Younger patients with dry skin and
no complexion problems generally prefer a moderately sheer moist semimatte finish. A more matte type
finish can be achieved by applying a loose transparent
powder over the foundation. Mature patients with dry
skin need a moderate coverage moisturizing foundation that will not accentuate wrinkling. Patients with
normal skin that has become dry due to topical medication may also need to use an oil-containing foundation.
Ottobre 2005
DRAELOS
Patients with extremely dry skin may feel that they
need to use a cream or soufflé foundation. These
products do contain more oils; however, they also
yield a thicker appearance. If the skin surface is perfectly smooth, a thicker foundation can be attractive;
if the skin surface is wrinkled or has other surface
irregularities, the thicker foundation will accentuate
the underlying contour. An alternative is use of a
heavy cream moisturizer under the foundation followed by application of a thinner, less moisturizing
foundation.
COMBINATION SKIN FACIAL FOUNDATIONS
Combination skin presents a challenge in terms of
selecting a facial foundation. This is probably the
most common skin type encountered by the dermatologist. These patients are generally women between
the ages of 25 and 45 who have an oily central forehead, nose, and medial cheek. This area is also known
as the T zone. Occasionally, the perioral and central
chin areas are similarly affected. The more lateral
facial areas are usually dry. These patients also tend
to have acne, which may fluctuate with ovulation and
menstruation.
No foundation has been developed to date that can
supply the proper amount of moisturizing to dry areas
and absorb the proper amount of sebum from oily
areas. A product marketed by some cosmetic companies to deal with this problem is known as an oilcontrol foundation. These products contain starch,
clay, polymers or additional talc to aid in oil absorption. They usually contain a fair amount of oil and fit
best under cosmetics for normal skin, since their high
oil content makes them unsuitable for acne patients
and their oil absorbing capabilities makes them unsuitable for patients with a dry complexion. Even patients
with normal skin make sebum, and oil-control foundations can wear longer than average on these individuals.
Patients with combination skin seem to do best with
foundations that will not accentuate complexion problems. However, they must carefully prepare the face prior to application of the foundation. All oily areas should
be treated with an astringent and all dry areas should
be treated with an oil-free moisturizer. The aim is to
create an even oil distribution. Foundation is then
applied to the entire face and a loose transparent powder applied to central facial areas. Most patients prefer a moderate coverage foundation.
Vol. 140 - N. 5
Combination skin may tend to be drier in the winter and oilier in the summer. All patients should be
encouraged to match their foundation to their complexion needs. Some patients may need to change
foundations depending on the season, just as they
change their wardrobes.
FACIAL FOUND FOUNDATIONS FOR SCARRED SKIN
All of the foundations appropriate for facial scarring
are oil-containing or silicone derivative-containing,
since oil is required for increased coverage and extended wear. It is also desirable for these foundations to be
somewhat waterproof, which necessitates a low to
absent water content. These products provide increased
coverage by containing higher amounts of titanium
dioxide, but are not intended to be opaque. One disadvantage of these foundations is the tendency to
accentuate facial surface irregularities.
Conclusions
This review article has covered the basic steps in a
skin care regimen designed to maintain healthy skin.
Cleanser selection is important to insuring good
hygiene without damage to the skin barrier. Any skin
barrier damage that is present can be minimized with
occlusive moisturizers that retard transepidermal
water loss and humectant moisturizers that attract
water. Moisturizers can also be sun protective through
the incorporation of sunscreen actives into the formulation, preventing aging of the skin and skin cancer. Lastly, facial foundations can be used to provide
better skin cosmesis while improving the condition
of the skin in a variety of complexion types.
Cleansers, moisturizers, sunscreens, and facial foundations are part of the maintenance phase of dermatologic treatment following disease resolution. The
dermatologist should master their use in clinical practice.
Riassunto
Dermatologia cosmetica: una revisione della cura della cute
Il trattamento dermatologico prevede due fasi: quella terapeutica e quella di mantenimento. La fase terapeutica del
trattamento dermatologico comprende la diagnosi della condizione e lo sviluppo di un piano per alleviare i segni e sin-
527
DRAELOS
tomi della malattia. La fase di mantenimento del trattamento dermatologico comprende la raccomandazione di una
cura costante della cute che prevenga la ricorrenza della
patologia, dal momento che il fallimento nell’identificazione ed eliminazione dei fattori causali porteranno alla ricorrenza della malattia. Questo articolo di revisione tratta della fase di mantenimento del trattamento dermatologico discutendo l’utilizzo di detergenti, creme, protezioni solari e
cosmetici per il viso per mantenere l’integrità della barriera
cutanea e in definitiva la salute della cute.
PAROLE CHIAVE: Dermatologia - Cosmesi - Cute.
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Clinical and molecular aspects of Kindler syndrome
J. E. LAI CHEONG, V. WESSAGOWIT, H. FASSIHI, J. A. MCGRATH
Kindler syndrome is a rare autosomal recessive genodermatosis
that was first described in 1954. Clinically, the early features
consist of trauma-induced skin blisters but later there is photosensitivity and poikiloderma. For years, this poorly understood condition was thought to be a variant of dystrophic epidermolysis bullosa or a congenital poikiloderma, or possibly the
simultaneous occurrence of both disorders. In 2003, however,
the genetic basis of Kindler syndrome was established. This
inherited skin disease is caused by ablation of a novel protein,
kindlin-1 (also known as kindlerin), encoded by the gene KIND1
on 20p12.3, and 19 different mutations (nonsense, frameshift
or splice-site) have now been reported. Kindlin-1 is a 677-amino
acid protein that is predominantly expressed in basal keratinocytes, where it provides a link between the actin microfilaments and the extracellular matrix, and forms part of focal
contact junctions at the dermal-epidermal junction. The protein has functional domains involved in adhesion (via β integrin),
other integrin-mediated cell activities such as cell spreading
β), and cell sig(e.g. in response to transforming growth factor-β
nalling (possibly via PI3 kinase), although just how loss of
kindlin-1 leads to the photobiological abnormalities seen in
patients with Kindler syndrome is still a mystery. Nevertheless, the recent molecular findings have confirmed that Kindler
syndrome is indeed a specific genodermatosis, and one which
represents the first inherited skin disorder to involve a primary abnormality of the actin cytoskeleton-extracellular matrix
complex. This review describes the clinico-pathological feaFundings. Support for several of the original studies referred to in this
review was kindly provided by the Dystrophic Epidermolysis Bullosa
Research Association (DebRA, UK), the Barbara Ward Children's
Foundation, and Action Medical Research.
Address reprint requests to: J.A. McGrath, Genetic Skin Disease Group,
St John's Institute of Dermatology, St Thomas` Hospital, Lambeth Palace Road, London, UK. E-mail: [email protected]
Vol. 140 - N. 5
Genetic Skin Disease Group
St John's Institute of Dermatology, The Guy's, King's College
and St Thomas' Hospitals' Medical School, London, UK
tures of Kindler syndrome, the identification of the KIND1
gene, and the biological significance of the kindlin-1 protein.
KEY WORDS: Skin diseases, genetics - Blister - Poikiloderma congenitale - Actins - Cytoskeletal proteins - Cell adhesion - Keratinocytes - Integrins.
Historical background to Kindler syndrome and
related disorders
J
ust over 50-years ago, Theresa Kindler, a German
paediatrician, reported a case of a 14-year old
Caucasian girl who had congenital trauma-induced
blistering (mainly on the extremities) as well as mottled pigmentation on her cheeks.1 She also sun-burnt
easily and developed progressive skin atrophy with
worsening of the condition during summer months.
Collectively, there were features of both a sub-type of
epidermolysis bullosa and a form of inherited poikiloderma, but the precise nature of the disorder, and
the mode of inheritance, could not be established. In
1971, however, Weary et al. reported a family with
what appeared to be an overlapping syndrome.2 Inheritance was clearly autosomal dominant but the clinical features were very variable (vesicopustules,
eczema, poikiloderma and acral keratotic papules).
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CLINICAL AND MOLECULAR ASPECTS OF KINDLER SYNDROME
Figure 1.—Clinical appearances of Kindler syndrome. (A) A 12-year-old Indian boy showing blisters and crusts around his neck as well as signs of
poikiloderma on his face and sun-exposed neck and upper trunk (figure courtesy of Dr G. Sethuraman, New Delhi). (B) A 34-year-old Italian man with
marked skin atrophy on the hands (figure courtesy of Dr G. Zambruno, Rome).
Nevertheless, the similarities between the Weary
pedigree and the Kindler case report led to the introduction and widespread use of the term "WearyKindler syndrome". A further similar autosomal dominant pedigree was reported by Larregue et al. in
1981.3 However, evidence for distinct recessive disease that more closely resembled the Kindler case
was published in 1985.4 Thereafter, case reports have
tended to separate autosomal recessive Kindler syndrome from autosomal dominant Weary syndrome,
but less than 100 cases of Kindler syndrome have
been published.5 Moreover, in recent years, characterisation of the molecular basis of a number of genodermatoses that partly resemble Kindler syndrome
has provided further evidence that it is indeed a distinct disorder (OMIM 173650). Notably, the
mechano-bullous disease, dystrophic epidermolysis
bullosa, has been shown to result from mutations in
the type VII collagen gene, COL7A1, on 3p21, a locus
that has been excluded in Kindler syndrome linkage
analyses.6, 7 Likewise, Kindler syndrome does not
map to the poikiloderma Rothmund-Thomson syndrome gene, RECQL4, on 8q24.3 or other known
DNA helicase genes.5 Another genodermatosis that
overlaps with Kindler syndrome, epidermolysis bullosa simplex with mottled pigmentation, has been
found to result from a specific mutation in keratin 5,
thus also distinguishing this genodermatosis from
true Kindler syndrome.8
532
Clinical features of Kindler syndrome
Kindler syndrome is characterised by congenital
acral blistering, most frequently on the hands and feet
and particularly at sites of trauma and sun exposure
(Figure 1A).1 Blistering tends to diminish around the
first decade but may still persist during adulthood and
indeed very occasionally can worsen in later life.
Another important feature is photosensitivity, which
starts at infancy but which may lessen with age, paralleling the decrease in blister formation.9 Poikiloderma, which is characterised by telangiectasia, skin
atrophy and abnormal pigmentation, occurs during
the first few years of life, affecting both sun-exposed
and non-sun-exposed areas and persists throughout
life. The associated skin atrophy is often parchmentlike and predominantly involves the dorsa of the hands
and feet, leading to wrinkling (Figure 1B). There is
also frequent involvement of the anal, urethral,
oesophageal and genital mucosae leading to stenosis.1, 10 In addition, there is a high incidence of oral
disease in Kindler syndrome. Affected individuals
have early-onset periodontitis, which can be a destructive process leading to loss of teeth. The gingivae are
fragile and bleed spontaneously.11, 12 Other associated
features include webbing of the fingers, contractures,
nail abnormalities and palmoplantar keratoderma. The
pseudo-syndactyly of the fingers may resemble dystrophic epidermolysis bullosa and surgical release of
Ottobre 2005
LAI CHEONG
Figure 2.—Light microscopy of skin from a Pakistani patient with Kindler syndrome skin. Biopsy reveals features of a poikiloderma with hyperkeratosis, epidermal atrophy, focal vacuolar changes/blistering close to the
dermal-epidermal junction and pigmentary incontinence. (Bar= 50 µm).
the contractures may be needed.13 The risk of mucocutaneous malignancy is increased, possibly due to a
combination of longstanding inflammation and ultraviolet irradiation-induced DNA damage. Squamous
cell carcinoma involving the hard palate, lip and hand,
as well as transitional cell carcinoma of the bladder
have all been reported as complications of Kindler
syndrome.14, 15
Skin biopsy findings in Kindler syndrome
Light microscopy of a skin biopsy from a bullous
lesion in Kindler syndrome shows cleavage close to the
dermal-epidermal junction, whereas sampling an
atrophic area may reveal features consistent with poikiloderma, with orthokeratosis, epidermal atrophy and
focal vacuolisation of the basal keratinocyte layer (Figure 2).16 In the papillary dermis, melanophages and
colloid bodies may be found.17 Transmission electron
microscopy findings in Kindler syndrome are very
variable and multiple planes of cleavage have been
demonstrated. In non-blistered skin there is reduplication of the lamina densa (Figure 3A), focal disruption of the lamina densa at the dermal-epidermal junction and areas of collagen lysis and disrupted elastic tissue in the dermis.6, 16, 17 The level of blister formation
can occur below the lamina densa (Figure 3B) but tissue separation in the lamina lucida or within basal
keratinocytes (Figure 4) has been documented in many
cases. Often, there is evidence for multiple planes of
blister formation in the same patient. Duplication of the
Vol. 140 - N. 5
Figure 3.—Transmission electron microscopy of Kindler syndrome skin.
(A) In intact skin there is extensive reduplication of lamina densa within
the dermis (arrows). (B) In areas of blister formation the plane of cleavage
(asterisk) is below the lamina densa. (Bar= 2 µm).
lamina densa is a useful clue to the diagnosis of Kindler
syndrome and skin immunohistochemistry with an
anti-type IV or VII collagen antibody may illustrate the
features of basement membrane reduplication with
broad patchy staining within the upper dermis (Figure
5). This finding is not pathognomonic for Kindler syndrome but is a helpful diagnostic finding.
Discovery of the Kindler syndrome gene
The gene for Kindler syndrome was discovered
simultaneously by 2 groups in 2003. Both sets of investigators used genome wide mapping with DNA
obtained from large consanguineous families.18, 19 The
gene responsible for Kindler syndrome mapped to
chromosome 20p12.3 and was initially termed
c20orf42, but subsequently renamed KIND1. This new
gene contains 15 exons, with the initiating methionine
in exon 2, spans 48.5kb of genomic DNA and encodes
a 677-amino acid protein, known as kindlin-1 (or kindlerin). KIND1 is highly expressed in keratinocytes,
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LAI CHEONG
Figure 4.—Transmission electron microscopy of Kindler syndrome skin.
In this biopsy there is an indistinct plane of cleavage (asterisk) close to the
dermal-epidermal junction. In the superficial dermis there is extensive
reduplication of lamina densa (arrows). (Bar= 2 µm).
Figure 5.—Immunofluorescence microscopy of skin sections labelled
with an anti-type IV collagen antibody. (A) In normal skin there is linear
labelling at the dermal-epidermal junction as well as immunostaining
around the dermal blood vessels. (B) In Kindler syndrome skin, there are
additional foci of positive anti-type IV collagen labelling within the dermis. (Bar= 25 µm).
with lower expression in prostate, ovary, colon, kidney,
and pancreas and weak expression in spleen, thymus,
testis, heart, brain, placenta, lung, liver, and fibroblasts. Alternative splicing is predicted to lead to an
additional larger transcript in colon and several further
splice variants in skin and other tissues, although the
biological significance of these different isoforms is not
yet known.15 Individuals with Kindler syndrome have
low or undetectable KIND1 gene expression.15 Database searches reveal that there are 2 other human genes
with homology to KIND1, namely MIG-2 and
MGC10966, but tissue expression is different and neither is relevant to the pathogenesis of Kindler syndrome.
patients with Kindler syndrome.19 Both these mutations occur at hypermutable CpG dinucleotides.22 In
contrast, mutated ancestral KIND1 alleles include
676insC, IVS7-1G>A, E304X and W616X that have
been detected in several Pakistani, Italian, British Caucasian and Omani patients, respectively.15 Identification
of these recurrent mutations is useful in optimising
mutation detection strategies. Sequencing of the KIND1
gene in some patients with Kindler syndrome, however, has failed to identify any pathogenic mutation(s),18,
19 and it is, therefore, likely that there is genetic heterogeneity, with mutations in one or more as yet unidentified genes accounting for the molecular pathology of
up to 25% of Kindler syndrome cases.
KIND1 gene mutations in Kindler syndrome
Kindlin-1 protein expression in skin
Since the identification of the KIND1 gene, 19 different loss-of-function mutations have been identified
in patients with Kindler syndrome.20, 21 These comprise 9 nonsense, 8 frameshift and 2 splice site mutations (Figure 6). Most mutations are specific to individual families, but recurrent hotspot and founder effect
mutations have been identified. Specifically, the mutation R271X has been demonstrated in Panamanian,
Caucasian American, and Omani subjects, whereas
R288X has been found in UK Caucasian and Turkish
Kindlin-1 is found mainly within the epidermis,
particularly in basal keratinocytes and at the dermalepidermal junction (Figure 7A). By immunohistochemistry, it is barely detectable in dermal fibroblasts
and is not present around blood vessels. Within keratinocytes, kindlin-1 co-localises with the ends of actin
microfilaments and vinculin in focal contact junctions.
This distribution is consistent with a role for kindlin1 in anchorage of the actin cytoskeleton to the dermal-epidermal junction/extracellular matrix. The lack
534
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LAI CHEONG
Figure 6.—Database of pathogenic KIND1 gene mutations in patients with Kindler syndrome. Nineteen different nonsense, frameshift or splice site
mutations have now been identified.
of kindlin-1 expression in the dermis, however, fails to
account for the histological features of collagen lysis
and elastic tissue disruption seen in Kindler syndrome.
Labelling with an anti-kindlin-1 antibody in Kindler
syndrome skin shows a marked reduction or absence
of immunostaining, consistent with the loss-of-function mutations identified in genomic DNA and the
lack of KIND1 mRNA on Northern blotting. This lack
of kindlin-1 labelling, therefore, provides the basis
for a new, rapid diagnostic test in Kindler syndrome
(Figure 7B). This is very useful since Kindler syndrome can be very difficult to diagnose in early life, and
early recognition of the disorder will have important
implications for clinical management of patients in
anticipating, and hopefully preventing, some of the
specific complications of Kindler syndrome.
notype (paralysed arrested elongation at twofold, pat) due
to loss of adhesion of muscle and the extracellular matrix
during embryo unfolding.23 UNC-112 binds to a molecule with potential adapter and signalling functions,
namely PAT-4, the worm homologue of integrin-linked
kinase (ILK).25 During assembly of junctions of muscle
cells to the extracellular matrix, PAT-4/ILK and UNC112
function after integrins have marked the sites of the
nascent junctions and before deposition of vinculin and
attachment of actin filaments.23, 25 The loss of cytoskeletal-extracellular matrix adhesion noted in nematodes
carrying mutations in UNC-112 would seem to be analogous to the adhesive defect observed in the skin of
patients with Kindler syndrome. Collectively, these data
provide new insight into mechanisms of epidermal-dermal adhesion in human skin.
Kindlin-1 homology with other proteins
Functional domains of kindlin-1
The kindlin-1 protein is highly conserved throughout
evolution, with closely related homologues in fish
(Onchorhynchus), flies (Drosophila), and worms
(Caenorhabditis elegans) suggesting that it has an important function across species.19, 23, 24 Kindlin-1 is the
human homologue of the Caenorhabditis elegans protein, UNC112, which has been shown to be a critical
component of a chain of molecules linking the actin
cytoskeleton to the extracellular matrix.19 In worms, loss
of the UNC112 protein leads to a paralysed lethal phe-
Computer database searches reveal that kindlin-1 has
homology to talin, a cytoskeletal protein of 270-kDa
thought to link the cytoplasmic domains of various β
integrin subunits to the actin cytoskeleton.26 Recently,
Kloeker et al. showed that, using integrin tail binding
assays and Chinese hamster ovary cells transfected with
FLAG-kindlerinCMV2, kindlin-1 binds to β1 and β3
integrin subunits.27 Both kindlin-1 and talin share a FERM
domain (F for Four point one protein, E for Ezrin, R for
Radixin and M for Moesin). FERM domains are impor-
Vol. 140 - N. 5
535
LAI CHEONG
Figure 7.—Immunofluorescence microscopy of skin sections labelled
with an anti-kindlin-1 antibody. (A) In normal skin, there is labelling close
to the dermal-epidermal junction as well as within the epidermis, particularly in basal keratinocytes. (B) In contrast, in Kindler syndrome, skin
labelling is almost completely absent (dotted line indicates the dermal-epidermal junction). (Bar=25 µm).
tant in the localisation of proteins to the plasma membrane
28 and are usually located at the N terminus in the majority of FERM-containing proteins whereas in kindlin it
is located near the centre of the protein’s primary structure and is bipartite. The bipartite FERM domain in
kindlin-1 is separated by a pleckstrin homology domain,
which is a module of 100-200 amino acids. Pleckstrin
homology domains are found in a large number of proteins involved in intracellular signalling or as constituents
of the cytoskeleton and potentially bind to various phospholipids, which in turn regulate several important proteins such as gelsolin and cofilin involved in cell turnover
dynamics and signal transduction.29, 30 The organisation
of the FERM and pleckstrin homology domains in
kindlin-1 is unusual but has also been found in the
Caenorhabditis elegans protein, UNC112. These data
suggest that kindlin-1 is a focal contact protein mediating actin cytoskeleton-extracellular matrix linkage. This
protein may also be involved in signal transduction via its
pleckstrin homology domain and in the recruitment of other binding partners through its bipartite FERM domain.
The role of kindlin-1 in human skin
More than 50 different proteins have been shown
to associate with focal contact junctions. These proteins
536
perform structural and/or signalling functions.31 For
kindlin-1, it remains to be seen whether the main function this new protein is primarily one of structural tethering or whether its main role might be in regulating
or recruiting of other molecules mediating actin-extracellular matrix adhesion. Identification of protein and
phospholipid binding partners of kindlin-1 by yeast
two-hybrid analysis, co-immunoprecipitation, and blot
overlays will now be the logical next step in elucidating the precise function of kindlin-1. Some clues to the
nature of potential kindlin-1 binding partners, however, have been derived from recent protein-protein
interaction studies on the kindlin-1 homologous protein, MIG-2. These studies showed that MIG-2 binds
to migfilin and filamin before linking to actin microfilaments, and that migfilin also binds to β-catenin.32
Kindlin-1 does not bind to migfilin (Irwin McLean,
unpublished data) and therefore it is likely that further, currently unidentified, proteins may be involved
in kindlin-1 binding to actin. It is also plausible that
these proteins may be relevant to the pathogenesis of
some cases of Kindler syndrome (or similar disorders)
for which no abnormality in KIND1 has been identified on gene sequencing.
A role for kindlin-1 in malignancy
Increased levels of kindlin-1 may be detected in
certain human malignancies. Notably, upregulation
of KIND1/kindlin-1 expression has been found in
~70% of colon cancers and ~60% of lung cancers,
with expression in several lung tumours increased by
more than sixty-fold.33 The significance of these findings is not fully known, but microarray analysis has
identified KIND1 as a potential transforming growth
factor-beta (TGF-β) inducible gene. Kindlin-1 expression is increased after treatment with TGF-β, and it
is possible that it may mediate TGF-β signalling in
tumour progression via contributions to integrin-dependent cellular processes, given that kindlin-1 forms
complexes with β1 and β3 integrins in focal contacts.27
The enhanced invasiveness of tumour cells is attributed
to epithelial to mesenchymal transition, a normal biological process that is critical for wound healing and
development. Epithelial to mesenchymal transition is
characterised by a change in cell shape from a polarised
epithelial cell to a flattened fibroblast-like morphology, a decrease in cell-cell junctions concurrent with a
decrease in E-cadherin expression, and an increase in
Ottobre 2005
cell motility. Kindlin-1, therefore, clearly has a critical role in cell spreading and the pathophysiology of
tumours, with in vitro data suggesting that kindlin-1
may be involved in the downstream events of the TGFβ response.27
Conclusions
Recent studies have now determined that Kindler
syndrome is a distinct genodermatosis caused by lossof-function mutations in a new structural/signalling
protein called kindlin-1, encoded by the KIND1 gene
on chromosome 20p12. Kindler syndrome is the first
inherited skin disorder to arise from abnormalities in
actin cytoskeleton-extracellular matrix adhesion, in
contrast to other forms of epidermolysis bullosa that
involve disruption of keratin filament-extracellular
matrix adhesion. Blistering in Kindler syndrome may
be explained by a lack of kindlin-1 binding to β-integrins in focal contacts, but the other clinical features
such as poikiloderma, photosensitivity and the risk of
mucocutaneous malignancy remain poorly understood.
Moreover, it is likely that Kindler syndrome is genetically heterogeneous and that other associated focal
contact proteins that bind to kindlin-1 may be further
candidate genes for this disorder. Identification and
characterisation of the kindlin-1 protein also provides
new insight into other complex biological processes,
including cell spreading and malignancy, and a fresh
source of intrigue for both basic science and clinical
medicine.
Riassunto
Aspetti clinici e molecolari della sindrome di Kindler
La sindrome di Kindler è una rara dermatosi autosomica
recessiva che è stata descritta per la prima volta nel 1954. Clinicamente le prime manifestazioni sono lesioni cutanee di tipo
bolloso indotte da traumi, ma successivamente si manifesta
fotosensibilità e poichiloderma. Per anni queste manifestazioni non venivano diagnosticate e si pensava potesse essere una variante della epidermolisi distrofica bollosa o una poichiloderma congenita o una combinazione di entrambe queste patologie. Nel 2003 si è riusciti a scoprire le basi genetiche della sindrome di Kindler. Si tratta di una patologia
cutanea ereditaria causata dalla ablazione di una proteina di
recente scoperta, la kindlina-1 (conosciuta anche come kinderlina) codificata dal gene KINDI situatati su 20p12.3 e
sono state descritte 19 relative mutazioni (nonsense, frame-
Vol. 140 - N. 5
LAI CHEONG
shift o splice-site). La kindlina-1 è una proteina di 677 aminoacidi che è espressa prevalentemente nei cheratinociti
basali dove garantisce un legame tra i filamenti di astina e la
matrice extracellulare e contribuisce a formare le giunzioni
di contatto dello strato dermico-epidermico. Tale proteina possiede dei domini funzionali coinvolti nei meccanismi di adesione (mediante delle β integrine), in altre funzioni cellulari mediate sempre da delle integrine come la diffusione delle cellule (ad esempio in risposta ai fattori di crescita e trasformazione di tipo β) e in nei processi di comunicazione tra
le cellule (principalmente mediante la chinasi P13) sebbene
sia difficile capire il meccanismo per cui si verificano le
manifestazioni cliniche della sindrome di Kindler quando
viene a mancare la kindlina-1. Le recenti scoperte nel campo molecolare hanno confermato che questa sindrome è tipo
genetico e rappresenta il primo disordine cutaneo di tipo
ereditario la cui anomalia primaria risiede a livello del complesso tra citoscheletro actinico e matrice extracellulare.
Questo articolo descrive le caratteristiche cliniche della sindrome di Kindler, l'identificazione del gene KINDI ed il
significato biologico della proteina kindlina-1.
PAROLE CHIAVE: Cute patologie, genetatica - Poichiloderma Actina - Citoscheletro - Cellule di adesione - Cheratinociti - Integrine.
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Ottobre 2005
Intradermal delivery of active principles:
field of dermatological research
P. SANTOIANNI
Various aspects have been established of how a drug or an
active principle can cross the barrier of the corneous layer,
and how this limiting factor can be influenced to obtain better
functional and therapeutical effects. In spite of the wide variety of the methods studied in order to increase transdermal
vehiculation to obtain systemic effects, the applicability in this
field remains limited. Attention to the problem of overcoming
the epidermal barrier has come mostly from dermocosmetic
studies. Dermatological research is mainly concerned today
with dedicating clinical and experimental studies to methods of
intradermal penetration. The cutaneous bioavailability of most
commercialized dermatological formulations is low. The strategies to increase delivery can be of a chemical, biochemical or
physical order. The chemical enhancers: a) increase the diffusibility of the substance across the barrier; b) increase their
solubility in the vehicle; c) improve the partition coefficient.
Moreover, the methods able to interfere with the biosynthesis
of some lipids by altering the structure of the barrier increase
penetration. Of dermatological interest are the physical mechanisms that increase the cutaneous penetration of substances:
iontophoresis (that increases the penetration of ionized substances), electroporation (that uses currents to create new ways
through the barrier), and sonophoresis, based on ultrasounds
ranging from 20 to 25 KHz that induce alterations and overcoming of the horny barrier. This paper reports the recent
development of these methods, and stress is laid on the importance and role of vehicles and excipients, that determine effects
of partition and diffusion, differences in the entity of the absorpReceived September 13, 2004.
Accepted for publication October 21, 2005.
Address reprint requests to: Prof. P. Santoianni, Dipartimento di Patologia Sistematica, Sezione di Dermatologia, Università degli Studi di
Napoli Federico II, Via Pansini 5, 80131 Napoli.
Vol. 140 - N. 5
Unit of Dermatology, Department of Systematic Pathology
University of Naples Federico II, Naples, Italy
tion and complex interactions between substance, vehicle and
skin, conditioning the specific effects; for which reason useful
formulations cannot be established on an extemporaneous
basis.
KEY WORDS: Drugs - Skin - Farmacology.
Intradermal and transdermal delivery
of active principles
T
he skin constitutes an important barrier to the penetration of exogenous substances in the body, and,
on the other hand, a potential way for the transport of
functional active principles or for therapeutic purposes in the skin and/or the body. Numerous recent studies have shown the modalities through which these
molecules cross the horny layer, which represents the
major limiting factor in the process of diffusion and
penetration, and how the penetration of pharmacologically useful substances could be increased.1
The horny layer has a very peculiar structure, with
a heterogenous organization like bricks and concrete,
determining barrier function. The corneocytes (the
539
SANTOIANNI
INTRADERMAL DELIVERY OF ACTIVE PRINCIPLES
bricks: about 85% of the horny mass) and intercellular lipids (15%) are arranged in approximately 15-20
layers.2 It is made upapproximately of 70% proteins,
15% lipids and only 15% water.3
Keratin and filagrin and demolition products are
packed into the corneocytes. The corneocyte lacks
lipids, but is rich in proteins. Lipids are contained
inside extracellular spaces, in bilayer organization surrounding the corneocytes.4 The very low permeability of the horny layer to hydrosoluble substances is due
to this extracellular lipidic matrix. This limits the cutaneous penetration of hydrophilic substances, due to
the convoluted and tortuous route, and for the extreme
hydrophobicity of 3 lipidic constituents: ceramids,
cholesterol, free fatty acids in molar ratio: 1: 1: 1
(weight ratio: ceramids 50%, cholesterol 35-40%, free
fatty acids 10-15%).5 This molar ratio is critical: the
diminution of the concentration of one of these types
of lipids, by altering the molar ratio functional to the
normality of the barrier, compromises its integrity.6
The variations in this lamellar structure and/or its
lipidic composition constitute the structural and biochemical bases for the variations ini permeability related to the body area (together with the thickness of the
horny layer, which depending on the body area differs regarding accumulation of filaments of keratin in
the corneocytes, their filagrin content, and number of
corneodesmosomes).7 The extracellular matrix also
forms the so-called reservoir of the horny layer (for
some substances that remain partially in the corneous
layer, from which they are slowly yielded).
The cutaneous penetration of substances comprises
various processes that are carried out serially or in
parallel. The substances may cross the horny layer via
the intercellular or transcellular route. Moreover there
are possible ways through the pilosebaceous units and
the eccrine glands.
Many efforts have also been made to obtain therapeutical effects in tissues far from the skin. According
to the definition of Flynn and Weiner we have: topical
administration with a pharmacological effect limited
to the skin structures, with some unavoidable systemic
adsorption; loco-regional delivery, when the therapeutic effect should be obtained in tissues beneath the
skin more or less deeply (generally muscles, articulations, vessels, etc.) with more or less limited systemic
adsorption.8 Doubtful is the efficacy of this type of
administration (performed with creams, ointments,
pomades, plasters, etc.). Transdermic delivery on the
540
other hand aims to obtain, through the application of
suitable preparations on the skin, pharmacologically
active levels for the treatment of systemic diseases.
From many years it has been attempted to obtain this
adsorption through the skin vascular network of substances with systemic effects, using various methods for
increasing the penetration. Transdermic (or percutaneous) delivery has proved useful in some conditions
and for some drugs, but has noteworthy difficulties: 1)
there are few suitable substances, and various parameters influence the diffusion; 2) metabolic processes may
be involved in the skin and the drugs may be metabolized before reaching the circulation. Moreover the
drug can accumulate in the skin and the bioavailability is determined by several other elements; 3) the transdermal penetration can vary from individual to individual and in connection with the age of the subject.
In topical administration the active principle must
viceversa obtain maximal local effectiveness with minimal systemic absorption. In the dermatological field
(as in the orthopedic field, ophthalmological, etc.)
great experience has been gained of the preparations
for local pharmacological action; results which can
be reached by an expert combination of suitable properties of the active substance with the appropriate vehicle (cream, gel, ointment, etc.)
Horny layer barrier and intradermal delivery
Penetration through the horny layer involves repeated phenomena of partition of molecules of the substances between the lipophilic and hydrophilic compartments. For many substances the penetration takes
place through an intercellular route, rather than transcellular, diffusing around the keratinocytes.
Intercellular way
The lipidic lamellae of the intercellular spaces –
each one including 2 or 3 bilayers, and made up mainly of ceramids, cholesterol and free fatty acids - form
the intercellular structure of the horny layer, with its
main role in the barrier function. The main part of the
solute substances crosses through the intercellular
lipidic ways, non-polar or polar. The permeability of
very polar solutes is constant and similar to the transport of ions (e.g. potassium ions). For the lipophilic
solute there is permeability that increases according to
their lipophilic property.
Ottobre 2005
Transcellular way
In the corneous layer the intracellular components
are devoid of lipids and there is no functional lipid
matrix around the filaments of keratin and keratoialin;
the result is almost impenetrability of corneocytes.9
An intercellular penetration is formed from the
degradation of the corneodesmosomes, and leads to the
formation of a continuous lacunar dominion (aqueous pore). Normally the lacunae formed from the
degradation of corneodesmosomes are scattered and
not continuous, but - as a result of a stimulation
(hydratation by occlusion, ionophoresis, ultrasound
waves, etc.) – they may become larger and connect in
a net forming a pore-way, returning afterwards to the
base condition. This recent data is particularly interesting working with various methods to obtain the
permeability increment.6, 10
Transport through follicular and gland structures
A limited diffusion way is that through hair follicles,
pilosebaceous units and eccrine glands. The orificies
of the pilosebaceous units represent about 10% in
areas where their density is high (such as the face and
scalp) and only 0.1% in areas where the density of
this unit is low. It is therefore possible selectively to
address the action of some drugs to follicles and sebaceous glands. The follicular route may be influenced
by the sebaceous secretion, which favors the absorption of substances soluble in lipids. Penetration through
the pilosebaceous units is dependent on the characteristics of the substance and on the type of preparation.
In the methods of transdermic penetration to obtain
systemic effects, this route is utilized initially, while the
intercellular one, which is obtained subsequently, is the
principal one.
PHARMACOKINETIC
PARAMETERS.
VEHICLE/CORNEOUS
LAYER PARTITION
For the study of the transport mechanisms and skin
barrier functions, this can be considered as a membrane, or a cluster of membranes (for the study of
which mathematical principles can also be applied).11
On the whole its transport through the corneous layer
is mainly a molecular passive diffusion process. The
physico-chemical and structural properties of the substance determine the capacity of diffusion and penetration through the membrane: important determinants
are solubility and diffusibility.
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SANTOIANNI
The relative solubility of a solute,—that is the equilibrium between the solubility of the substance in the
corneous layer in relation to that in the vehicle—determines its partition coefficient. This means, i.e., the
ability of the substance to separate itself from the vehicle towards the external layer of the horny layers, with
the possibility therefore of absorption from the keratinocytes.
The diffusibility and the ability of a solute to penetrate through the barrier is influenced by several factors, including the tortuosity of the intercellular route.
This passive process of absorption is ruled by Fick’s
law of diffusion: the velocity of absorption—flow—
is proportional to the difference of concentration of
the substance in relation to that within the barrier. It can
finally be cited that the permeability coefficient relates
flow and concentration, and results from partition
coefficient, diffusion coefficient, and length of diffusion route.12
ROLE OF THE VEHICLE AND EXCIPIENTS AND INTERACTION
WITH THE ACTIVE PRINCIPLES
A vehicle is defined by the type of preparation
(cream, ointment, gel, etc.) and by the excipients (water,
paraffin, propylene glycol, etc.), and the terms “vehicle” and “excipient” refer to different entities.
Vehicle and excipients deeply influence the velocity and intensity of absorption and, consequently,
bioavailability and efficacy. The following are of critical importance: 1) solubility of the substance in the
vehicle; 2) partition coefficient (which determines the
entity of transfer of the substance from the vehicle in
the horny layer). The excipients of the vehicle modulate the effects of partition and diffusion in the horny
layer.
A lipid preparation, by occluding, may enhance the
penetration of the drug, but fat vehicles and ointments
are not always more powerful than creams; and creams,
gels and solutions may be formulated so as to obtain
power equal to that of the ointments. For instance for
topical corticosteroids of different classes of potency
the same activity may result when formulated in different vehicles.13 A gel preparation of kellin, obtaining
better penetration, has demonstrated better results in
the treatment of vitiligo.14 Also the transfollicular penetration is influenced by vehicle and excipients; and,
in this case, better results are given by lipophilic and
alcoholic vehicles and dimension and charge of the
molecules of the solute are relevant.9
541
SANTOIANNI
All the reported studies underline the absolute relevance of the composition of vehicle and formulations, which determine the effects of partition and diffusion, differences in the entity of absorption and complex interaction among substance, vehicle and skin,
and consequently the specific effects. Vehicles and
formulations are specifically studied in relation to the
variable elements reported and cannot be extemporaneously formulated.
CONDITIONS WHICH MODIFY THE BARRIER FUNCTION
During hydratation the greater part of the water is
associated with intracellular keratin; the natural factor
of hydratation (NMF) absorbs a considerable amount
of water (10% of the weight of the corneocyte).15 Corneocytes swell and the barrier property of the horny layer is deeply altered (in the intercellular regions the
small amounts of water linked to polar groups by
hydratation do not provoke reduction in the organization of lipids and of permeability).16 The effect of the
hydratation, however, proves discontinuous: for some
substances the increment is 10 times and for others it
is very limited.17
The occlusion partially hinders the loss of humidity of the skin and increases the water content of the
horny layer. However, the NMF level in the horny layer is almost zero: it seems, therefore, that there is a
homeostatic mechanism that prevents hyperhydratation
of the skin.9 The occlusion may increase the absorption
several times, especially for hydrophilic compounds,
or in some conditions the formation of the reservoir
effect.
The acidity of the cutaneous surface, controlling
homeostasis and enzymatic activities, influences their
permeability;18 and the metabolic activity of the skin
(enzymatic oxidoreductive processes, etc.) may modify the substances applied, influencing permeability and
effects.
The barrier function is altered in some pathological
conditions, like the alterations inintercellular lipid barrier properties—due to a deficit in essential fatty acids,
the accumulation of cholesterol sulfate, abnormal intercellular deposition of lipids—determining “dry skin”
(with abnormal desquamation and reactive hyperproliferation); and gene alterations in some genetic diseases, codifying for keratin filaments (e.g. Thost-Unna
disease) or for the structural proteins of the corneous
envelope or for enzymes (e.g. glutaminase I in lamellar ichthyosis). Nevertheless, it is not always possible
542
to establish their influence. In the various pathological conditions there are differences in absorption, but,
in spite of the alterations to the barrier, do not lack
obstacles to the penetration of substances applied on
the skin.6
The influence on absorption of the variations pertinent to cutaneous regions and anatomical sites is very
important. As regards the regions, the absorption diminishes from the palpebral to the plantar skin.19
As for the influence of age, various biological activities are lower in the skin of the aged individual; therefore, in these subjects, the pharmacodynamic parameters and the effects on absorption are to be evaluated
according to the nature of active principles; as in the
premature and newborn babies, who have greater cutaneous permeability.20
There are no experimental data confirming the validity of the friction on transcutaneous absorption.6
Lastly the barrier alterations induce modifications in
transepidermal water loss (TEWL),9 and the horny
layer may be defined a biosensor: i.e. alterations in
external humidity regulate the proteolysis of filaggrin,
synthesis of lipids, of DNA etc. within epidermocytes,
which can also lead to inflammatory phenomena.21
BIOAVAILABILITIES
OF THE ACTIVE PRINCIPLES OF THE
FORMULATIONS
The cutaneous bioavailability of most commercialized dermatological formulations is low (within 1-5%
of the applied dose).8
The active substances of topical formulations are
generally absorbed in small amounts: only a reduced
fraction passes from the vehicle to the corneous layer.
The greater part remains on the skin, subject to loss
over time for a series of factors: sweat, chemical degradation, removal, etc. The entity of the absorption of the
drug is in the order of 1-5% of the applied dose; so
determining differences in bioavailability. Future standards would, therefore, have to comprise formulations
with a not high concentration, but pharmaceutically
optimized to an elevated (50-100%) bioavailability.8
This is because the marked variations regarding the
different cutaneous areas and conditions of the skin
make the therapeutic equivalence with other ways of
administration, obtainable in clinical conditions, uncertain.22
It has been authoritatively asserted that the standards concerning topical preparations—bioavailability, bioequivalence and therapeutic equivalence, meth-
Ottobre 2005
ods and protocols of appraisal, and biopharmaceutical parameters of the products—are 10-20 years
behind the standards which can be reached with other therapeutic modalities of administration, such as the
oral one.8
Biopharmaceutical appraisal
of topical formulations
For measuring drug release and absorption there
are models widely used in the development of topical
dermatological formulations; formed by synthetic
membranes or by animal or human skin studied in vitro (presenting, however, considerable variability in
barrier power).23 The in vivo methods include kinetic
and dynamic models. The former are based on: a)
selective removal of the corneous layer; b) skin dissection technics; c) methods of appraisal of indirect percutaneous absorption (in blood, secretions, etc.). The
dynamic models include: 1) determination of skin color variations by various instruments (Minolta, X-rite,
etc.) with which are also determined the provoked
degree of erythema or the variation of color after UVB
stimulation; 2) determination of the cutaneous blood
flow (monitored with optical Doppler Laser procedures); 3) appraisal of the UVA-induced neutrophil
infiltrate (e.g. in order to establish inflammatory corticosteroid activity); 4) animal models.24
Methods of modulation
of cutaneous permeability
When a substance is applied on the skin with a simple vehicle the therapeutic result can be unsatisfactory because of the insufficient concentration obtained
in the application area. In the last few years researches and strategies have been developed in order to
increase the vehiculation. The strategies may be ofa
chemical, biochemical or physical order.
Chemical enhancers
In order to increase penetration the vehicle may be
integrated with so called enhancers, some of which
interact with intercellular lipids and so improve the
diffusion coefficient of the substance in the corneous
layer. Chemical enhancers may: a) increase the diffusibility of the substance inside the barrier; b) increase
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SANTOIANNI
the solubility in the vehicle or both; or c) improve the
partition coefficient.
These substances may frequently have a non specific
action, implicating the penetration of irritating substances subsequently in probable contact with the skin.
Enhancers of this type, that, however, have not achieved
wide diffusion, are: Azone, SEPA, Dermac SR-38 and
several others;25 oleic acid is frequentely used. In some
cases, however, they also have an irritating effect, for
which reason their application is carefully evaluated in
the various preparations.26
Excipients like ethanol and propylene-glycol and
the dimethylsulfoxide (DMSO) may increase the diffusion 25 by altering the organization of lipids in the corneous layer and the structure of the barrier.27 Their
solvent properties influence positively the solubility of
the active substances in the vehicle and the partition
coefficient (however, with some possible unpleasant
effects, like stinging or irritative dermatitis). These
enhancers also increase the reservoir effect. Detergents and surfactants behave in an analogous way
extracting lipids.
Very interesting is the metabolic approach. The
strategies that interfere with the synthesis, organization,
formation, activation of extracellular lamellae may
increase the penetration interfering with the permeability of the barrier. Interference with the biosynthesis of some lipids may alter the structure of the barrier, and increase penetration. Experiments have also
been carried out with methods of interference with
secretion and organization of lipids (e.g. brefeldine
A, monetine and chloroquine) and enhancers which
alter the supramolecolar organization of the bistratified
lamellae (synthetic analogues of fats which induce
abnormalities in membrane organization; complex
precursors which can not be metabolized, etc.).6
These methods lead to alteration of the critical molar
ratio among ceramids, cholesterol and fatty acids: if
there is deletion or excess of one of these 3 key-lipids,
the lipidic component in excess cannot maintain the
lamellar organization. There may be separation of the
phases with more permeable interestitial spaces and
creation of a new way of penetration.
As a matter of fact every alteration of the components
and of the critical mixture of the 3 types of lipids,
leading to a greater than 3 times increase in one component delays the repair of the altered barrier.28
The efficacy of the enhancers may be increased by
inhibiting the metabolic reaction of repair, when bar-
543
SANTOIANNI
rier alteration has been obtained; inhibiting metabolic sequences that can rebuild and maintain the barrier
function. Inhibitors of enzymes with relevant functions (e.g. lovastatine) or specific inhibitors of enzymes
of the synthesis of ceramids or fatty acids, with alteration of the critical molar ratio of the types of lipids
mentioned, and discontinuity in the lamellar double layer system.29 An increment may be obtained due to
enhancers modifying the polarity.30
Recent studies propose using the methods discussed
in collaboration with physical methods.
In spite of the wide variety of methods experimented in order to increase transdermal vehiculation (for a
systemic action) their current application has been
very restricted, and the list of drugs for which it has
been possible to use the transdermic way and their
systemic use is very small (<10), and restricted to
lipophilic and low molecular weight substances: nicotinicacid, nitroglycerin, clonidine, steroid hormones,
scopolamine.31 Certainly the main difficulties encountered by these studies are that they are carried out not
on human skin but in vitro.
Carrier vesicular systems
Liposome formulations have proved very effective,
however; they probably increase the penetration only
through the transappendigeal way, their capacity to penetrate the corneous layer not having been demonstrated.32
Also worthy of mention are: niosomes, whose formulation comprises non ionic surfactants, and transferosomes, formed by modified liposomes (phosphatidylcholine, sodium cholate, ethanol, etc.), systems based on
the possibility of vesicles crossing the corneous layer
whole, because of the osmotic gradient between external and internal layers of the barrier. These are flexible
vesicles able to transport their content through the tortuous intercellular route of the corneous layer.
Physical systems for vehiculation increment
Several methods have been experimented to increase
the penetration of active substances in the general circulation to obtain a systemic action, avoiding a traumatic introduction, like the injection; or also for a
loco-regional effect; systems aiming to reach the target area with adequate drug concentration at some
depth, and in a selective way without dispersion in the
blood (transdermic systems).33
544
Iontophoresis and electroporation represent electrically assisted physical methods increasing the vehiculation of drugs/substances through the corneous layer. Sonophoresis exploits low frequency ultrasounds.
Sonophoresis
Sonophoresis (or phonophoresis, or ultrasonophoresis) appears as the more promising strategy
to enhance penetration of drugs and other active principles used in the dermatological field.34 Ultrasounds
(US) produce alterations in the structure of the corneous layer and permeabilization.35
Useful frequencies have long been uncertain: those
used in diagnostic procedures (1- 4 MHz) are insufficiently effective, while the very high ones (10-20 MHz)
increase the penetration;36 but those at low frequency
between 20 and 25 KHz have recently proved interesting in dermatology.37
Recent studies have evidenced the dependency on
various parameters of the effect at 20 KHz:38-40 the
lower barrier function appears to be mainly responsible for the phenomenon of cavitation,41 though the
increase in temperature induced by US has been
assigned a role around 25% in some experiments,42-44
and transport by convection and mechanical impact
are involved.45 Possible ways of penetration have been
assigned to the formation of transitory modifications
(pores approximately 20 µm),46 which prove sufficient
for the penetration of high molecular weight drugs in
experimental conditions.47, 48
The combination of US with surfactants (sodiolauril-sulfate) increases transport, and demands less
energy.49 Sonophoretic absorption varies according
to the different drugs.50 For instance, the 20 KHz frequency can increase the absorption of a corticosteroid
as in vitro experiments with clobetasol-17-propionate
have shown.40 The biological effects on human skin
and the possible applications have also been studied.44
The activation by US of drugs opens an interesting
perspective in the skin cancer field.51
Ionophoresis and iontophoresis
Ionophoresis increases the penetration of ionizable
substances, using the force of a weak electric field
(the direct applied or galvanic current ranges from 0 to
250 microamperes=0.25 mA. The ions transport the
applied current from an electrode to a second indif-
Ottobre 2005
ferent electrode).52 In dermatology it is widely used in
hyperhidrosis with various equipment.53
The time necessary to allow the penetration of an
ionized substance through the barrier is approximately 25-30 min in some applications, but in some transdermic applications it lasts several hours (for a systemic or loco-regional action on the osteotendinous
apparatus).
Conventional ionophoresis shows low efficiency
for transdermic transport, distributing the active agent
mostly on the surface of the skin: the absorption in
deeper tissues or for a systemic action takes place in
minimal part. If ionophoresis is prolonged for a transdermic effect it may provoke side-effects due to the
direct current (polar chemical burns) or possible allergic reactions to the drug. There are some contraindications (subjects with pacemaker, cutaneous lesions,
neurological seizures).
Iontophoresis utilizes bipolar electric fields with
alternating current, allowing the transport of ionizable substances, including macromolecules, and also
non ionizable substances and only polar molecules.
The transport takes place in the corneous layer, interstitially through the aqueous pores, and also extracellularly at lacunar level (and less frequently at appendigeal level).10 The iontophoresis of ionizable drugs may
increase penetration by 20-60 times compared to simple topical application. The amount of the drug is proportional to the intensity of the applied current.
The 2 main mechanisms responsible according to
Santi 54 are: ionophoresis, in which ions are rejected by
an electrode of the same charge; electrosmosis, convective movement of a solvent which takes place
through a charged pore, against the preferential passage
of counterions, under the influence of the electric field.
The iontophoresis increases transport through the barrier with 3 mechanisms: a) interaction between ions and
electrical field supporting ionic transfer; b) increment
of the permeability of the skin-membrane provoked by
the flow of electric current; c) electrosmosis, by which
phenomenon there is a massive flow of ionized solvent
which transports ionized species as well as neutral
ones.55 Because the skin above pH 4 has a negative
charge, the counterions are positive ions and the electrosmotic flow goes from anode to cathode. Therefore anodic transport is favored by electrosmosis, while
cathodic transport is delayed. Electrosmotic flow
becomes elevated with an increase in the dimension of
ionized molecules. Because of electrosmosis the trans-
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SANTOIANNI
port of anionic large molecules (including proteins)
from the anodic compartment can be more effective
than that from the cathodic compartment.
The phenomenon of electrosmosis probably increases in particular the anodic transport of drugs positively charged and of large dimensions (whose iontophoretic transport is often very small), and promotes
the transdermic migration of molecules not charged but,
however, polar, molecules whose passive permeation
is generally very reduced.56
As for the side-effects, in iontophoresis the direct
current (galvanic) applied through electrodes to a part
of the body, induces the formation of a unidirectional
electric field; moreover heating of the tissue, erythema and also burning, tingling of the area where electrodes are applied. Viceversa in iontophoresis the
numerous interruptions of the current (very high frequencies) eliminate the overheating produced by the
galvanic current.
As reported before the possibility of iontophoretic
transport also of macromolecules has been demonstrated.56
Combining iontophoresis and passive diffusion an
increase of the penetration of kelline at idoneous pH
7 through the corneous layer has been observed, even
if the drug is not ionized.57
It has been demonstrated 58 that for small molecules
the transport in general diminishes with the increase in
size, and the molecular structure represents a key element for the transport through iontophoresis. The
chemical sequence within the molecular structure (as
demonstrated for oligonucleotides) may also be important.
Santi et al.59 thinks that iontophoresis may be an
interesting alternative to parenteral administration in
a possible systemic therapy, also for peptides and for
macromolecular substances in an ionized state at physiological pH (these substances being poorly absorbed
through other ways and degraded by proteolytic
enzymes). A treatment for some cutaneous diseases
(like herpes simplex and psoriasis) would also be possible.60
It has also been reported that iontophoresis may
allow penetration of the drug at the depth of 1 cm or
more. Once the current flow has transported the ions
of the drug through the first 2 mm of the corneous layer, there would be a reservoir effect above the microcirculation area.
It is also believed thatthe drug spreads from the
545
SANTOIANNI
reservoir in the dermis and is transported from the
capillary network in the blood flow (with reduction,
therefore, of the gradient necessary for the penetration towards the deep underlying tissues). The local
administration of a drug through iontophoresis would
therefore reach a depth equal at least to that of the vascular network (superficial vascular plexus).
In iontophoresis the conventional units of administered drug are indicated by mA/min and are calculated
like thjis: mA (applied current) X time (minutes application).
Iontophoresis has a long history, having been for
many years suggested for several therapies in various
fields of medicine, in physical therapy and also in
odontostomatology, however, it has limited efficiency
in systemic therapy, and, therefore, the transfer of
active principles has been studied mostly for locoregional therapy (e.g. in the treatment of muscle-skeletal disorders) or for a few local treatments. Many substances have been tested: lidocaine, epinephrine,
methylprednisolone, desametazone, antivirals, antibiotics.31
The usefulness of iontophoresis in dermatology has
been recognised by Gangarosa for local anesthesia,
for postherpetic neuralgia, and for topical corticosteroid therapy.61 The anesthesia up to 1 cm depth or
more has been studied in double blind conditions,
proving effective for surgery of the eyelids and cutaneous biopsies (shave).
In conclusion, iontophoresis is particularly indicated for short applications in which it turns out to be
safe and effective. It may be improved by modern electronic instrumentation and by some devices (hydrogel
pads).62
Recent experimental studies have shown new perspectives, introducing substances functioning as cooperators, such as iontophoresis with hydrogel. In a study
by Fang using formulations with several polymers,
hydrogels have shown a capacity of transport greater
than solutions,63 results attributed by the author to the
antinucleant ability of polymers, which increment the
thermodynamic activity of the active substance in the
formulation. Moreover, in this study, pretreatment
with isopropylmiristate, enhancer of lipofilic substances, improved the penetration, both passive and
iontophoretic.
Autonomous systems of iontophoresis which are
based on vehiculation with very low current intensity
have recently been proposed: in practical terms, on
546
the application of a plaster (inside which is lodged an
ultrathin battery) through which the ions penetrate the
skin and underlying tissues with an intensity of 80
mA/min and for 24 h.
The 24 h migration makes it possible to create a
subcutaneous deposit of the drug in the target zone,
obtaining therapeutic effects higher than the traditional ones. Moreover, the low intensity current largely avoids the risks of skin irritation, and this system
allows wide freedom of movement to the patient.64
Recently iontophoresis has been proposed in combination with low frequency ultrasounds.48
Some interesting applications are under study in
order to obtain the extraction of analytes from the
venous circulation through the skin (inverse iontophoresis).
Electroporation
With this modality a reversible electrical field, with
short and high voltage pulses, electropores the double
layer lipid barrier and leads to the formation of non
lamellar lipid phases and subcellular pores, with greater
speed of substance transport.65 Electroporation uses
low intensity currents from an electrode of the same
charge of net polarity of the drug, transporting the
molecules through the corneous layer and utilizing
extracellular routes (probably also transappendigeal).
Electroporation with the purpose of increasing the
permeability of the skin for drug penetration is in the
first few steps of development;66, 67 in vitro studies
still have to be integrated with in vivo and clinical
studies, performed with new instruments. According
to some authors, a greater increment could be attained
by the combination of electroporation with iontophoresis and ultrasounds, also for macromolecules.
Electroporation probably creates new routes and consequently a more uniform distribution of the electric
charge; therefore, probably with less cutaneous irritation.
It is to be expected that in the near future increasing
researches will be devoted to further develop physical
methods for active cutaneous penetration, modifying
the present methods of iontophoresis and/or with ultrasound methods (sonophoresis).
Cryoelectrophoresis
Some problems due to iontophoresis may be overcome if the pad of the active electrode is replaced by
Ottobre 2005
an ice-block produced by freezing the drug solution.
Cryoelectrophoresis is a technique of vehiculation of
frozen drugs and active principles, that allows the penetration of hydrosoluble substances, better if ionizable. It allows their transdermic penetration, but also
that of substances with different physico andchemical
properties, reaching deep level (6-8 cm) in amount
greater than other methods, with very reduced systemic impact (0.04%). It utilizes alternating electric current, with frequency and polarity according to the
drug; and with appropriate variation of intensity.68 The
transport of the drug/substance probably takes place
according to the following mechanisms and physicochemical phenomena: a) diffusion (strongly enhanced
by the poration effect due to the oscillating current
used); b) electrosmosis. Through this phenomenon
are transported water as well as dissolved substances;
when the drug is not dissociated or when the active
ion is positive, electroosmosis strengthens the diffusion
and also the electrophoretic action;69 c) electrophoresis, that increases the diffusion of ionized drugs. It
demands the electrolytic dissociation of the drug while
the aforementioned other mechanisms also allow the
passage of neutral molecules.68 The alternating current also probably has a poration effect.
The active substance is dissolved in ice, and the
electrodes are positioned on both sides of the areas to
be treated. The electric current passes essentially
through the liquid solution originating from the fusion
of the ice, and permeates the tissues moving towards
the other electrode and transporting the ions of the
active substance. The modification of the parameters
of the current produces a definite direction of the ionic flow toward the deep tissues with only local high
concentration. According to Aloisi 68 depth may be
reached (e.g. articular caps) not only by anti-inflammatory molecules of low molecular weight (M=302.68
d) but also by mucopolysaccharides (mol.wt. 5 to 10
kd).
This modality may increase the maximum intensity and respectively the total intensity of the current
many times (200-300%) without damage and/or
unpleasant sensations: the negative effects of the cold,
of the electrical current and of the drug become mutually neutralized. The provoked vasocostrictive effect
given by the low temperature prevents an unwanted
removal of the drug by local blood flow.
Aloisi et al. have calculated formulas concerning
the quantity of drug that can penetrate. It turns out
Vol. 140 - N. 5
SANTOIANNI
that the drug that can be administered is not dependent
on the dissolved quantity in the solution but only on the
electric charge in the circuit and on the molar fraction
f.68 The main applications (for which specific instruments have been created) are apparently the following:
corticosteroids in high local concentration without
systemic effects; local therapy for local circulation
deficit; venous insufficiency.68 According to Vazharov
this technique is useful to treat pain, edema, trophic disturbances, and post-traumatic conditions of the muscle-skeletal system, arthritis, hematoma, etc. In dermatology this complex instrumental technique does
not seem to have effective future possibilities.
Hydroelectrophoresis
This different method of iontophoresis uses pulsed
currents, of different shape and frequency, with gel
formulations. An important function is assigned to the
gel (e.g. agarose gel) in which the active agent is dispersed with a mobility enhancer.
The gel improves migration during the electric field
and the solution with the enhancer creates the appropriated ionic strength for the type of active agent transported. This may be ionized or not. Hydroelectrophoresis allows the active agent to penetrate the tissues by 0.5 to 10 cm approximately, avoiding its premature dispersion and creating a directional flow,69
which allows loco-regional therapy. A computerized
instrument supplies electric waves of different shape
and frequency (limited, however, to some predefined
possibilities), adjusted to the depth to be reached by the
active agent. Hydroelectrophoresis might be utilized in
the management of pain, with applications lasting 1530 min, and possibility to focus on the regions interested.
Laser waves
The fotomechanical compression obtained by laser
(Q-switched rubin laser) has also been recently experimented for modulating the permeation of the corneous layer.70 The material (polystirene) including the
solution with the active principle (e.g. δ-amino-levulinic acid) absorbs the radiation provoking its ablation,
while the solution increases propagation in the corneous layer of the stress induced by laser pulses. The
penetration route is apparentely extracellular as for
sonophoresis and ionophoresis. The effect is only temporary and the barrier function is restored.6
547
SANTOIANNI
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Veicolazione intradermica e ricerca dermatologica
L
a pelle costituisce, da un lato, un’importante barriera
verso l’ingresso di sostanze estranee nel corpo e, dall’altro, una via potenziale utilizzabile per il trasporto di principi attivi funzionali o a scopo terapeutico in essa e nell’organismo. Da numerosi recenti studi sono state rese note le
modalità con le quali queste molecole possono attraversare
lo strato corneo, che costituisce il più importante fattore limitante del processo di diffusione e penetrazione, e con le quali possa essere incrementata la penetrazione di sostanze farmacologicamente utili 1.
Lo strato corneo presenta una struttura unica, con organizzazione eterogenea a mattoni e cemento, determinante
nella funzione di barriera. I corneociti (i mattoni, circa l’85%
della massa del corneo) e i lipidi intercellulari (il 15%) sono
disposti in circa 15-20 strati 2. Esso si compone all’incirca di:
70% di proteine, 15% di lipidi e solo 15% di acqua 3.
Nei corneociti sono addensate cheratina e filaggrina e prodotti di scissione. Il corneocita è privo di lipidi, ma ricco
proteine. I lipidi sono contenuti all’interno degli spazi extracellulari, organizzati in lamelle a doppio strato che circondano
i corneociti 4. La bassissima permeabilità del corneo a sostan-
Vol. 140 - N. 5
ze idrosolubili è legata a questa matrice extracellulare lipidica. Questa limita la penetrazione cutanea di sostanze idrofile, per il percorso convoluto e tortuoso, e per l’estrema
idrofobicità dei 3 diversi lipidi: ceramidi, colesterolo, acidi
grassi liberi in rapporto molare 1: 1: 1 (rapporto in peso:
ceramidi 50%, colesterolo 35-40%, acidi grassi saturi 1015%) 5. Questo rapporto molare è critico: la diminuzione
della concentrazione di uno di questi tipi di lipidi, alterando
il rapporto molare funzionale alla normalità della barriera, ne
compromette l’integrità 6. Le variazioni di questa struttura
lamellare e/o della sua composizione lipidica costituiscono
la base strutturale e biochimica delle variazioni di permeabilità correlata alla sede corporea (insieme allo spessore del
corneo, che, in rapporto alla sede corporea, differisce per
addensamento dei filamenti di cheratina nei corneociti, loro
contenuto in filaggrina, nonché numero di corneodesmosomi) 7. La matrice extra-cellulare rappresenta anche il cosiddetto reservoir dello strato corneo (per alcune sostanze che
rimangono relativamente bloccate nello strato corneo, da cui
vengono lentamente cedute).
La penetrazione cutanea di sostanze comprende diversi
549
SANTOIANNI
processi che si svolgono in serie o in parallelo. Le sostanze
possono attraversare il corneo attraverso la via intercellulare e quella transcellulare. Inoltre sono possibili vie attraverso le unità pilosebacee e le ghiandole eccrine.
Si è provato a raggiungere un obiettivo terapeutico anche
in tessuti lontani dalla cute. Secondo le definizioni di Flynn
e Weiner si ha somministrazione topica per un effetto farmacologico limitato alle strutture cutanee, con qualche assorbimento sistemico inevitabile; somministrazione loco-regionale, quando l’effetto terapeutico deve essere raggiunto in
tessuti al di sotto della cute più o meno profondamente (in
genere muscoli, articolazioni, vasi, etc.) con assorbimento
sistemico, più o meno limitato 8. Notevoli sono le perplessità
riguardo all’efficacia di questo tipo di somministrazione (realizzata con creme, unguenti, pomate, cerotti, etc.). La somministrazione transdermica è diretta invece a ottenere, attraverso l’applicazione di una preparazione sulla cute, livelli
farmacologici attivi per il trattamento di malattie sistemiche.
Da molti anni si è cercato di ottenere quest’assorbimento attraverso la rete circolatoria cutanea di sostanze ad azione sistemica, utilizzando metodi diversi per incrementarne la
penetrazione. La somministrazione transdermica (o percutanea) si è dimostrata, in alcune condizioni e per alcuni farmaci, vantaggiosa, ma presenta notevoli difficoltà: 1) non
sono molte le sostanze idonee e una serie di parametri ne
influenzano la diffusione; 2) possono essere coinvolti processi
metabolici nella cute e i farmaci metabolizzati prima di raggiungere il circolo. Inoltre si può avere accumulo nella cute
del farmaco e biodisponibilità determinata da vari altri elementi; 3) la penetrazione transdermica può variare da individuo a individuo e in rapporto all’età del soggetto.
Nella somministrazione topica il principio attivo deve
viceversa ottenere massima efficacia locale con minimo
assorbimento sistemico. In campo dermatologico (come in
campo ortopedico, oftalmologico, etc.) vi è lunga e ampia
esperienza delle preparazioni per un’azione farmacologica
locale; questi risultati sono raggiungibili dall’esperta combinazione di opportune proprietà della sostanza attiva con
appropriate forme di veicoli (gel, creme unguenti, etc.)
Barriera dello strato corneo
e penetrazione intradermica
La penetrazione attraverso il corneo comprende ripetuti
fenomeni di partizione delle molecole delle sostanze tra i
compartimenti lipofilo e idrofilo del corneo. Per molte sostanze avviene per una via intercellulare, piuttosto che transcellulare, con diffusione intorno ai cheratinociti.
Via intercellulare
Le lamelle lipidiche degli spazi intercellulari — ciascuna
comprendente 2 o 3 doppi strati lipidici e costituita principalmente da ceramidi, colesterolo e acidi grassi liberi —
compongono la struttura intercellulare del corneo, con ruolo
550
principale nella funzione di barriera. La maggior parte dei
soluti attraversa vie intercellulari lipidiche, non polari o polari. La permeabilità di soluti molto polari è costante e simile
al trasporto di ioni (ad esempio ioni potassio). Per i soluti
lipofili vi è permeabilità che cresce con la loro lipofilia.
Via transcellulare
Nel corneo, tra i componenti intracellulari non vi sono
più presenti lipidi e non vi è matrice funzionale lipidica
intorno ai filamenti di cheratina e a cheratoialina; ne risulta
una quasi impenetrabilità dei corneociti 9.
Una via di penetrazione attraverso spazi interstiziali è
costituita dalla degradazione dei corneodesmosomi e porta
alla formazione di un dominio lacunare continuo (poro acquoso). Normalmente le lacune formate dalla degradazione di
corneodesmosomi sono disseminate e discontinue ma, in
seguito a stimolo (idratazione per occlusione, ionoforesi,
ultrasuoni e altri), possono ampliarsi e connettersi in rete
formando una via-poro, ritornando poi alla situazione di
base. Questo recente dato è particolarmente interessante nello studio dell’incremento della permeabilità ottenibile con
metodi diversi 6, 10.
Trasporto attraverso strutture follicolari e ghiandolari
Una via a limitata diffusione è quella attraverso follicoli
piliferi, unità pilosebacee e ghiandole eccrine. Gli orifici
delle unità pilosebacee rappresentano circa il 10% in aree
dove presentano elevata densità (come viso e cuoio capelluto)
e solo lo 0,1% in aree a bassa densità di unità. È, così, possibile selettivamente indirizzare l’azione di alcuni farmaci a
follicoli e ghiandole sebacee. La via follicolare può essere
influenzata dalla secrezione sebacea, che favorisce l’assorbimento di sostanze solubili in lipidi. La penetrazione attraverso le unità pilosebacee rimane dipendente dalle caratteristiche della sostanza e dal tipo di preparazione. Nei metodi di penetrazione transdermica per l’ottenimento di effetti
sistemici, questa via è utilizzata inizialmente, mentre quella intercellulare, raggiunta in tempi più lunghi, rimane dominante.
PARAMETRI
FARMACOCINETICI.
PARTIZIONE VEICOLO/STRATO
CORNEO
Per lo studio dei meccanismi di trasporto e delle funzioni di barriera della cute questa può essere considerata come
una membrana o un insieme di membrane (per il cui studio
sono anche applicabili principi matematici) 11.
Nel suo complesso il trasporto attraverso lo strato corneo
è fondamentalmente un processo di diffusione passiva molecolare. Le proprietà fisicochimiche e strutturali della sostanza sono determinanti ai fini della capacità di diffusione e
penetrazione attraverso la membrana: determinanti importanti
sono solubilità e diffusibilità.
La solubilità relativa di un soluto, — ossia l’equilibrio
tra la solubilità della sostanza nel corneo relativamente a
Ottobre 2005
quella nel veicolo — determina il suo coefficiente di partizione. Questo indica la capacità della sostanza di separarsi
dal veicolo verso lo strato esterno del corneo, con possibilità,
quindi, di assorbimento dagli epidermociti.
La diffusibilità è la capacità di un soluto di attraversamento della barriera ed è influenzata da vari fattori, compresa la tortuosità del percorso intercellulare. Questo processo
passivo di assorbimento è governato dalla legge di diffusione di Fick: la velocità di assorbimento — flusso — è proporzionale alla differenza di concentrazione della sostanza
rispetto a quella nella barriera. Può essere, infine, ricordato che
il coefficiente di permeabilità mette in relazione flusso e concentrazione e risulta da coefficiente di partizione, da coefficiente di diffusione e lunghezza della via di diffusione 12.
RUOLO DI VEICOLO ED ECCIPIENTI E INTERAZIONE CON I PRINCIPI ATTIVI
Un veicolo è definito dal tipo di preparazione (crema,
unguento, gel, etc.) e dagli eccipienti (acqua, paraffina, glicole propilenico, etc.), e i termini «veicolo» ed «eccipiente»
hanno riferimenti diversi.
Veicolo ed eccipienti influenzano profondamente velocità e intensità di assorbimento e, quindi, biodisponibilità
ed efficacia. Sono di importanza critica: 1) solubilità della
sostanza nel veicolo; 2) coefficiente di partizione (che determina l’entità del trasferimento della sostanza dal veicolo nel
corneo). Gli eccipienti del veicolo modulano gli effetti di
partizione e diffusione nel corneo.
Una preparazione grassa, occludendo, può realizzare un
incremento della penetrazione del farmaco, ma veicoli grassi e unguenti non sono sempre più potenti delle creme e creme, gel e soluzioni possono essere formulati in modo da
ottenere potenza uguale a quella degli unguenti. Ad esempio,
per i corticosteroidi topici, la stessa attività può risultare per
differenti classi di potenza se formulate in veicoli differenti 13. Una preparazione in gel di kellina, assicurando migliore penetrazione, ha dimostrato migliori risultati nel trattamento di vitiligine 14. Anche la penetrazione transfollicolare è influenzata da veicolo ed eccipienti e, in questo caso,
migliori risultano alcuni veicoli lipofili o alcolici e sono
importanti dimensione e carica delle molecole del soluto 9.
Tutte le osservazioni riportate sottolineano l’assoluta
importanza di composizione di veicolo e formulazioni, che
determinano effetti di partizione e diffusione, differenze nell’entità dell’assorbimento e interazioni complesse tra sostanza, veicolo e cute e, di conseguenza, gli effetti specifici. Veicoli e formulazioni sono opportunamente e specificamente
studiati in rapporto agli elementi variabili riportati e non
possono essere stabiliti estemporaneamente.
SANTOIANNI
profonda delle proprietà di barriera del corneo (nelle regioni intercellulari le piccole quantità di acqua legate a gruppi
polari per l’idratazione non provocano riduzione dell’organizzazione lipidica e permeabilità 16). L’effetto dell’idratazione, tuttavia, risulta discontinuo: incremento di 10 volte per
alcune sostanze e per altre molto limitato 17.
L’occlusione previene la perdita di umidità della cute e
aumenta il contenuto di acqua del corneo, tuttavia il livello
di NMF nel corneo rimane pressoché nullo: sembra, così, che
vi sia un meccanismo omeostatico che impedisce l’iperidratazione della cute 9. L’occlusione può incrementare l’assorbimento di varie volte, particolarmente di composti idrofili, o, in alcune condizioni, la formazione dell’effetto reservoir.
L’acidità della superficie cutanea, controllando omeostasi e attività enzimatiche, influenza la permeabilità 18 e l’attività metabolica propria della cute (processi enzimatici,
ossidoriduttivi etc.) può modificare le sostanze applicate,
influenzando permeabilità ed effetti.
La funzione di barriera è alterata in alcune condizioni patologiche, come le alterazioni delle proprietà di barriera lipidica
intercellulare — per deficit di acidi grassi essenziali, accumulo
di colesterolo solfato, abnorme deposizione lipidica intercellulare — che portano a cute secca (con abnorme desquamazione e iperproliferazione reattiva) e le alterazioni in alcune malattie genetiche legate a geni che codificano per i filamenti della cheratina (ad esempio malattia di Thost-Unna) o
per le proteine strutturali dell’involucro corneo, per enzimi (ad
esempio glutaminasi I nell’ittiosi lamellare). Tuttavia non
sempre è possibile stabilirne l’influenza. Nelle diverse condizioni patologiche si hanno differenze di assorbimento, ma,
nonostante le alterazioni della barriera, non mancano ostacoli
alla penetrazione di sostanze applicate sulla cute 6.
È importante l’influenza sull’assorbimento legata alle
diverse regioni cutanee e alle variazioni tra i diversi siti anatomici. Per quanto riguarda le sedi, l’assorbimento va a diminuire dalla cute palpebrale a quella plantare 19. Per quanto
concerne l’influenza dell’età, diverse attività biologiche sono
diminuite nella cute dell’individuo anziano; pertanto, in questi soggetti, i parametri farmacodinamici e gli effetti sull’assorbimento vanno valutati in rapporto ai principi attivi;
come nel prematuro e nel neonato, per i quali la permeabilità cutanea è maggiore 20.
Non vi sono elementi sperimentali che confermino la validità della frizione sull’assorbimento percutaneo 6.
Infine, le alterazioni della barriera producono modificazioni
della perdita di acqua per via transepidermica 9 e lo strato corneo può essere definito un biosensore: ad esempio, alterazioni
dell’umidità esterna regolano proteolisi di filaggrina, sintesi negli epidermociti di lipidi, di DNA, etc., per cui possono
essere innescati anche fenomeni infiammatori 21.
CONDIZIONI CHE MODIFICANO LA FUNZIONE DI BARRIERA
Nell’idratazione la maggior parte dell’acqua è associata alla
cheratina intracellulare; assorbe notevolmente acqua il fattore di idratazione naturale (NMF) (10% del peso del corneocita) 15. Si ha rigonfiamento dei corneociti e alterazione
Vol. 140 - N. 5
BIODISPONIBILITÀ
DEI PRINCIPI ATTIVI DELLE FORMULAZIONI
La biodisponibilità cutanea della maggior parte delle formulazioni dermatologiche commercializzate è bassa (1-5%
della dose applicata) 8.
551
SANTOIANNI
Le sostanze attive di formulazioni topiche sono in generale assorbite in piccola quantità: soltanto una ridotta frazione passa dal veicolo nello strato corneo. La maggior parte rimane sulla cute, soggetta a perdita nel tempo per una
serie di fattori: sudore, degradazione chimica, rimozione,
etc. L’entità dell’assorbimento del farmaco di solito è nell’ordine dell’1-5% della dose applicata; determinando differenze della biodisponibilità. Futuri standard dovrebbero,
perciò, comprendere formulazioni a concentrazione non elevata, ma farmaceuticamente ottimizzate a elevata (50-100%)
biodisponibilità 8, anche perchè le marcate variazioni in rapporto alle aree cutanee e alle condizioni della cute danno
poca certezza dell’equivalenza terapeutica con altre vie di
somministrazione ottenibile in situazioni cliniche 22.
È stato autorevolmente affermato che gli standard sui topici — per quanto riguarda biodisponibilità, bioequivalenza ed
equivalenza terapeutica, metodi e protocolli di valutazione
e parametri biofarmaceutici dei prodotti — sono di 10-20
anni indietro rispetto agli standard raggiunti in altre modalità terapeutiche come quella orale 8.
Valutazione biofarmaceutica di formulazioni topiche
Per la misura della velocità di rilascio e per l’assorbimento di farmaci vi sono modelli largamente adoperati nello sviluppo di formulazioni dermatologiche topiche, costituiti da
membrane sintetiche artificiali o da cute in vitro animale o
umana (che, tuttavia, presenta notevole variabilità del potere di barriera) 23. I metodi in vivo comprendono modelli cinetici e dinamici. I primi si basano su: a) rimozione selettiva dello strato corneo; b) tecniche di dissezione della cute; c) metodi di valutazione dell’assorbimento percutaneo indiretti (in sangue, secrezioni, etc.). I modelli dinamici comprendono: 1)
determinazione delle variazioni di colore della cute con strumenti diversi (Minolta, X-rite, etc.) con i quali vengono determinati anche il grado di eritema provocato o la variazione di
colore dopo fotostimolo UVB; 2) determinazione del flusso
ematico cutaneo (monitoraggio con procedure ottiche laser
Doppler); 3) valutazione dell’infiltrato neutrofilo UVA-indotto (ad esempio per stabilire l’attività infiammatoria di corticosteroidi); 4) modelli su animali 24.
Metodi di modulazione della permeazione cutanea
Quando si applica sulla cute una sostanza con un veicolo
semplice, il risultato terapeutico ottenuto può essere insufficiente per la scarsa concentrazione che si realizza nell’area
di applicazione. Da alcuni anni si sono sviluppate ricerche e
strategie per incrementare la veicolazione. Le strategie possono essere di ordine chimico e biochimico o fisiche.
Enhancer chimici
Per aumentare la penetrazione può essere integrato il veicolo con sostanze dette enhancer, alcuni dei quali, intera-
552
gendo con i lipidi intercellulari, migliorano il coefficiente di
diffusione della sostanza attiva nel corneo. Gli enhancer chimici possono: a) incrementare la diffusibilità della sostanza
all’interno della barriera; b) aumentare la solubilità nel veicolo o entrambe; c) migliorare il coefficiente di partizione.
Queste sostanze tendono ad avere un’azione non specifica,
con implicazioni per la penetrazione di sostanze irritanti
che possano venire in contatto successivamente con la cute.
Enahncer di questo tipo, che, tuttavia, non hanno raggiunto ampia diffusione, sono: Azone, SEPA, Dermac SR-38 e
numerosi altri 25. Molto usato è l’acido oleico. In alcuni
casi, tuttavia, hanno anche un effetto irritante, per cui la
loro applicazione viene attentamente valutata nelle diverse
preparazioni 26.
Eccipienti come etanolo e propilenglicole e il dimetilsolfossido (DMSO) possono, alterando l’organizzazione dei
lipidi dello strato corneo e la struttura della barriera 27,
aumentare la diffusione 25. Le loro proprietà solventi influenzano positivamente la solubilità delle sostanze attive nel veicolo e il coefficiente di partizione (tuttavia con qualche possibile effetto spiacevole come bruciore o dermatite irritativa).
Questi enhancer incrementano anche l’effetto reservoir.
Estraendo i lipidi, detergenti e surfattanti agiscono in modo
analogo.
Interessante è l’approccio metabolico. Le strategie che
interferiscono con sintesi, organizzazione, creazione, attivazione delle lamelle extracellulari possono incrementare
la penetrazione interferendo con la permeabilità della barriera.
L’interferenza con la biosintesi di alcuni dei lipidi può alterare la struttura della barriera e incrementare la penetrazione. Sono anche stati provati metodi di interferenza con la
secrezione e l’organizzazione dei lipidi (come brefeldina A,
monetina o clorochina) ed enhancer che alterano l’organizzazione sopramolecolare delle lamelle bistratificate (analoghi sintetici dei grassi che inducono anomalie nell’organizzazione delle membrane; precursori complessi non metabolizzabili ecc.) 6.
Questi metodi portano all’alterazione del rapporto molare critico tra ceramidi, colesterolo e acidi grassi: se si ha
delezione o eccesso di uno dei 3 lipidi chiave, il componente lipidico in eccesso non può consevare l’organizzazione
lamellare. Si ha separazione delle fasi con interstizi più permeabili e creazione di ulteriore via di penetrazione.
In realtà, ogni alterazione dei componenti e della miscela critica dei 3 tipi di lipidi, che possa portare ad aumento
maggiore di 3 volte di un componente, ritarda la riparazione della barriera alterata 28.
L’efficacia degli enhancer può essere aumentata inibendo
la reazione metabolica di riparazione, quando si è ottenuta
l’alterazione della barriera; inibendo sequenze metaboliche
che tendono a riformare e a mantenere la funzione di barriera;
inibitori di enzimi con rilevanti funzioni (ad esempio la lovastatina) o, specificamente, di enzimi della sintesi di ceramidi o di acidi grassi, con alterazione del rapporto critico molare dei tipi di lipidi sopra menzionati, e discontinuità nel sistema lamellare a doppio strato 29. Un incremento può essere
raggiunto attraverso modifica della polarità, indotta da enhan-
Ottobre 2005
cers 30. Recenti studi propongono la possibilità di adoperare i metodi sopra citati in collaborazione con i metodi fisici.
Nonostante l’ampia varietà dei metodi sperimentati per
aumentare la veicolazione transdermica (per obiettivo sistemico), l’applicazione è stata minima, e l’elenco dei farmaci per i quali è stato possibile utilizzare la via transdermica
e l’uso sistemico è breve (<10) e limitato a sostanze lipofile e di basso peso molecolare: acido nicotinico, nitroglicerina,
clonidina, ormoni steroidi, scopolamina 31. Certamente le
grosse difficoltà di questi studi sono rappresentate dal fatto
che sono stati condotti non su cute umana, ma in vitro.
Sistemi carrier vescicolari
Formulazioni liposomiche sono risultate considerevolmente efficaci, tuttavia incrementerebbero la penetrazione soltanto attraverso una via transappendigeale, poichè non vi è
evidenza che possono penetrare lo strato corneo 32. Vanno
anche citati i niosomi, la cui formulazione comprende surfattanti non ionici, e i trasferosomi, formati da liposomi
modificati (fosfatidilcolina, colato sodico, etanolo, etc.) che
si basano sulla possibilità delle vescicole di attraversare intere il corneo, per azione del gradiente osmotico tra gli strati
esterni e interni del corneo. Sono vescicole flessibili capaci
di trasportare il contenuto attraverso la tortuosa via intercellulare del corneo.
Sistemi fisici di incremento della veicolazione
Alcuni metodi strumentali sono stati sperimentati per
incrementare la penetrazione di sostanze attive in circolo
per un’azione sistemica, evitando una traumatica introduzione, come quella attraverso aghi, o anche per ottenere un
effetto loco-regionale; si tratta di sistemi che hanno l’obiettivo di raggiungere l’area da trattare con adeguata concentrazione di farmaco a una qualche profondità e in modo selettivo, senza dispersione in circolo (sistemi transdermici) 33.
La iontoforesi e l’elettroporazione rappresentano metodi
fisici elettricamente assistiti per aumentare la veicolazione
di farmaci/sostanze attraverso il corneo. La sonoforesi utilizza
ultrasuoni con modalità a bassa frequenza.
Sonoforesi
La sonoforesi (o fonoforesi o ultrasuonoforesi) costituisce la strategia più promettente per ottenere incremento degli
effetti di farmaci e altri principi attivi utilizzati in campo dermatologico 34. Gli ultrasuoni (US) producono alterazioni nella struttura dello strato corneo e permeabilizzazione 35.
Le frequenze utili sono state a lungo incerte: quelle adoperate nelle indagini diagnostiche (1-4 MHz) sono scarsamente efficaci, mentre quelle molto elevate (10-20 MHz)
incrementano la penetrazione 36; in dermatologia, recentemente, sono risultate interessanti quelle a bassa frequenza,
tra i 20 e i 25 KHz 37.
Vol. 140 - N. 5
SANTOIANNI
Recenti studi hanno messo in evidenza la dipendenza dell’effetto a 20 KHz da vari parametri 38-40: responsabile della diminuzione della funzione di barriera appare principalmente il fenomeno di cavitazione 41, anche se all’aumento di
temperatura indotto da US è stato assegnato in alcuni esperimenti un ruolo intorno al 25% 42-44 e vengono coinvolti
trasporto per convezione e impatto meccanico 45. Possibili vie
di penetrazione sono attribuite alla formazione di transitorie
modificazioni (pori di circa 20 m) 46, che risultano, in condizioni sperimentali, sufficienti per la penetrazione di molecole di farmaci di alto peso molecolare 47, 48.
La combinazione di US con surfactanti (sodiolauril-solfato) accresce il trasporto e richiede minore energia 49.
L’assorbimento sonoforetico varia in rapporto ai diversi farmaci 50. Ad esempio, la frequenza a 20 KHZ può incrementare l’assorbimento di un cortisonico (esperimenti in vitro con
clobetasolo 17-propionato) 40. Sono stati studiati anche effetti biologici su cute umana e possibili applicazioni 44. L’attivazione mediante US di farmaci apre un’interessante prospettiva in campo oncologico cutaneo 51.
Ionoforesi e iontoforesi
La ionoforesi incrementa la penetrazione di sostanze ionizzabili, adoperando la forza di un debole campo elettrico (la
corrente continua o galvanica applicata va da 0 a 250
microampere=0,25 mA. Gli ioni trasportano la corrente
applicata tra un elettrodo e un secondo elettrodo indifferente) 52. In dermatologia è largamente adoperata con varie
apparecchiature nell’iperidrosi 53. Il tempo necessario per consentire che la sostanza ionizzata attraversi la barriera è, in
alcune applicazioni, di circa 25-30 min, ma raggiunge diverse ore in applicazioni transdermiche (per effetto sistemico o
loco-regionale sull’apparato osteotendineo).
La ionoforesi convenzionale presenta scarsa efficienza
del trasporto transdermico, distribuendo l’agente attivo prevalentemente nelle aree superficiali cutanee: l’assorbimento verso tessuti profondi o per ottenere azione sistemica
avviene in minima parte. Se la ionoforesi è prolungata per
ottenere un effetto transdermico, si possono avere effetti collaterali legati alla corrente continua (ustioni chimiche polari) e/o possibili reazioni allergiche al farmaco. Presenta,
inoltre, alcune controindicazioni (soggetti con pace-maker,
lesioni cutanee in atto, epilessia).
La iontoforesi utilizza campi elettrici bipolari con corrente alternata, consentendo il trasporto di sostanze ionizzabili, incluse macromolecole, ma anche di non ionizzate e
solo polari. Il trasporto è a livello interstiziale nel corneo, attraverso pori acquosi, o ancora livello extracellulare, a livello
lacunare (meno frequentemente appendigeale) 10. La iontoforesi di farmaci ionizzati potrebbe dar luogo a un aumento di 20-60 volte della penetrazione rispetto alla sola applicazione topica. La quantità del farmaco è proporzionata
all’intensità della corrente applicata.
I 2 principali meccanismi responsabili secondo Santi 54
sono: ionoforesi, nella quale gli ioni sono respinti da un elettrodo della stessa carica; elettrosmosi, movimento convetti-
553
SANTOIANNI
vo di un solvente che ha luogo attraverso un poro caricato,
in risposta al passaggio preferenziale di contro-ioni, sotto
l’influenza del campo elettrico. La iontoforesi incrementa il
trasporto attraverso la barriera con 3 meccanismi: a) interazione ioni-campo elettrico che supporta la conduzione ionica; b) incremento di permeabilità della membrana-cute provocata dal flusso di corrente elettrica; c) elettro-osmosi,
fenomeno per il quale si ha flusso massivo di solvente che trasporta specie sia ionizzate sia neutre 55. Poichè la cute al di
sopra del pH 4 ha carica negativa, i controioni sono ioni
positivi e il flusso elettroosmotico va dall’anodo al catodo.
Pertanto il trasporto anodico è favorito dall’elettro-osmosi,
mentre quello catodico è ritardato. Il flusso elettro-osmotico diviene elevato con l’aumento di dimensione delle molecole ionizzate. Per l’elettro-osmosi il trasporto di grosse
molecole anioniche (anche proteine) dal compartimento anodico può essere più efficace di quello dal compartimento
catodico.
Questo fenomeno di elettro-osmosi incrementerebbe, in
particolare, il trasporto anodico di farmaci caricati positivamente e di grosse dimensioni (il cui trasporto iontoforetico è spesso ridottissimo) e promuove la migrazione transdermica di molecole non caricate ma tuttavia polari, molecole la cui permeazione passiva è caratteristicamente molto ridotta 56.
Per quanto riguarda gli effetti collaterali, nell’ionoforesi
la corrente continua (o galvanica), applicata tramite elettrodi in una parte del corpo, dà luogo alla formazione di un
campo elettrico unidirezionale; inoltre, può essere indotto
riscaldamento del tessuto, eritema e anche ustione, nonché
formicolio nell’area di applicazione degli elettrodi. Viceversa, nella iontoforesi, con le numerose interruzioni della
corrente (frequenze altissime), si eliminerebbe il surriscaldamento cutaneo dato dalla corrente galvanica.
Come già riportato, è stata dimostrata la possibilità di trasporto iontoforetico anche di macromolecole 56.
Combinando iontoforesi e diffusione passiva è stata osservato aumento della penetrazione attraverso lo strato corneo
di kellina a opportuno pH 7, anche se il farmaco non è ionizzato 57.
È stato dimostrato 58 che per molecole piccole il trasporto in generale diminuisce con l’aumento della grandezza e la
struttura molecolare costituisce un elemento chiave per il
trasporto da iontoforesi. Può avere importanza, inoltre, la
sequenza chimica nella struttura molecolare (come dimostrato per gli oligonucleotidi).
Santi 59 ritiene che la iontoforesi possa essere un’interessante alternativa alla via parenterale in una possibile terapia
sistemica, anche per peptidi e per sostanze macromolecolari in stato ionizzato a valori di pH fisiologico (essendo queste per altre vie poco assorbite e assai degradate da enzimi
proteolitici). Sarebbe anche possibile un trattamento in alcune patologie cutanee (come l’herpes simplex e la psoriasi) 60.
È stato riportato che la iontoforesi può far penetrare il
farmaco a una profondità di oltre 1 cm. Una volta che il flusso di corrente ha trasportato gli ioni del farmaco attraverso
il corneo nei primi 2 mm di tessuto, inizialmente vi sarebbe
554
un effetto deposito al di sopra dell’area di microcircolo. Poi
il farmaco diffonderebbe dal deposito nel derma e dalla rete
capillare sarebbe trasportato nella corrente ematica (riducendosi così il gradiente per una penetrazione verso i tessuti profondi sottostanti). La somministrazione locale di un
farmaco tramite iontoforesi avverrebbe così a una profondità
pari almeno a quella della rete vascolare (plesso vascolare
superficiale).
Nella iontoforesi le unità convenzionali di farmaco in
somministrazione sono rappresentate da mA/minuti; e vengono così calcolate: mA (di corrente applicata) X tempo (di
applicazione in minuti).
La iontoforesi ha una lunga storia, essendo stata suggerita per varie terapie da molti anni in diversi campi della medicina, in terapia fisica e anche in campo odontoiatrico. Tuttavia
presenta anche essa efficienza limitata per effettuare terapia sistemica, per cui il trasferimento di principi attivi è stato studiato prevalentemente per terapia loco-regionale (ad
esempio nel trattamento di forme muscoloscheletriche) o
per qualche trattamento locale. Molte le sostanze provate:
lidocaina, epinefrina, metilprednisolone, desametazone, antivirali, antibiotici 31.
L’utilità della iontoforesi in campo dermatologico è stata preconizzata da Gangarosa per l’anestesia locale, per la
neuralgia posterpetica, e per la terapia con corticosteroidi
topici 61. L’anestesia fino a una profondità di 1 cm o più è stata studiata in doppio cieco; risultando efficace per chirurgia
delle palpebre e per biopsie cutanee (shave).
In conclusione, la iontoforesi risulta particolarmente indicata per brevi applicazioni nelle quali risulta sicura ed efficace. Essa potrà essere migliorata da moderne strumentazioni elettroniche e da dispositivi (cuscinetti di idrogel) 62.
Studi sperimentali molto recenti indicano nuove prospettive introducendo sostanze con funzione di cooperazione:
iontoforesi a idrogel. In uno studio di Fang con vari polimeri incorporati in formulazioni, gli idrogel hanno dimostrato maggiore capacità di trasporto delle soluzioni 63. L’Autore attribuisce questo risultato all’abilità antinucleante dei
polimeri che incrementano l’attività termodinamica della
sostanza attiva nella formulazione. Inoltre, in questo studio,
il pretrattamento con isopropilmiristato, enhancer di sostanze lipofile, migliorava la penetrazione sia passiva sia iontoforetica.
Recentemente sono stati proposti dei sistemi autonomi
di iontoforesi che si basano sulla veicolazione di farmaci
con corrente bassissima: in pratica, sull’applicazione di un
cerotto (al cui interno è presente una batteria ultrasottile)
attraverso cui gli ioni penetrano la cute e i tessuti sottostanti con un’intensità di 80 mA/min e per una durata di 24 h. La
migrazione nelle 24 h permette di creare nella zona di interesse un deposito di farmaco sottocutaneo, con effetti terapeutici maggiori rispetto a quelli tradizionali. Inoltre la bassa intensità di corrente evita in maniera considerevole i rischi
di irritazione della pelle; questo sistema consente al paziente ampia libertà di movimento 64. Recentemente la iontoforesi è stata proposta in combinazione con gli ultrasuoni a
bassa frequenza 48.
Ottobre 2005
Alcune interessanti applicazioni sono in studio per ottenere
l’estrazione dal circolo venoso attraverso la cute di analiti
(iontoforesi inversa).
Elettroporazione
In questa modalità un campo elettrico reversibile, con
impulsi brevi e di alto voltaggio, elettropora le barriere lipidiche a doppio strato e porta alla formazione di fasi lipidiche
non lamellari e di pori subcellulari, ottenendo maggiore
velocità di trasporto delle sostanze 65. L’elettroporazione
utilizza correnti di bassa intensità da un elettrodo della stessa carica della polarità netta del farmaco, conducendo le
molecole attraverso lo strato corneo e utilizzando vie extracellulari (e probabilmente anche transappendigeali).
L’elettroporazione per incrementare la permeabilità della cute verso la penetrazione di farmaci è nelle prime fasi di
sviluppo 66, 67 e gli studi in vitro dovranno essere integrati con
studi in vivo e clinici, messi a punto con nuovi opportuni
strumenti. Secondo qualche Autore, un maggior incremento potrebbe essere raggiunto dalla combinazione di elettroporazione con iontoforesi e ultrasuoni, anche per macromolecole. Con l’elettroporazione si creerebbero nuovi percorsi e, conseguentemente, più uniforme distribuzione della carica elettrica e, pertanto, possibile minore irritazione
cutanea.
È prevedibile che nel prossimo futuro molte nuove ricerche saranno dedicate all’ulteriore sviluppo di metodi fisici per
l’attivazione della penetrazine cutanea con modificazioni
degli attuali metodi di iontoforesi e con metodologie a ultrasuoni (sonoforesi).
Crioelettroforesi
Alcuni problemi dati dalla iontoforesi possono essere
essere eliminati se il «pad» dell’elettrodo attivo è sostituito
da un blocco di ghiaccio prodotto dal congelamento del farmaco nella soluzione. La crioelettroforesi è una tecnica di veicolazione di farmaci e principi attivi congelati, che introduce sostanze medicamentose idrosolubili, ancor meglio se
ionizzabili. Consente la penetrazione transdermica di queste,
ma anche di sostanze con caratteristiche fisico-chimiche
diverse, a livelli profondi (6-8 cm) e in quantita più elevate
che con altri metodi, con impatto sistemico di entità molto
ridotta (0,04%). Utilizza corrente elettrica alternata, di frequenza e polarità a seconda del farmaco; con opportuna
variazione dell’intensità 68. Il trasporto del farmaco/sostanza avrebbe luogo per i seguenti meccanismi e fenomeni fisico-chimici: a) diffusione (fortemente potenziata dall’effetto di porazione della corrente oscillante adoperata); b) elettrosmosi. Per questo fenomeno vengono trasportate sia l’acqua sia le sostanze disciolte; quando il farmaco non si dissocia
o quando lo ione attivo è positivo, l’elettrosmosi rafforza
sia la diffusione sia l’azione elettroforetica 69; c) elettroforesi,
che rafforza la diffusione dei farmaci ionizzabili. Soltanto l’elettroforesi richiede la dissociazione elettrolitica del farmaco: gli altri consentono il passaggio anche alle molecole
Vol. 140 - N. 5
SANTOIANNI
neutre 68. La corrente alternata realizzerebbe anche un effetto di porazione.
La sostanza attiva è sciolta in ghiaccio che è messo in
contatto diretto con la cute e con gli elettrodi da entrambe le
parti della regione da trattare. La corrente passa essenzialmente attraverso la soluzione liquida che origina dalla fusione del ghiaccio e attraversa i tessuti verso l’altro elettrodo trasportando gli ioni della sostanza attiva. Modificando i parametri della corrente si avrebbe una definita direzione del
flusso ionico nei tessuti profondi con alta concentrazione
solo locale. Secondo Aloisi 68 non solo una molecola antiinfiammatoria di basso peso molecolare (M=302,68 d) ma
anche un mucopolisaccaride (mol.wt.da 5 a 10 kd) può giungere in profondità (ad esempio sino alle capsule articolari).
Questa modalità di iontoforesi rende possibile l’aumento
di molte volte (200-300%) dell’intensità massima e, rispettivamente, dell’intensità totale della corrente senza danni e
sensazioni spiacevoli: gli effetti negativi del freddo, della
corrente elettrica e del farmaco vengono neutralizzati reciprocamente. L’effetto vasocostrittore provocato dalla bassa
temperatura impedisce una non desiderata rimozione del
farmaco tramite il flusso ematico locale.
Sono state calcolate formule da Aloisi et al. circa la quantità di farmaco che può passare. Da queste risulta anche che
il farmaco somministrato non dipende dalla quantità disciolta nella soluzione ma soltanto dalla carica elettrica che passa nel circuito e dalla frazione molare f 68. Le principali
applicazioni (per la quale sono stati creati strumenti specifici)
sembrano queste: corticosteroidi in alta concentrazione locale senza effetti sistemici; terapia locale in caso di deficit della circolazione locale; insufficienza venosa 68. La tecnica è
utilizzabile secondo Vazharov per il dolore, risoluzioni di
edemi, disturbi trofici e condizioni post-traumatiche del
sistema muscolo scheletrico, artriti, strappi, ematomi, ecc. In
campo dermatologico questa complessa tecnica strumentale non sembra presentare reali possibilità.
Idroelettroforesi
Costituisce un diverso metodo di iontoforesi che adopera correnti pulsate, di diversa forma e frequenza, con formulazioni gel. Una funzione importante è affidata al gel (ad
esempio gel di agaroso) nel quale è disperso l’agente attivo
con un enhancer di mobilità. Il gel migliora la migrazione
durante l’azione del campo elettrico e la soluzione con
l’enhancer crea la forza ionica appropriata per il tipo di agente attivo trasportato. Questo può essere ionizzato o non. L’idroelettroforesi consente all’agente attivo di penetrare nei tessuti da 0,5 fino a circa 10 cm, evitando la dispersione precoce
e creando un flusso direzionale 69, per una terapia loco-regionale. Uno strumento computerizzato fornisce onde elettriche
di diversa forma e frequenza (tuttavia limitate ad alcune possibilità predefinite), rapportate alla profondità che deve raggiungere l’agente attivo. L’idroelettroforesi sarebbe utilizzabile nella terapia del dolore, con applicazioni della durata di 15-30 min e la possibilità di focalizzare le regioni affette.
555
SANTOIANNI
Onde laser
oggi a dedicare studi sperimentali e clinici ai metodi di penetrazione intradermica.
La biodisponibilità cutanea della maggior parte delle formulazioni dermatologiche commercializzate risulta bassa. Le
strategie per incrementare la veicolazione possono essere chimiche, di ordine biochimico o fisiche. Gli enhancer chimici a)
incrementano la diffusibilità della sostanza all’interno della
barriera; b) ne aumentano la solubilità nel veicolo; c) migliorano il coefficiente di partizione. Inoltre, metodi capaci di
interferire con la biosintesi di alcuni lipidi alterando la struttura
della barriera incrementano la penetrazione. Di interessante
approfondimento dermatologico sono i meccanismi fisici che
consentono aumento della penetrazione cutanea di sostanze:
la iontoforesi (che incrementa la penetrazione di sostanze
ionizzate), l’elettroporazione (che utilizza correnti creando
nuovi percorsi attraverso la barriera), e la sonoforesi, metodologia a ultrasuoni tra i 20 e i 25 KHz che producono alterazioni e superamento della barriera rappresentata dal corneo.
In questo lavoro è riportato il recente sviluppo di questi
metodi ed è sottolineta l’importanza di veicolo ed eccipienti che determinano effetti di partizione e diffusione, differenze
nell’entità dell’assorbimento e interazioni complesse tra
sostanza, veicolo e cute, condizionando gli effetti specifici;
pertanto formulazioni utili non possono essere estemporaneamente stabilite.
Parole chiave: Farmaci - Cute - Somministrazione intradermica e transdermica di principi farmacologicamente attivi.
La compressione fotomeccanica con laser (laser a rubino
Q-switched) è stata anche sperimentata ultimamente per
modulare la permeazione del corneo 70. Il materiale (polistirene) includente la soluzione con il principio attivo (ad
esempio acido δ-aminolevulinico) assorbe la radiazione che
ne provoca ablazione, mentre la soluzione incrementa la
propagazione nel corneo dello stress indotto dagli impulsi
laser. La via di penetrazione sarebbe extracellulare come
per la sonoforesi e ionoforesi. L’effetto è solo temporaneo e
la funzione di barriera viene a ricostituirsi 6.
Riassunto
Diversi aspetti sono stati chiariti riguardo al modo in cui
un farmaco o un altro principio attivo possa attraversare la barriera costituita dallo strato corneo e al modo in cui questo fattore limitante possa essere influenzato in modo da ottenere
migliori effetti funzionali o terapeutici. Nonostante l’ampia
varietà dei metodi studiati per aumentare la veicolazione
transdermica per ottenere effetti sistemici, l’applicabilità in
questo campo è rimasta limitata. La ricerca dermocosmetica si è concentrata prevalentemente sul problema del superamento della barriera epidermica. La ricerca dermatologica, anche per i numerosi aspetti applicativi, è interessata
556
Ottobre 2005
Biological agents in the treatment of psoriasis
A. RAUSIN, A. HENNO, M. DE LA BRASSINNE
Psoriasis is a disease affecting about 2% of the world caucasian
population. Progress in the understanding of psoriasis as a Tcell mediated disease and advances in molecular biology and
technology has led to the development of new classes of therapeutic agents. These so-called biologics are in form of fusions
proteins, monoclonal antibodies, cytokines and are designed to
block specific molecular steps important in the pathogenesis of
psoriasis (reducing the number of pathogenic T-cells, blocking their migration to the skin, antagonizing the effector
cytokines they secrete). This article will discuss the most promising and advanced agents along with their interactions on cellular and humoral systems in psoriasis.
KEY WORDS: Psoriasis, immunology - Biology - T lymphocytes.
P
soriasis is a very common disease affecting about
2% of the world Caucasian population.
It is considered as a chronic immune-mediated
inflammatory skin disorder that is still presently without a permanent cure. A genetic susceptibility has
been demonstrated and 7 genes are suspected, but
the concordance rate between monozygotic twins
only reaches 72%. It is generally accepted that patients
inherit a predisposition to psoriasis but that the disease
is only expressed after being triggered by certain
environmental or antigenic factors. Psoriasis can have
a major negative effect on the quality of life. Up to
40% of patients with psoriasis also develop psoriatic arthritis.1 Approximately 20% of patients have
Address reprint requests to: Prof. M. de la Brassinne, Department of Dermatology, CHU B35, Domaine Universitaire du Sart-Tilman, B-4000 Liège, Belgium. E-mail: [email protected]
Vol. 140 - N. 5
Department of Dermatology,
University of Liege, Liege, Belgium
moderate to severe disease and require phototherapy,
systemic therapy or both. While effective, currently
available systemic therapies are not ideal for all
patients with psoriasis due to drug interactions, contraindications with concurrent illness, short-term
adverse effects or long-term toxicity (for instance,
cyclosporin can induce hypertension and nephropathies and methotrexate is hepatotoxic and can cause
lung fibrosis).2
Based on the continuous progresses in psoriasis
research (detailed knowledge of psoriasis as a T-cell
mediated disease with a special type 1 cytokine pattern) and advances in molecular biology and technology, a new class of agents has emerged. These
biological medications are designed to block specific molecular steps important in the pathogenesis
of psoriasis without global immunosuppression and
are in form of fusion proteins, monoclonal antibodies, and cytokines.3, 4 These new approaches may
reduce the number of pathogenic T-cells, block their
migration to the skin, or antagonise the effector
cytokines they secrete.There are currently several
products approved or under evaluation at various
stages of development.5 We will only discuss the
most promising and advanced agents along with
557
RAUSIN
BIOLOGICAL AGENTS IN THE TREATMENT OF PSORIASIS
their interactions on cellular and humoral systems in
psoriasis.
Focusing on cellular immunomodulation
An increased level of activated T lymphocytes present in psoriatic skin plaques and blood of patients
supports the evidence that activated T-cells play an
important role in the pathogenesis of psoriasis.1 Some
of these new biological agents are able to reduce the
number of pathogenic T-cells or block T-cell migration
and adhesion. The most advanced agents that have
been developped for this therapeutic purpose are efalizumab and alefacept.
Efalizumab (Raptiva™) is a humanized monoclonal antibody directed against CD11a, the α subunit of
leukocyte function associated antigen-1 (LFA-1), preventing its binding to intercellular adhesion molecule
type 1 (ICAM-1). It inhibits the costimulation of T-cell
activation and is also thought to prevent T cells from
entering psoriatic lesions by blocking the interaction
with ligands on endothelial cells, fibroblasts, and keratinocytes.4 Efalizumab was approved for the treatment of psoriasis in 2003 by the US Food and Drug
Administration (FDA) and received a marketing authorization application for psoriasis in the European Union
by the European Agency for the Evaluation of Medical
Products in 2004. Efalizumab is given once a week
subcutaneously.
The most frequently reported adverse events are
influenza-like symptoms. Rare observations of thrombocytopenia have been mentioned and a thrombocyte
check-up is advised.3
Alefacept (Amevive™) is a soluble recombinant
human fusion protein, consisting of the external domain
of LFA-3 and the Fc portion of human IgG. It binds to
CD2 on activated T-cells preventing costimulatory
signals delivered by LFA-3 and has also been shown
to produce a dose-dependent reduction in circulating
memory T-cells, but not naïve T-cells, possibly by
inducing apoptosis of T-cells expressing CD2.5 Alefacept was approved in 2003 by the FDA for psoriasis
as weekly intramuscular or intravenous administration. The most frequent side-effects include mild fluelike symptoms and injection site reactions and the
CD4 count should be regulary watched.4
These treatments have proven to be effective and
safe in short- and intermediate-term administration
but long-term safety (related to the risk of immuno-
558
supression and development of infection or malignancy) and efficacy outcomes need to continue to be
observed and accumulated.
Other molecules with the same target are under
investigation as spilizumab (anti CD2, Medi-507™),
OKTcdr4a (anti CD4, Imuclone™), daclizumab (anti
CD25, Zenaprax™), basiliximab (anti CD25,
Simulect™)...4
Focusing on humoral immunomodulation
A lot of cells involved in the psoriatic reaction are
able to produce T helper type 1 (Th 1) cytokines,
including tumour necrosis factor-α (TNF-α), interleukin (IL)-1, -2, -3, -6, -8 and interferon-γ. Restoring
the Th1/Th2 cytokine profile balance became therefore
a potential approach of psoriasis treatment.
It is now admitted that TNF-α plays a fundamental
role in the pathogenesis of psoriasis.
As a result of overproduction by keratinocytes and
inflammatory cells, the concentrations of TNF-α are
clearly elevated in psoriatic lesions as compared to
patient uninvolved or normal people skin. These concentrations decrease parallel to clinical improvement.6
TNF-α is involved in the production of other proinflammatory cytokines and receptors through the activation of nuclear factor-κB and in trafficking of leucocytes to the lesions by increasing cell adhesion molecules on endothelial cells and keratinocytes. Those
cells can then amplify local inflammation and keratinocyte proliferation by producing further interferon-γ and TNF-α. The latter also stimulates migration
of Langerhans’cells to lymph nodes as well as their
ability to present antigens to lymphocytes.4 The inhibition of TNF-α activity could block this cycle of
inflammation, making TNF-α an excellent target for
treatment strategies.
The best-characterized TNF-α inhibitors that have
been developed for these therapeutic purposes are
infliximab and etanercept.
Infliximab (Remicade™) is a human-mouse
chimeric monoclonal antibody that can neutralize soluble TNF-α and bind to the membrane-bound cytokine,
leading to complement- and cell-mediated cell lysis.4
It is currently approved in Europe and in the United
States for the treatment of rheumatoid arthritis and
Crohn’s disease but not yet for the treatment of cutaneous psoriasis. Adverse events that have been reported include infections, immediate and delayed hyper-
Ottobre 2005
sensitivity events, appearance of antinuclear and antids-DNA antibodies, anaphylaxis, cardiac decompensation, upper respiratory tract infections and symptoms such as headache, nausea and palpitations.4
Etanercept (Enbrel™) is a soluble, dimeric fusion
protein consisting of 2 molecules of the ligand-binding
portion of the human TNF-α receptor (p75) fused to the
Fc portion of human IgG1. It binds to soluble and membrane-bound TNF-α and has the ability to bind to 2 molecules of TNF-α. This molecule was approved for the
treatment of psoriasis by the European Commission in
2004 with a dose-regimen twice a week subcutaneously. Treatment with etanercept is generally well tolerated,
mild injection site reactions being the most common
adverse event reported. Rare cases of demyelinisation or
opportunistic infections have, however, been reported
during etanercept treatment as well as aplastic anemia,
pancytopenia and lupus-like syndromes.7
The efficacy of these treatments has been reported
as very good, although better with infliximab. This
could be a result of the intravenous route of administration of the latter, to its ability to lead to cell lysis or
to a more stable complex with TNF-α. The cessation
of treatment seems not to be followed by a rapid relapse
but further studies need to be realised to confirm the
long-term benefit of these medications. Other TNF-α
inhibitors are under investigation, among them Onercept (recombinant soluble type I TNF-α receptor) and
adalimumab (Humira™) (human monoclonal antibody).
Special caution must be taken with those inhibitors
concerning the possibility of infections (among them
tuberculosis) and neoplastic diseases. This kind of
treatment should be restricted to patients severely
affected and without additional organic disease.
Another approach targeting the humoral component of psoriatic inflammation is the application of
recombinant Th2 cytokines deficient in psoriatic
lesions. Those exogenous cytokines could help to
restablish the Th1/Th2 balance by deviating the differentiation of Th1 cells to Th2. Several molecules
are under investigation as IL 4, and IL11. As for IL10,
whose development has been discontinued, IL11 displays only moderate efficacy, making them a possible future “maintenance treatment”.
Dermatologists need to familiarize with those new
biological psoriasis treatments, but until now, long
term safety data are still missing. As such, classical
treatments should not lose their place. The so-called
Vol. 140 - N. 5
RAUSIN
biologics aren’t indeed devoid of known or potential
secondary effects. Their efficacy can vary with the
importance of the step they target in the pathogenesis
of psoriasis and is sometimes comparable (or higher?) to classical systemic treatments, with a remaining
(and varying) part of patients being non responders.
This can be understood as psoriasis is a multifactorial and polygenic disease.4 These biologicals could be
used as monotherapy or as combination therapy. As any
others, this kind of treatment has to be adapted to each
psoriatic patient and the available components of this
new treatment generation can be considered as important new therapeutic weapons. From our point of view,
their use should be restricted to severely affected
patients and to recalcitrant types of psoriasis as several
published data and case reports have underlined their
efficacy in these cases as well as in pustular psoriasis.8
As they target different specific steps of the psoriasis
cascade, their use in combination could also be conceivable provided that they have complementary
actions. This could also help to reduce side effects by
diminishing the dose of each of these biologics.
Riassunto
Agenti biologici nel trattamento della psoriasi
La psoriasi è una malattia che colpisce circa il 2% della
popolazione di razza caucasica in tutto il mondo.
Il progresso nella comprensione che la psoriasi è una patologia mediata dalle cellule T e gli sviluppi della biologia
molecolare e della tecnologia hanno consentito di sviluppare nuove classi di agenti terapeutici. Quelli cosiddetti «biologici» sono rappresentati da proteine di fusione, anticorpi
monoclonali, citochine, e sono stati concepiti per bloccare
specifici meccanismi molecolari importanti per la patogenesi
della psoriasi (riducendo il numero delle cellule T patogene,
bloccando la loro migrazione verso la cute, antagonizzando
le citochine effettrici che secernono).
Questo articolo si interesserà degli agenti più promettenti e avanzati, considerando anche le loro interazioni sui sistemi cellulare e umorale nella psoriasi.
Parole chiave: Psoriasi, immunologia - Biologia cellulare Linfociti T.
References
1. Kormeili T, Lowe NJ, Yamauchi PS. Psoriasis:immunopathogenesis
and evolving immunomodulators and systemic therapies; U.S. experiences. Br J Dermatol 2004;151:3-15.
2. Mendonça CO, Griffiths CEM. Side effects of systemic treatment for
psoriasis. Retinoids 2004;20:98-101.
559
RAUSIN
3. Sterry W, Barker J, Boehncke WH, Bos JD, Chimenti S, Christophers
E et al. Biological therapies in the systemic management of psoriasis:
international consensus conference. Br J Dermatol 2004;151 Suppl
69:3-17.
4. Schleyer V, Landthaler M, Szeimies R-M. Novel pharmacological
approaches in the treatment of psoriasis. J Eur Acad Dermatol Venereol 2005;19:1-20.
5. Mendonça CO, Burden AD. Current concepts in psoriasis and its
treatment. Pharmacol Ther 2003;99:133-47.
560
6. Leonardi CL, Powers JL, Matheson RT, Goffe BS, Zitnik R, Wang A
et al. Etanercept as monotherapy in patients with psoriasis. N Engl J
Med 2003;349:2014-22.
7. Krueger G, Callis K. Potential of tumor necrosis factor inhibitors in psoriasis and psoriatic arthritis. Arch Dermatol 2004;140:
218-25.
8. Benoit S, Toksoy A, Bröcker EB, Gillitzer R, Goebeler M. Treatment
of recalcitrant pustular psoriasis with infliximab: effective reduction
of chemokine expression. Br J Dermatol 2004;150:1009-12.
Ottobre 2005
Therapeutic uses of botulinum toxin
D. ROY 1, N. S. SADICK 2
Botulinum toxin is a powerful medication that has been used
to treat various conditions in humans for over 2 decades.
In the last few years, there has been an explosion in the use
of this medication for cosmetic purposes. Botulinum toxin
treatments have become on of the most common non-surgical procedures performed by dermatologists and plastic surgeons. Dynamic rhytides of the upper, middle and lower
face and neck have been treated successfully with botulinum
toxin. Treatment of hyperhidrosis of the axillae, hands and
feet has also become established. Botulinum toxin may be
employed as the primary method of facial rejuvenation or
can be an adjunct to other surgical and non-surgical modalities. Millions have benefited from the therapeutic effects
of botulinum toxin, with very few patients experiencing
adverse effects. In this paper will review the mechanism of
action, therapeutic uses and possible complications of botulinum toxin therapy.
KEY WORDS: Botulinum toxins - Skin - Cosmetics.
B
otulinum toxin (BTX) is a powerful medication that has been used to treat various conditions in humans for over 2 decades. In the last
few years, there has been an explosion in the use
of this drug for cosmetic purposes. Millions have
benefited from the therapeutic effects of BTX,
with very few patients experiencing adverse
effects. In this paper we will be discussing the
various therapeutic uses of BTX in the field of
cosmetic dermatology.
Address reprint requests to: D. Roy, MD, Sadick Aesthetic Surgery &
Dermatology, P.C., 772 Park Avenue, New York, NY 10021.
Vol. 140 - N. 5
1Department
of Facial Plastic and Reconstructive Surgery
Lenox Hill Hospital, New York City, NY, USA
2Unit of Dermatology, Cornell University Medical College
New York City, NY, USA
Mechanisms of action
The mechanism of action of BTX has been well
established.1-4 Protein interactions at the neuromuscular junction prevent the release of acetylcholine.
There are 7 known antigenically distinct serotypes of
BTX (A-G). The most widely used and studied is botulinum toxin Type A (BTX-A). This protein irreversibly
binds and cleaves the SNAP-25 protein. There are currently 2 available preparations of BTX-A on the American market, Botox and Reloxin. Botulinum toxin type
B (BTX-B) binds to synaptobrevin. BTX-B is available
commercially as Myobloc. The temporary nature of
therapeutic BTX therapy is due to axonal sprouting
at the motor end plate of the neuromuscular junction
as well as the development of extrajunctional acetylcholine receptors.5 This occurs over several months
after BTX therapy.
Cosmetic use
Cosmetic BTX treatments have become on of the
most common non-surgical procedures performed
by dermatologists and plastic surgeons. Cosmetic
561
ROY
THERAPEUTIC USES OF BOTULINUM TOXIN
Figure 1.—Dynamic rhytides of 3 upper facial areas. A) Forehead. B)
Glabella. C) Lateral periorbital area (Crow’s feet). D) Corresponding
mimetic muscle of the upper face.
use of BTX can be performed on the entire face and
neck, although current Food and Drug Administration
(FDA) guidelines are for use in the glabella (Botox
Cosmetic) and in the neck for cervical dystonia
(Myobloc). Casual users limit their BTX treatments
to the upper face. Advanced users address the midface, lower face and neck as well. BTX treatments
may be employed as the primary method of facial
rejuvenation or can be an adjunct to other surgical and
non-surgical modalities.
is muscle. We recommend using an average of 15 to 30
U of BTX-A for treatment of the frontalis. Vertically
oriented rhytides of the glabella are due to contraction of the procerus, depressor supercilii, and medial
portions of the orbicularis oculi. We use an average
of 15 to 30 U of BTX-A to treat the glabellar complex. Horizontal rhytides of the lateral peri-orbital area
(crow’s feet) are due to contraction of the lateral orbicularis oculi muscle. We use an average of 8 to 15 U of
BTX-A to treat each side of the face.
Dynamic rhytides of the upper face
Dynamic rhytides of the mid face
Facial wrinkles involving the forehead, glabella,
and lateral peri-orbital regions are a common aesthetic problem (Figure 1). These wrinkles are a direct
result of hyperactivity of the underlying muscles of
expression.6 The horizontal rhytides of the central and
lateral forehead are due to contraction of the frontal-
The midface is not an area commonly treated with
BTX. Dynamic rhytides of the midface are usually
centered about the nose, due to the lack of muscles of
facial expression laterally.
Subciliary rhytides caused by contraction of the
medial portion of the lower orbicularis oculi muscle are
562
Ottobre 2005
ROY
change the rotation of the nasal tip and decrease nasal
flaring.
Dynamic rhytides of the lower face
Figure 2.—”Bunny lines” caused by contraction of the transverse head of
the nasalis muscle.
treated by advanced BTX users. A few well-placed
injections of 2 U of BTX-A each are used to treat this
area. It is important to avoid the pre-tarsal portion of
the muscle.
Perinasal rhytides are due to contraction of several
small muscle groups. The most commonly injected
area is the upper nasal sidewall, where contraction of
the nasalis muscle causes the so-called “bunny lines”,
the oblique rhytides of the upper nose (Figure 2). This
usually requires 5 to 10 U of BTX-A per side.Treatment
of the lower nose is usually not directed toward the
ablation of rhytides, but to change static or dynamic
aspects of the nasal tip. BTX treatment can be used to
The lower face is not a common area treated with
BTX. Dynamic rhytides of this region are in the perioral area. Extreme caution must be used when treating
this area with BTX, since diffusion of toxin and injection of adjacent musculature can result in functional
deficits. The so-called “lipstick lines”, vertical peri-oral
rhytides caused by contraction of the orbicularis oris
are the most commonly treated in the lower face. Several well placed injections of 2 U of BTX-A along the
edge of the upper and lower lips are used to treat this
area. A down-turned corner of the mouth is treated
with injections into the depressor anguli oris muscle.
The average dose is between 2 and 8 U of BTX-A per
side. Dimpling of the central chin is treated with injection into the mentalis muscle, using 5 to 10 U of BTXA in total (Figure 3).
Treatment of neck rhytides
Cosmetic use of BTX in the neck is primarily for the
treatment of platysmal banding. These are vertical
bands usually found in the central neck. There are also
horizontal rhytides caused by contraction of the platysma muscle, usually seen laterally, which can be treated with BTX injections (Figure 4). The platysma is a
thin muscle, and carefully placed injections are crucial
to avoid effecting the deeper neck musculature, which
Figure 3.—A) Red arrow indicates the anatomical location of the depressor anguli oris muscle. B) Treatment of chin dimpling caused by contraction
of the mentalis muscle.
Vol. 140 - N. 5
563
ROY
need for repeat treatments, and found that the mean
time between treatments was 29 weeks, and that repeated injections was an effective treatment.8 In a multicenter, randomized, double-blinded trial Heckmann
et al. demonstrated that the rate of sweat production
responded to BTX treatment in a dose-dependant manner.9
Injection technique for the face
Figure 4.—Vertical bands and horizontal neck rhytides caused by contraction of the platysma muscle.
can cause dysphagia, hoarseness and neck weakness.
Dosing varies greatly depending on the area treated, but
multiple injections of 2 to 5 U of BTX-A are used.
Other cosmetic uses
Hyperhidrosis can also be treated with BTX. The
paralytic effect of BTX on the eccrine glands is less
studied than the effect on the mimetic muscles of the
face. The areas most commonly treated are the axilla,
palms and soles of the feet. In a randomized, doubleblinded, placebo-controlled parallel group study, Naumann et al. treated each axilla with 50 U of BTX-A,
and found that the majority of patients had at least a
50% reduction in sweat production, even 18 weeks
after the initial injection.7 Lowe et al. also studied the
Before any injections are performed, the facial musculature is evaluated thoroughly. Muscle activity is
graded as mild, moderate, or severe (Figure 5). This
helps to decide how many units of BTX to use to
achieve the desired effect. Pre-treatment photos are
taken and the face is appropriately prepped and marked.
Precise delivery of small volumes is the best way to
achieve great results with a low incidence of side
effects.
We prefer to use the 1cc Braun Injekt Tuberkulin
Solo syringe with a 30g, 1/2 inch needle (Figure 6).
We do not use any anesthesia for the injections, but prefer the Zimmer Cryo 5 cold air device (Figure 7). Firm
digital pressure is used after each injection (Figure 8).
Occasionally, ice packs are applied for several minutes
after the injection. We counsel patients to avoid vigorous massaging of the areas injected, but all other
light activity is permitted. We usually perform other
facial procedures (such as light, laser, or radiofrequency treatments or superficial peels) prior to injecting with BTX.
The proper amount of toxin to administer should be
determined by the injecting physician based on the
inherent muscle activity, the amount of wrinkling
present, and the desired result. Precisely titrating the
Figure 5.—Pretreatment assessment of dynamic rhytides formed by frontalis muscle activity as A) mild; B) moderate; C) severe.
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Ottobre 2005
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Figure 6.—Syringe and needle used for most BTX treatments. 1cc Braun
Injekt Tuberkulin Solo, 30 gauge, 1/2 inch.
Figure 8.—Bi-manual injection technique with simultaneous skin cooling.
produce a similar effect as 1 000-2 500 U of BTX-B.
For the lateral orbicularis (Crow’s feet) 10 to 15 U of
BTX-A are equivalent to 750-1 500 U of BTX-B.10
Figures 9 through 12 outline the suggested areas to
inject, amount of toxin to deliver, and the corresponding results for the most common treatment scenarios.
Injection effect
Figure 7.—Zimmer Cryo 5 cold air device.
correct amount of BTX per injection site is a skill
that develops with experience. Provisional dosing
guidelines for BTX A and B used in the upper face
provided by Sadick and Matarasso suggest the following conversions: when using 20 to 30 U of BTXA to treat the glabellar complex, one would need to
use 2 000-3 000 U of BTX-B to achieve a similar
effect and duration. Their numbers for treatment of the
forehead are slightly different: 20 to 30 U of BTX-A
Vol. 140 - N. 5
From numerous studies of both BTX-A and BTXB, the average length of onset of therapeutic effect
and its duration have been determined. These numbers vary slightly from patient to patient and are dose
dependent. In general, a similar therapeutic effect is
seen with both products, although the onset of activity of BTX-B seems to be faster. The duration of action
seems to be longer for BTX-A, although with larger
doses, the BTX-B duration can be similar to BTXA.10 Sadick et al. also discuss that there is an increased
“smoothness” to the flaccid paralysis obtained with
BTX-B when compared to BTX-A. Overall, the 2 are
interchangeable.
Complications
Complications are not common. There are those
that are technique-related, and usually occur with casual users and novices. Other complications are due to the
local and systemic actions of the toxin itself.
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Figure 9.—Treatment of forehead and glabellar complex. A) Suggested treatment sites and BTX-A doses. B) Patient frowning before and C) one month
after treatment.
Figure 10.—Treatment of the forehead and glabellar complex. A) Suggested treatment sites and BTX-A doses. B) Patient frowning before and C) two
months after treatment.
Figure 11.—Treatment of the Crow’s feet area. A) Suggested treatment sites and BTX-A doses. B) Patient smiling before and C) two months after treatment.
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Figure 12.—Treatment of forehead. A) Suggested treatment sites and BTX-A doses. B) Patient raising eyebrows before and C) three months after treatment.
usually consists of supportive care. Isolated cases of
distal or generalized muscle weakness have been
reported, but these are rare cases and involve patients
being treated for neurologic diseases.12 When BTX is
used for cosmetic treatments, immunoresistance and
unusual or uncategorized reactions are extremely rare
and should be thoroughly investigated and reported.
Conclusions
Figure 13.—Complications of the BTX treatment. Left eyelid ptosis, treated with Apraclonidine drops b.i.d.
Technique-related
Asymmetry can occur anywhere and is the most
common complication. This can be easily remedied
with additional BTX injections. Improper technique
can cause cosmetic as well as functional problems by
inadvertently effecting adjacent musculature. In the
upper face, brow ptosis, blepharoptosis, dry eye and
diplopia can result from poor injection technique or diffusion of toxin (Figure 13). In the lower face, slurred
speech and oral incompetence can result. Discussing
possible complications prior to the procedure is an
important part of obtaining informed consent. After a
complication has occurred, it is important that it be
identified and treated as soon as possible.
Toxin-related
A wide range of complaints have been reported after
BTX treatments. The most common are headache,
nausea, and malaise.11 Treatment for these complaints
Vol. 140 - N. 5
The aforementioned therapeutic uses of BTX have
expanded the ability for cosmetic surgeons and dermatologists to treat the signs of aging. By specifically targeting the dynamic rhytides of the face, BTX
has allowed us to address a problem that we could not
in the past. Whether used as a primary mode of treatment or as an adjunct to volume-replacement or tissuetightening procedures, BTX is a great addition to the
anti-aging armamentarium.
Riassunto
Impiego terapeutico della tossina botulinica
La tossina botulinica è una molecola molto potente, che da
oltre 2 decenni è stata utilizzata sull’uomo per trattare diversi problemi. In questi ultimi anni si è avuta un’esplosione del
suo impiego a scopi cosmetici. I trattamenti a base di tossina botulinica sono diventati una delle procedure non chirurgiche più comuni eseguite dai dermatologi e dai chirurghi
plastici. Le rughe della parte superiore, media e inferiore del
volto e del collo sono state trattate con successo con la tossina botulinica. Anche il trattamento della iperidrosi dell’ascella, delle mani e dei piedi è diventato comune. La tossina botulinica può essere impiegata quale primo metodo per
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il ringiovanimento del viso o può essere associata ad altre
modalità chirurgiche e non chirurgiche. Sono ormai milioni le persone che hanno tratto beneficio dagli effetti terapeutici della tossina botulinica e solo in rari casi si sono avuti effetti collaterali. In questo lavoro verranno presi in considerazione il meccanismo d’azione, l’utilizzo terapeutico e
le possibili complicanze della terapia con tossina botulinica.
PAROLE CHIAVE: Tossina botulinica - Cute - Cosmesi.
5.
6.
7.
8.
References
1. Sakaguchi G. Clostridium botulinum toxins. Pharmacol Ther
1983;19:165-94.
2. Carruthers JDA, Carruthers JA. Treatment of glabellar frown lines
with C botulinum-A exotoxin. J Dermatol Surg Oncol 1992;18:17-21.
3. Keen M, Blitzer A, Aviv J, Binder W, Prystowsky J, Smith H et al. Botulinum toxin A therapy for hyperkinetic facial lines: results of a double-blind, placebo-controlled study. Plast Reconstr Surg 1994;94:
94-9.
4. Hambleton P. Clostridium botulinum toxins: a general review of
568
9.
10.
11.
12.
involvement in disease, structure, mode of action and preparation for
clinical use. J Neurol 1992;239:16-20.
de Paiva A, Meunier FA, Aoki KR, Dolly JO. Functional repair of
motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of synaptic activity between nerve sprouts and their parent
terminals. Proc Natl Acad Sci USA 1999;96:3200-5.
Pierard GE, Lapiere CM. The microanatomical basis of facial frown
lines. Arch Dermatol 1989;125:1090-2.
Naumann M, Lowe NJ. Botulinum toxin type A in treatment of bilateral primary axillary hyperhidrosis: randomised, parallel group, double blind, placebo controlled trial. BMJ 2001;323:596-9.
Lowe PL, Cerdan-Sanz S, Lowe NJ. Botulinum toxin type A in the
treatment of bilateral primary axillary hyperhidrosis: efficacy and
duration with repeated treatments. Dermatol Surg 2003;29:545-8.
Heckmann M, Ceballos-Baumann AO, Plewig G; Hyperhidrosis Study
Group. Botulinum toxin A for axillary hyperhidrosis. N Engl J Med
2001;344:488-93.
Sadick NS, Matarasso SL. Comparison of botulinum toxins A and B
in the treatment of facial rhytides. Dermatol Clin 2004;22:221-6.
Botox Cosmetic Package insert, Allergan, Inc. 2005
Bhatia KP, Munchau A, Thompson PD, Houser M, Chauhan VS,
Hutchinson M et al. Generalised muscular weakness after botulinum
toxin injections for dystonia: a report of three cases. J Neurol Neurosurg
Psych 1999;67:90-3.
Ottobre 2005
CLINICAL CASES
The use of Imiquimod 5% cream for the treatment
of basal cell carcinomas in a Gorlin’s syndrome
A. DI LANDRO, L. NALDI, L. MARCHESI
Imiquimod 5% cream has been used effectively to treat basal
cell carcinomas (BCCs). The purpose of this study was to examine the effectiveness and tolerability of Imiquimod 5% cream
in treating superficial non-facial BCCs in a patient with basal
cell nevus syndrome or Gorlin's syndrome and to follow the
patient over time for any signs of relapse. Five superficial BCCs
in the mammarian region of a woman suffering psychologically due to multiple scars resulting from many BCC removals,
were treated for 18 weeks with a once-a-day application of
Imiquimod 5% cream. A biopsy was performed before and
after the treatment. The 5 BCCs regressed completely without any sign of histological persistence. Topical therapy caused
irritation in the first weeks of treatment, which resolved after
a reduction in the number of applications. A three-year follow
up confirmed that no relapse had occurred and no scarring
was present at the sites of treatment; only the appearance of a
new small papular lesion was noted slightly above the treated
area. Imiquimod 5% cream can be considered an effective
treatment with good cosmetic results and absence of long term
recurrence for multiple BCCs in basal cell nevus syndrome.
KEY WORDS: Basal cell carcinoma - Imiquimod, administration and
dosage - Basal cell nevus syndrome - Gorlin’s syndrome.
N
aevoid basal cell carcinoma (BCC) syndrome or
Gorlin's syndrome is a rare hereditary disease characterised by the presence of many BCC, odontogenetic
keratocysts, tumours and systemic anomalies.1 Clinical
Received: May 9, 2003.
Accepted for publication: October 14, 2005.
Address reprint requests to: Dott.ssa A. Di Landro, Clinica Dermatologica, Ospedali Riuniti, L.go Barozzi 1, 24100 Bergamo, Italy.
E-mail: aislalomb@ tiscalinet.it
Vol. 140 - N. 5
Department of Dermatology, University of Milano-Bicocca
Ospedali Riuniti, Bergamo, Italy
history is usually characterised by the appearance of
many BCC on the face, neck and upper trunk, although
they can appear anywhere on the body. Exposure to sunlight, radiotherapy and immunodepression (HIV) are
considered to be aggravating factors. The disease affects
1 subject in 56 000 and is linked to a mutation in a tumour
suppressor gene on chromosome 9q22,3-q31. The treatment of multiple BCCs, even if it can be successfully performed through several surgical or cryotherapic techniques, has economical, physical and psychological
implications for the patient, primarly because of the risk
of scarring of reoccurrence. Imiquimod is a topical
immune response modifier that has been proven to induce
a local immune response.2 It is an approved treatment for
external genital and perianal warts,3 but also for different types of neoplastics and preneoplastic lesions, as
BCCs, superficial and nodular types,4-9 in situ epitheliomas 10 and actinic keratosis.11 We report our experience
in treating superficial non-facial BCCs in a patient with
basal cell nevus syndrome.
Case report
A 54-year old Caucasian woman with basal cell nevus
syndrome has been followed in our clinic for over 17 years.
She has had more than 430 BCCs removed resulting in
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DI LANDRO
THE USE OF IMIQUIMOD 5% CREAM FOR THE TREATMENT OF BASAL CELL CARCINOMAS IN A GORLIN’S SYNDROME
Figure 3.—The histopatological picture after 16 week of treatment: no
evidence of BCC (hematoxylin-eosin).
Figure 1.—Multiple basal cell carcinoma before the cycle of topical therapy with 5% Imiquimod cream.
Figure 2.—The histopatological picture of one superficial BCC before
the treatment (hematoxylin-eosin).
Figure 4.—The same area after 3 years from the therapy: absence of relapsing.
innumerable surgical scars and changes in skin pigmentation, particularly on the face, which have resulted in a severe psychological disturbance and the refusal of further surgery or cryotherapy for the remaining numerous BCCs present at the last follow up. As a result we started treatment
with Imiquimod 5% cream (Aldara, 3M Pharmaceuticals,
St. Paul, Minnesota) after obtaining informed consent from
the patient. Five large superficial lesions localised in the
mammary area (Figure 1) were treated with a daily application of Imiquimod 5% cream for 16 weeks. One of the
selected BCCs was biopsied before starting the treatment
and was confirmed to be a superficial BCC (Figure 2). At
the 4th week of treatment the patient complained a burning sensation in the areas of application, which were irritated and inflamed. The patient was advised to suspend
treatment for 1 week and then she was told to apply the
cream 3 times a week for 2 weeks and then once a day for
the rest of the treatment cycle. At the end of treatment (16
weeks), the patient was re-evaluated; we noted the complete
resolution of the lesions at the BCC sites with only slight
erythema. A new biopsy was negative for the presence of
BCC cells (Figure 3). A complete regression of the erythema was noted at a three-month follow up visit. The patient
attended follow up visits every 6 months without any signs
of BCC relapsing at treated sites, even though new lesions
appeared at other cutaneous sites. No reoccurrences or
scarring were observed at the three-year follow up (Figure 4). In particular, the treated BCC lesions were in complete
remission without any signs of scarring; only a new, small
papular BCC was noted slightly above the area.
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Ottobre 2005
Imiquimod is an immune response modifier that
favours the production of interferons (IFN) and other
cytokines related to local, cell-mediated immune
response effecting antiviral and antitumour properties.2 One of its action mechanisms depends on the
capacity of linking to the Toll like membrane receptors,
part of the innate immune response, in particular to
Toll-like 7 (TLR-7); experiences done in macrophages
of rats with a TLR-7 deficit or of metabolic via MyD88
related to it, have demonstrated that the administration
of Imiquimod or its derived resiquimod lacked to
induce cytokines.12 The link through Imiquimod and
TLR-7 has been shown also at plasmocitoid dendritic cells, which cause the greater production of plasmatic
IFN after a viral infection.13-16 Skin biopsies from hairless mice treated with 5% Imiquimod highlighted an
up regulation of interferon-α (IFN-α), IFN-β, tumour
necrosis factor-α (TNF-β), IL-1α, IL-1β, IL-6 and IL12 production.2 More recent experiences have demonstrated that tumour cells treated with Imiquimod
become more susceptible to apoptosis through
decreased Bcl-2 expression with an inflammatory infiltrate developing rapidly (within 3 to 5 days after treatment initiation), associated with the enhanced expression of ICAM-1. The infiltrate is made up of
macrophages and lymphocytes with the production of
IFN-α by CD4 and CD8 lymphocytes.17 The experience of Uhoda 18 has proved that Imiquimod can control epidermal field cancerogenesis and that dermal
dendrocytes may play a pivotal role in this regression
phenomenon. The use of Imiquimod in the treatment
of superficial BCCs has been documented 4-8 and
recently also in nodular BCCs.9 A dose-dependent
inflammatory response was reported in most patients
treated, but is generally considered tolerable, even
though it may affect patient compliance; out of 99
patients treated by Marks,6 only 1 abandoned treatment because of a skin reaction. There are few clinical experiences in the treatment of BCC in the context
of basal cell nevus syndrome;19-22 Kagy et al. examined
the effectiveness, tolerability and compliance in the
treatment of 3 BCCs in a 49-year old patient affected
by the syndrome during an eighteen-week course of
daily treatment.19 Despite a complete regression of
treated lesions and good tolerability profile, the patient
decided to stop treatment due to the time required and
also the appearance of local irritation. Micali et al.21
used Imiquimod to treat 17 BCCs (superficial and
Vol. 140 - N. 5
DI LANDRO
nodular) of 4 patients, with 3-5 applications per week
for 8-14 weeks obtaining a complete resolution of 13
lesions. In another case of Gorlin's syndrome, a facial
nodular BCC located under the eyelid was treated with
a complete remission after 3 months of therapy,22 a
good compliance on the part of the patient and absence
of relapsing after 6 months. Our patient, who was psychologically frustrated due to innumerable scars, many
of which on the face, resulting from surgical BCC
removals, went into complete remission for the 5 BCCs
treated with no signs of relapsing at 3 years.After the
irritation of the first weeks, the treatment was followed
by the patient with good compliance and the erythema
regressed 1 month after completing the treatment,
without scarring. This positive outcome can highlight
the importance of its good cosmetic value in a psychologically distressed subject like our patient. Up to
now our experience seems to be the one with the
longest follow-up (3 years) with the appearance of
new BCCs, but without any relapsing of previously
treated lesions. Topical therapy with 5% Imiquimod can
be considered an effective treatment for basal cell
nevus syndrome and an alternative to other treatments
(cryotherapy, electrosurgery, etc.) in general.
References
1. Miroswki GW, An-ti Liu A, Parks ET, Caldemeyer KS. Nevoid basal
cell carcinoma syndrome. J Am Acad Dermatol 2000;43:1092-3.
2. Slade HB, Owens ML, Tomai MA, Miller RL. Imiquimod 5% cream
(Aldara). Exp Opin Invest Drugs 1998;7:437-49.
3. Edwards L, Ferenczy A, Eron L, Baker D, Owens ML, Fox TL et al.
Self-administered topical 5% imiquimod cream for external anogenital warts. Arch Dermatol 1998;134:25-30.
4. Beutner KR, Geisse JK, Helman D, Fox TL, Ginkel A, Owens ML.
Therapeutic response of basal cell carcinoma to the immune response
modifier imiquimod 5% cream. J Am Acad Dermatol 1999;41:
1002-7.
5. Hannuksela-Svahn A, Nordal E, Christensen OB. Treatment of multiple basal cell carcinomas with imiquimod 5% cream. Acta Derm
Venereol 2000;80:381-2.
6. Marks R, Gebauer K, Shumack S, Amies M, Bryden J, Fox TL et al.
Imiquimod 5% in the treatment of superficial basal cell carcinoma:
results of a multicenter 6-week dose-response trial. J Am Acad Dermatol 2001;44: 807-13.
7. Chen TM, Rosen T, Orengo I. Treatment of a large superficial basal
cell carcinoma with 5% Imiquimod: a case report and review of the literature. Dermatol Surg 2002;28:344-6.
8. Drehs MM, Cook-Bolden F, Tanzi E, Weinberg JM. Successful treatment of multiple superficial basal cell carcinomas with Imiquimod:
case report and review of the literature. Dermatol Surg 2002;28:427-9.
9. Shumack S, Robinson J, Kossard S, Golitz L, Greenway H, Schroeter
A et al. Efficacy of topical 5% Imiquimod cream for the treatment of
nodular basal cell carcinoma. Arch Dermatol 2002;138:1165-71.
10. Mackenzie-Wood A, de Kossard S, Launey J, Wilkinson B, Owens ML.
Imiquimod 5% cream in the treatment of Bowen's disease. J Am Acad
Dermatol 2001;44: 462-70.
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DI LANDRO
11. Korman N, Moy R, Ling M, Matheson R, Smith S, McKane S et al.
Dosing with 5% imiquimod cream 3 times per week for the treatment of Actinic keratosis. Arch Dermatol 2005;141:467-73.
12. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K et al.
Small antiviral compounds activate immune cells via the TLR7 MyD88dependent signalling pathway. Nat Immunol 2002;3:196-200.
13. Gibson SJ, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE
et al. Plasmocytoid dendritic cells produce cytokines and mature in
response to the TLR7 agonists, Imiquimod and Resiquimod. Cell
Immunol 2002;218:74-86.
14. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2002;85:85-95.
15. Sauder DN. Immunomodulatory and pharmacologic properties of
imiquimod . J Am Acad Dermatol 2000;43 (1 Pt 2): S6-11.
16. Schon M, Bong A, Drewniok C, Herz J, Geilen CC, Reifenberger J et
al. Tumor-selective induction of apoptosis and the small-molecule
immune response modifier imiquimod. J Natl Cancer Inst
2003;95:1138-49.
17. Urosevic M, Maier T, Benninghoff B, Slade H, Burg G, Dummer R.
18.
19.
20.
21.
22.
Mechanisms underlying imiquimod-induced regression of basal cell
carcinoma in vivo. Arch Dermatol 2003;139:1325-32.
Uhoda I, Quatresooz P, Piérard- Franchimont C, Pierard GE. Nudging epidermal field cancerogenesis by imiquimod. Dermatology
2003;206:357-60.
Kagy MK, Amonette R. The use of Imiquimod 5% cream for the
treatment of superficial basal cell carcinomas in a basal cell nevus
syndrome patient. Dermatol Surg 2000;26:577-8.
Stockfleth E, Ulrich C, Hauschild A, Lischner S, Meyer T, Christophers
E. Successful treatment of basal cell carcinomas in a nevoid basal
cell carcinoma syndrome with topical 5% imiquimod. Eur J Dermatol 2002;12:569-72.
Micali G, Lacarrubba F, Nasca MR, De Pasquale R.. The use of
imiquimod 5% cream for the treatment of basal cell carcinoma as
observed in Gorlin's syndrome. Clin Exp Dermatology 2003;28 (Suppl 1):19-23.
Vereecken P, Monsieur E, Petein M, Heenen M. Topical application
of imiquimod for the treatment of high-risk facial basal cell carcinoma in Gorlin syndrome. J Derm Treatment 2004;15:120-1.
Uso dell’Imiquimod al 5% in crema nel trattamento
dei carcinomi basocellulari nella sindrome di Gorlin
L
a sindrome del nevo basocellulare o sindrome di Gorlin
è una malattia rara di origine ereditaria caratterizzata
dall'associazione di carcinomi basocellulari (basal cell carcinoma, BCC) multipli, cisti odontogeniche della mandibola, depressioni palmo-plantari, tumori e altre anomalie sistemiche 1. La storia clinica è caratterizzata dalla comparsa di
basaliomi multipli fin dalla pubertà e dalla terza decade di
vita, localizzati soprattutto al viso, al collo e al tronco, ma tutte le sedi cutanee possono essere interessate e i basaliomi possono diventare molto numerosi, anche centinaia. L'esposizione solare, la radioterapia e l'immunodepressione (HIV)
sono considerati fattori peggiorativi. La malattia interessa
1 soggetto ogni 56 000 ed è legata alla mutazione del gene
soppressore tumorale sul cromosoma 9q22,3-q31. Il trattamento dei BCC multipli, anche se efficace e attuabile con le
varie terapie in uso, chirurgica, crioterapica, ha un'implicazione fisica, psicologica ed economica per il paziente, soprattutto per gli esiti cicatriziali e per il rischio di recidive. L'Imiquimod è un farmaco immunomodulatore 2 che si è dimostrato efficace nel trattare le verruche ano-genitali 3 e differenti tipi di lesioni neoplastiche e preneoplastiche, come i
basaliomi, sia superficiali sia nodulari 4-9, gli epiteliomi in situ
10, e le cheratosi attiniche 11. Nella sindrome di Gorlin l'Imiquimod è stato descritto come un farmaco alternativo alle
classiche tecniche chirurgiche per trattare i BCC multipli.
Caso clinico
Una paziente di razza bianca di 54 anni, portatrice della sindrome del nevo basocellulare, era seguita nella nostra clini-
572
ca da più di 17 anni e aveva subito l'asportazione di circa
430 BCC, con innumerevoli esiti cicatriziali e alterazioni
nella pigmentazione cutanea, soprattutto del viso, che avevano causato problemi psicologici e il rifiuto di sottoporsi a
ulteriori trattamenti chirurgici o crioterapici dei nuovi BCC
evidenziati all'ultimo controllo clinico. Si decideva di iniziare
terapia con Imiquimod 5% in crema (Aldara, 3M Pharmaceuticals, St. Paul, Minnesota) dopo che la paziente rilasciava il consenso informato. Venivano sottoposte a terapia
5 grandi lesioni di tipo superficiale localizzate alla regione
mammaria (Figura 1) con l'applicazione giornaliera della
crema per 16 settimane. Uno dei BCC veniva biopsiato prima del ciclo di terapia per la conferma della diagnosi clinica (Figura 2). Al controllo clinico, dopo 4 settimane, la
paziente lamentava la comparsa di bruciore nelle sedi di
applicazione, che apparivano irritate e infiammate. Le veniva consigliato di sospendere per una settimana il ciclo terapeutico e di riprenderlo applicando il topico 3 volte la settimana per 2 settimane e in seguito di riprendere le applicazioni
giornaliere per il restante periodo di trattamento. Al followup a 16 settimane si notava la risoluzione dei BCC con la persistenza di un lieve eritema. Una nuova biopsia risultava
negativa per la presenza di cellule basaliomatose (Figura 3).
Una regressione completa dell'eritema senza alcun esito
cicatriziale era presente al follow-up a 3 mesi. La paziente
veniva in seguito seguita ogni 6 mesi senza mostrare recidive dei BCC già trattati anche se comparivano altre nuove
lesioni in varie sedi cutanee. In particolare, al follow-up a 3
anni, si osservava una completa remissione dei BCC senza
esiti cicatriziali; solo superiormente all'area era presente un
nuovo piccolo BCC papuloso (Figura 4).
Ottobre 2005
Discussione e conclusioni
L'Imiquimod è un modificatore della risposta immunitaria che favorisce la produzione di interferon (IFN) e altre
citochine legate alla risposta locale cellulo-mediata esercitando un effetto sia antivirale sia antitumorale 2. Uno dei
suoi meccanismi d'azione è legato alla capacità di legarsi ai
recettori di membrana Toll-like, parte della risposta immunitaria innata, in particolare al Toll-like 7 (TLR-7); esperienze condotte nei macrofagi di ratti con deficit di TLR-7 o
della via metabolica MyD88 ad esso correlata hanno dimostrato che la somministrazione di Imiquimod o del suo derivato resiquimod non ha indotto la produzione di citochine 12.
Il legame tra Imiquimod e TLR-7 è stato evidenziato anche
a livello delle cellule dendritiche plasmocitoidi, responsabili
della maggior parte della produzione di IFN a livello plasmatico a seguito di un'infezione virale 13--16. Da biopsie
cutanee da topi glabri trattati con Imiquimod al 5% si è ottenuta una produzione aumentata di interferon-α (IFN-α),
IFN-β, tumor necrosis factor α (TNF-α), IL-1α, IL-1β, IL-6 e
IL-122. Altre esperienze più recenti 17 hanno dimostrato che
le cellule tumorali trattate con l'Imiquimod diventano più
suscettibili all'apoptosi con una diminuzione dell'espressione Bcl-2 e con un infiltrato infiammatorio che si sviluppa rapidamente (tra i 3-5 giorni dopo l'inizio della terapia) al quale si associa un'aumentata espressione di ICAM-1. L'infiltrato
è di tipo misto, costituito da macrofagi e linfociti con produzione di IFN-α da parte di linfociti CD4 e CD8. Inoltre
ricerche di Uhoda1 8 hanno evidenziato che l'Imiquimod è in
grado di controllare la cancerogenesi epidermica e che i dendrociti dermici possono giocare un ruolo molto importante
in questo fenomeno di regressione. L'utilità dell'Imiquimod
nel curare i BCC è stata documentata nelle varianti cliniche
superficiali 4-8 e, più recentemente, anche nelle nodulari 9.
Una risposta infiammatoria dose-dipendente è stata riportata in diversi dei pazienti trattati, ma è, in genere, considerata tollerabile, anche se può interessare la compliance del
paziente; del gruppo di 99 soggetti trattati da Marks 6 solo 1
ha abbandonato la terapia per la reazione cutanea. Per quanto riguarda il trattamento della sindrome del nevo basocellulare, le esperienze cliniche sono scarse 19-22; Kagy et al. hanno esaminato l'efficacia, la tollerabilità e la compliance nel
trattamento di 3 BCC in un paziente di 49 anni, portatore
della sindrome durante un ciclo terapeutico di 18 settimane
con applicazione giornaliera del farmaco 19. Malgrado una
regressione completa delle lesioni trattate e una buona tollerabilità, il paziente decideva di interrompere il trattamento per la durata e per la comparsa di irritazione locale. Micali et al.21 hanno usato l'Imiquimod per trattare 17 BCC (superficiali e nodulari) di 4 pazienti, con cicli di 3-5 applicazioni
a settimana per 8-14 settimane, ottenendo una risoluzione
completa di 13 lesioni. In un altro caso di sindrome di Gorlin è stato trattato un BCC nodulare sottopalpebrale con una
Vol. 140 - N. 5
DI LANDRO
remissione completa dopo 3 mesi di terapia 22 e una buona
compliance da parte del paziente e assenza di recidiva dopo
6 mesi. Nella nostra paziente, provata psicologicamente per
gli esiti cicatriziali multipli localizzati in particolare al viso
dai precedenti interventi di asportazione degli innumerevoli BCC, si è avuta una completa regressione dei 5 BCC trattati con la crema di Imiquimod al 5% e non si sono manifestate recidive al follow-up a 3 anni. Dopo la fase di irritazione
iniziale, la terapia è stata seguita con una buona compliance e si è notata la regressione dell'eritema dopo 1 mese dalla sospensione della terapia senza alcun esito cicatriziale. È
da sottolineare il buon esito ottenuto dal punto di vista cosmetologico, molto importante per un soggetto provato psicologicamente come la nostra paziente. A oggi, il nostro caso
risulta il più seguito a lungo nel tempo (3 anni) con la comparsa di nuovi BCC ma assenza di recidive dei BCC precedentemente trattati. La terapia topica con Imiquimod al 5%
è da considerarsi efficace per il trattamento dei BCC multipli della sindrome di Gorlin e può essere considerata una
buona alternativa ai trattamenti chirurgici in genere.
Riassunto
L'Imiquimod al 5% in crema ha dimostrato una buona
efficacia nel trattare i carcinomi basocellulari (basal cell
carcinoma, BCC). Lo scopo di questo studio è stato provare l'efficacia e la tollerabilità del prodotto nel trattare i BCC
superficiali non localizzati al viso in una paziente portatrice
della sindrome del nevo basocellulare o sindrome di Gorlin
e di seguirla nel tempo per il rischio di recidive.
Cinque BCC superficiali della regione mammaria in una
paziente già trattata per lesioni multiple e con gravi sequele psicologiche legate agli esiti cicatriziali sono stati trattati per 18 settimane con un'applicazione giornaliera della crema al 5% di Imiquimod. Un prelievo bioptico è stato eseguito
prima e dopo il ciclo di terapia.
I BCC sono regrediti in toto con un buon risultato cosmetologico. La terapia topica ha determinato un'irritazione nelle prime settimane di trattamento che è regredita con la riduzione del numero di applicazioni settimanali. Al follow-up
a 3 anni si è notata la completa remissione di tutti i BCC
trattati senza la presenza di esiti cicatriziali; si riscontrava solo
la comparsa di un nuovo piccolo elemento papuloso superiormente all'area trattata.
La crema a base di Imiquimod al 5% deve essere considerata un trattamento efficace per i BCC multipli della sindrome del nevo basocellulare con buoni esiti cosmetologici
e l'assenza di recidive a lungo termine.
PAROLE CHIAVE: Carcinoma basocellulare - Crema al 5% di
Imiquimod - Sindrome del nevo basocellulare - Sindrome di
Gorlin.
573
THERAPEUTICAL NOTES
Evaluation of the efficacy and tolerability
of a new intermittent treatment regimen
with cyclosporin A in severe psoriasis
G. A. VENA 1, N. CASSANO 1, A. GALLUCCIO 2, F. LOCONSOLE 1, C. COVIELLO 1, D. FAI 3,
S. DATTOLA 4, A. FERRARI 4, P. LIGORI 3, R. LOPREIATO 4, S. PELLÈ 3,
M. F. POSTORINO 4, F. RICCIUTI 5, G. VALENTI 6, C. MALVINDI 3, P. LUI 7
Aim. The aim of this study was to evaluate the efficacy and tolerability of a new intermittent treatment regimen with
cyclosporin A (CsA) in severe psoriasis.
Methods. Patients with severe chronic plaque psoriasis
(PASI≥12) were allocated to 2 different six-month duration
treatment regimens consisting of continuous administration
of CsA, 4 mg/kg/day (group A) or CsA administered for 4 consecutive days per week (group B). After the baseline, visits
were carried out after 2, 4 and 6 months of therapy, assessing
PASI and severity of pruritus graded with a five-point scale.
Results. Eligible patients were 203:101 subjects in group A and
102 in group B. Baseline PASI was significantly more severe in
group A than group B (30.12±12.74 versus 24.71±11.44, P=0.000),
as well as the severity of pruritus (1.92±1.15 versus 1.58±1.04,
P=0.057). Instead, at the baseline, concomitant controlled hypertension was more frequent in group B (47 subjects) as compared with group A (25 patients). Both treatment regimens
caused a significant improvement of PASI and pruritus at each
visit (P<0.05). Frequency of adverse events was 31% in group
A and 12% in group B; they caused premature treatment discontinuation in only 6 patients (4 in group A and 2 in group B).
Interestingly, abnormal pressure values were more frequently
detected in group A that in group B (21% versus 7%).
Conclusion. Our preliminary data suggest that this new schedule of intermittent treatment with CsA is effective in severe
psoriasis and appears to show a better tolerability profile than
continuous treatment.
KEY WORDS: Psoriasis, diagnosis - Cyclosporin A - Psoriasis, therapy.
Received: March 19, 2005.
Accepted for publication: September 30, 2005.
Address reprint requests to: Prof. G. A. Vena, Seconda Unità di Dermatologia, Università degli Studi di Bari, Policlinico, Piazza Giulio Cesare 11, 70124 Bari. E-mail: [email protected]
Vol. 140 - N. 5
1Second Unit of Dermatology
Department of Internal Medicine
Immunology and Infectious Diseases
University of Bari, Bari, Italy
2Division of Dermatology and Phototherapy
Ospedale Fatebenefratelli, Benevento, Italy
3Collegio Salentino di Dermatologia, CSD, Italy
4Associazione Dermatologi della Magna Grecia, ADMG, Italy
5Division of Dermatology, Ospedale San Carlo, Potenza, Italy
6Division of Dermatology
Ospedale di Catanzaro, Catanzaro, Italy
7Dermatology Service, ASL Mantova, Mantova, Italy
C
yclosporin A (CsA) is a well-established systemic
approach to severe psoriasis whose efficacy has
been proven by several randomized controlled studies.1-7 These studies have demonstrated that CsA
induces a rapid and sustained improvement of psoriasis and quality of life. In psoriasis clinical practice,
adverse events, principally blood hypertension and
renal toxicity, are generally mild to moderate, reversible
and manageable with dose reduction. A careful selection of patients, including assessment of general conditions, contraindications, and concomitant treatments,
may help to reduce the risk of toxic effects. However,
the strict dependence of unwanted events on CsA daily dose and duration of therapy has prompted the everincreasing adoption of short-term intermittent courses. Actually, intermittent treatment courses with CsA
used so far were performed with continuous adminis-
575
VENA
EVALUATION OF HE EFFICACY AND TOLERABILITY OF A NEW INTERMITTENT TREATMENT REGIMEN WITH CICLOSPORIN A
tration of CsA for short period of approximately 12
weeks up to 24 weeks.5, 8, 9
The aim of this pilot open experience was to evaluate the efficacy and tolerability of a new intermittent
regimen in severe psoriasis, consisting in the administration of CsA for 4 consecutive days per week.
Adult patients of both sex with active severe chronic plaque psoriasis (e.g. with psoriasis activity and severity index, PASI,10 of at least 12) were enrolled after oral
informed consent. Women of childbearing potential
accepted to use a medically approved method of contraception throughout the treatment period. Eligibility
was assessed after excluding any contraindications to the
use of CsA, including relevant renal impairment (serum
creatinine above 1.5 mg/dL and/or creatinine clearance
<60 mL/min/1.73 m3) and abnormal liver function
(bilirubin or liver enzymes twice the upper limit of the
normal range), uncontrolled blood hypertension, systemic infections, history of malignancy, clinically significant haematopoietic, immune, cardiovascular and/or
neurological abnormalities. Concomitant therapy with
medications apt to influence psoriasis or to interfere
with CsA metabolism, as well as nephrotoxic drugs,
was also excluded. All patients stopped receiving systemic therapy, phototherapy or PUVA for at least 4
weeks and topical therapy for a minimum of 2 weeks
whereas the use of biological agents had to be interrupted at least 3 months before enrolment. Eligible
patients were randomly allocated to one of the following treatment groups according to a 1:1 ratio:
— group A: CsA 4 mg/kg/day continuously for 6
months;
— group B: CsA 4 mg/kg/day for 4 consecutive
days followed by 3 day of suspension per week for 6
months.
In both groups, the daily dosage of CsA was administered in 2 divided doses, after meals.
After baseline, patients were visited at months 2, 4
and 6. A visit after the first month of therapy was considered optional, unless it was required for variable
reasons related to clinical response and/or safety; additional unscheduled visits were left at the discretion of
dermatologists or patients. During the scheduled visits, clinical evaluations, including physical examination, measurement of blood pressure and assessment
576
of PASI and pruritus severity, were carried out. The
intensity of pruritus was graded by patients using a
five-point scale (0=absent; 1=mild; 2=moderate; 3=
severe; 4=very severe).
For statistical purposes, Wilcoxon matched-pairs
signed-ranks test was used to analyze the variation of
PASI score and pruritus severity in each group (significance for P<0.05). The difference of these parameters between the 2 treatment groups at each visit
was evaluated by the Mann-Whitney test; differences
were considered significant for P values less than 0.05.
Safety assessments consisted of monitoring and
recording all adverse events, regular measurement of
vital signs and performance of physical examination,
regular monitoring of hematology, blood chemistry
(urea, creatinine, total bilirubin, AST, ALT, alkaline
phosphatase, magnesium, potassium, sodium) and urinanalysis. Laboratory examinations were performed at
monthly intervals. Patients were instructed to undergo monitoring of blood pressure and to communicate
any variation of laboratory parameters during the period between scheduled visits.
The safety assessment was mainly based on the frequency of adverse events and the number of abnormal laboratory values. Relevant changes of blood pressure, confirmed by repeated measurements, were
recorded. Blood pressure values were classified according to the Joint National Committee-6th report (JNC 6)
guidelines.11 Abnormal pressure values during CsA
treatment were defined as follows: development of
borderline hypertension (130-139/85-89 mmHg) or
proper hypertension (≥ 140-90 mmHg) in subjects
with baseline normal pressure; development of hypertension in patients with baseline borderline hypertension; relevant increase of pressure levels in already
hypertensive patients (i.e. responsible for the worsening of the hypertension stage or requiring the adjustment of the antihypertensive therapy).
Results
Study population was composed by 203 patients:
101 subjects (61 males and 40 females) with a mean age
of 42.8 (range: 25-83) in group A and 102 (57 males and
45 females) aged 27 to 85 (mean age, 44.6) in group B.
During the six-month observational period, 17 patients
prematurely withdrew from the study for administrative
reasons (3 in group A, 4 in group B), because adverse
events (4 in group A, 2 in group A) or inefficacy (1 in
Ottobre 2005
TABLE I.—Baseline relevant systemic disorders in the 2 treatment
groups.
Condition
Group A
(total: 101)
Group B
(total: 102)
10
15
19
28
7
8
2
3
5
4
Borderline hypertension
Hypertension I stage (systolic and/or
diastolic)
Cardiovascular and metabolic disorders
(diabetes mellitus, congestive cardiopathy)
Thyroid disorders
Gastric disturbances
TABLE II.—Change of PASI and pruritus severity over the study period
in the 2 treatment groups.
Group A
Mean±SD
PASI
Baseline
Month 2
Month 4
Month 6
Pruritus severity
Baseline
Month 2
Month 4
Month 6
Differences
between
group A
and group B
(MannWhitney test)
Group B
Mean±SD
30.12±12.74
17.83±10.17*
10.18±8.64*
5.41±6.17*
24.71±11.44
13.27±10.17*
7.27±8.47*
3.51±6.54*
P=0.000
P=0.002
P=0.012
P=0.011
1.92±1.15
0.88±0.93*
0.42±0.73*
0.20±0.40*
1.58±1.04
0.52±0.84*
0.29±0.59*
0.15±0.41*
P=0.057
P=0.008
n.s.
n.s.
*) P<0.05 versus baseline value (Wilcoxon Signed Ranks Test).
n.s.= not significant.
VENA
group A, 3 in group B). Only 55 patients underwent
clinical evaluation after 1 month; the low number of
analysable patients at this optional visit did not enable
statistical considerations. Baseline general conditions
of the eligible patients showed that controlled relevant
concomitant disorders were almost similar between
the 2 groups except for hypertension, which was more
frequent in group B (Table I). Of hypertensive subjects, 7 patients in group A and 10 in group B were
taking antihypertensive medications. Distribution of
PASI score between groups was rather non-homogeneous, with mean PASI value significantly higher in
group A than in group B (30.12±12.74 versus
24.71±11.44, P=0.000). Most patients (72% in group
A and 68% in group B) complained of pruritus of variable intensity; also this symptom was more severe in
group A as compared with group B (1.92±1.15 versus
1.58±1.04, P=0.057). Both treatment regimens caused
a significant improvement (P<0.05) of PASI and pruritus at each visit (Table II). Significant differences in
PASI scores were maintained throughout the study
period between group A and group B whereas differences in pruritus severity between the 2 groups did not
reach a statistical significance at both four-month and
six-month evaluations. A reduction of PASI of at least
50% from baseline was more frequent in patients receiving continuous therapy rather than the intermittent
treatment at 2 months (60% versus 43%) and, despite
a constant trend towards a superior activity of the con-
90
Group A
Group B
80
70
% patients
60
50
40
30
20
10
0
M2
M4
0-24%
M5
25-49%
M2
50-74%
M4
M5
At least 75%
Figure 1.—Distribution of PASI improvement (%) in the treatment groups.
Vol. 140 - N. 5
577
VENA
TABLE III.—Relevant adverse events in the 2 treatment groups.
Type
Group A
(N.)
Group B
(N.)
21
2
7
7
—
3
1
1
1
—
—
1
—
—
1
1
1
1
1
1
—
—
32*
12
Abnormal pressure values
— Premature discontinuation
Gastrointestinal disorders (abdominal pain,
vomitus, nausea, dyspepsia)
Angina pectoris
Skin rash (not specified)
Others (not responsible for withdrawal)
— Bronchitis
Leucocytosis
Migraine
Total
*Some patients experienced more than one adverse event.
tinuous treatment, the differences between the 2 groups
appeared to be gradually less pronounced with the prolongation of therapy (Figure 1). In fact, a ≥ 75% reduction of PASI was achieved by 84% and 78% of patients
in group A and group B, respectively, after 6 months.
Laboratory examinations did not reveal any clinically
significant changes in the study population, apart from
the finding of transient leucocytosis in a patient of
group A. Frequency of adverse events was 31% in
group A and 12% in group B (Table III); they were of
mild to moderate severity, transient or manageable in
the majority of cases, except for those cases in whom
premature treatment cessation was required. In group
A, there were a slight increase of occurrence of gastrointestinal complaints and, notably, a more frequent
recording of abnormal pressure values as compared
with patients treated with intermittent regimen (21%
versus 7%).
The usefulness of CsA for the treatment of chronic
inflammatory skin disorders, including psoriasis, is
well known and confirmed by numerous data from
clinical trials. The major limitation is the potential
toxicity, which is of particular concern in long-term use.
Safety considerations are even more important when
they refer to a chronic condition, like psoriasis, which
is not life-threatening and often affects otherwise
healthy people.
Numerous evidences indicate that adverse events,
578
and particularly renal impairment, are dose- and timedependent.12 CsA nephrotoxicity is reversible after
short-term courses; progressive renal structural injury
and stable reduction of glomerular filtration rate (GFR)
can develop after prolonged treatment courses.13, 14
Therefore, it is recommended that duration of continuous treatment should not exceed 2 years.9, 12 Several
studies have demonstrated the efficacy of short-term
intermittent courses for a maximum of 12 weeks in
severe psoriasis.15-18 In clinical practice, intermittent
courses of CsA up to six-month duration are usually
used 9, 12 allowing restoration of normal renal functions.8 Another common side effect of CsA is arterial
hypertension. It was found that short-term use of CsA
at a dose of 5 mg/kg/day for the treatment of psoriasis
induces a significant increase in blood pressure, but
only a transient mild non-significant reduction in GFR.19
Intermittent courses used in clinical setting consist
indeed of continuous administration of CsA for short
periods. In this study, we evaluated the effects of a
new intermittent regimen based on the administration
of CsA for 4 consecutive day followed by 3 treatmentfree days per week (4 on-3 off weekly treatment). The
results show the effectiveness of this regimen on both
skin lesions (PASI) and pruritus and urge us to make
some considerations. First of all, psoriasis characteristics of the patients enrolled in the 2 treatment groups
were significantly different, with a marked trend
towards the inclusion of more severe forms in the
group treated with continuous regimen. This group
was also characterized by a lower proportion of patients
with abnormal pressure values. This indicates that
most probably there was not a random allocation of participants and that the intermittent course was more
frequently chosen for patients with less severe forms
and worst general conditions. The imbalance of baseline PASI did not allow a direct comparison between
the treatment regimens over the study period. Instead,
the severity of pruritus, which was greater in group A
at the baseline and after 2 months, became similar in
the 2 groups at the following evaluations (P>0.05).
Overall safety data suggest that the intermittent
treatment has a better tolerability profile than the continuous therapy. Particularly, despite the higher proportion of hypertensive subjects, patients of group B
experienced less frequently relevant changes in blood
pressure. Therefore, it is likely that weekly drug holidays may improve the tolerability of CsA.
To our knowledge, previous attempts to use CsA in
Ottobre 2005
psoriasis with an intermittent intraweekly schedule
have been rarely reported. In an open-labelled study,20
CsA 5 mg/kg/day was administered continuously for
4 weeks followed by maintenance treatment at the
same dose every 4 days. In a randomised study,21 CsA
was administered 3 times weekly for 12 weeks as
maintenance after a twelve-week continuous therapy.
Goodman et al.22 studied the effects of oral doses of
CsA taken at 12-h intervals for 3 doses per week during a ten-week period. The rational basis for the use of
this schedule was that the cell cycle of psoriatic keratinocytes is of 36 h. Anyway, relevant side effects
were observed due to the high doses used (up to 10
mg/kg).
In conclusion, a new intraweek intermittent schedule with CsA in severe psoriasis, consisting of the
administration for 4 consecutive days per week, appears
to be effective and well-tolerated in severe psoriasis,
although randomised studies are needed to confirm
these preliminary results.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Acknowledgements.—The authors thank Dr. M. Carbonara (Bari, Italy)
for the support in the statistical analysis.
17.
References
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1. Griffiths CE, Clark CM, Chalmers RJ, Li Wan Po A, Williams HC. A
systematic review of treatments for severe psoriasis. Health Technol
Assess 2000;4:1-125.
2. Lebwohl M, Ellis C, Gottlieb A, Koo J, Krueger G, Linden K et al.
Cyclosporine consensus conference: with emphasis on the treatment
of psoriasis. J Am Acad Dermatol 1998;39:464-75.
3. Berth-Jones J. Current management of psoriasis. Cyclosporin. J Derm
Treat 1997;8:46-9.
4. Koo J. Neoral in psoriasis therapy: toward a new perspective. Int J Dermatol 1997;36 Suppl 1:25-9.
5. Ho VC. The use of ciclosporin in psoriasis: a clinical review. Br J
Dermatol 2004;150 Suppl 67:1-10.
6. Touw CR, Hakkaart-Van Roijen L, Verboom P, Paul C, Rutten FF,
Finlay AW. Quality of life and clinical outcome in psoriasis patients
using intermittent cyclosporin. Br J Dermatol 2001;144:967-72.
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Finzi AF. Individualized short-course cyclosporin therapy in psoriasis. Br J Dermatol 1996;135 Suppl 48:31-4.
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The sixth report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. Arch Intern
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Vena GA, Cassano N. Terapia sistemica nella psoriasi. In: Lotti MT,
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Zachariae H, Kragballe K, Hansen HE, Marcussen N, Olsen S. Renal
biopsy findings in long-term cyclosporin treatment of psoriasis. Br J
Dermatol 1997;136:531-5.
Powles AV, Hardman CM, Porter WM, Cook T, Hulme B, Fry L.
Renal function after 10 years’ treatment with cyclosporin for psoriasis. Br J Dermatol 1998;138:443-9.
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Atakan N et al. Intermittent short courses of cyclosporin (Neoral(R))
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Valutazione dell’efficacia e della tollerabilità di un nuovo schema
di terapia intermittente con ciclosporina A nella psoriasi severa
L
’efficacia della ciclosporina A (CsA) nella psoriasi severa è stata dimostrata in numerosi studi randomizzati
controllati 1-7, che hanno documentato anche un rilevante
effetto sulla qualità della vita. Gli eventi avversi che si verificano in corso di terapia con CsA, rappresentati principal-
Vol. 140 - N. 5
mente da ipertensione arteriosa e da alterazioni della funzionalità renale, sono, in genere, di entità lieve o moderata,
reversibili e controllabili con l’aggiustamento posologico. Il
rischio di tossicità può essere minimizzato da un’attenta
selezione dei pazienti, ovvero dall’accurata valutazione del-
579
VENA
le condizioni generali e dei trattamenti concomitanti e dall’esclusione di controindicazioni al trattamento con CsA. È,
altresì, noto che gli eventi avversi associati a CsA sono dipendenti non soltanto dal dosaggio quotidiano ma anche dalla
durata di somministrazione; di conseguenza, nell’approccio alla psoriasi si raccomanda il ricorso a cicli di terapia
intermittente di breve durata. Gli schemi di terapia intermittente finora utilizzati consistono, in realtà, nella somministrazione continua, a cadenza giornaliera, di CsA per periodi di tempo limitato, in genere di circa 12 settimane fino a un
massimo di 24 settimane 5, 8, 9.
Lo scopo di questa esperienza pilota è stato valutare l’efficacia e la tollerabilità di un nuovo schema di terapia intermittente con CsA nella psoriasi severa, effettuato con la
somministrazione di CsA per 4 giorni consecutivi a settimana.
Materiali e metodi
Sono stati selezionati pazienti adulti di entrambi i sessi
affetti da psoriasi a placche grave, definita da un valore del
psoriasis activity and severity index 10 (PASI) di almeno 12.
Durante il trattamento, veniva richiesto l’utilizzo di un adeguato metodo contraccettivo da parte delle donne in età fertile. In tutti i pazienti si è esclusa la presenza di controindicazioni all’uso di CsA: alterazioni rilevanti della funzionalità renale (creatininemia >1,5 mg/dl e/o clearance della
creatinina <60 ml/min/1,73 m3) ed epatica (bilirubina o enzimi epatici di almeno due volte maggiori rispetto al limite
superiore del range di normalità); ipertensione arteriosa non
controllata; infezioni sistemiche; neoplasie pregresse o in
atto; disordini rilevanti di tipo ematopoietico, immune, cardiovascolare e/o neurologico. Un altro criterio di esclusione
era l’uso concomitante di farmaci nefrotossici, di sostanze in
grado di influenzare la psoriasi e di interferire con il metabolismo di CsA. Prima dell’arruolamento, era previsto un
periodo di wash-out da precedenti trattamenti attivi sulla
psoriasi: di almeno 3 mesi per terapie con farmaci biologici, 4 settimane per terapia sistemica, foto- o PUVA-terapia,
e di almeno 2 settimane per terapie topiche.
Dopo aver ottenuto il consenso orale, i pazienti eleggibili venivano inseriti in maniera casuale in uno dei seguenti
gruppi di trattamento secondo un rapporto di 1:1:
— gruppo A: CsA 4 mg/kg/die in maniera continua (ogni
giorno) per 6 mesi;
— gruppo B: CsA 4 mg/kg/die per 4 giorni consecutivi
seguiti da 3 giorni di sospensione, a settimana, per 6 mesi.
In entrambi i gruppi, la dose giornaliera di CsA veniva
assunta in 2 somministrazioni, dopo i pasti principali.
Dopo l’arruolamento, i pazienti erano visitati a distanza di
2, 4 e 6 mesi dall’inizio del trattamento. Visite addizionali,
inclusa una alla fine del primo mese, venivano effettuate a
discrezione del dermatologo e/o del paziente per motivi legati alla risposta clinica e/o alla tollerabilità. Durante le visite
programmate, si eseguivano l’esame obiettivo generale, la
580
misurazione della pressione arteriosa, il calcolo del PASI e
la valutazione dell’intensità del prurito attraverso una scala
semiquantitativa (0=assente; 1=lieve; 2=moderata; 3=marcata; 4=molto marcata).
Per quanto concerne l’analisi statistica, è stato usato il
test di Wilcoxon per dati appaiati al fine di rilevare la significatività (P<0,05) della variazione del PASI e dell’intensità
del prurito nell’ambito di ciascun gruppo. La differenza di
questi indici tra i 2 gruppi, a ogni visita, è stata esaminata con
il test di Mann-Whitney (significatività per valori di P inferiori a 0,05).
I dati relativi alla tollerabilità sono stati riportati in maniera descrittiva e erano desunti dalla segnalazione di eventi
avversi e dal monitoraggio regolare delle funzioni vitali e dei
parametri di laboratorio. Gli esami di laboratorio, eseguiti con
frequenza mensile, includevano: esame emocromocitometrico
completo, creatininemia, bilirubina totale, transaminasi,
fosfatasi alcalina, magnesio, potassio e sodio. Eventuali altre
indagini laboratoristiche o strumentali venivano effettuate in
base alla necessità e in dipendenza dalle condizioni cliniche dei pazienti o dall’esperienza dei dermatologi. Ai pazienti veniva, inoltre, richiesto di misurare periodicamente la
pressione arteriosa e di comunicare eventuali variazioni dei
parametri di laboratorio e dei valori pressori. I valori della
pressione arteriosa registrati nella nostra popolazione sono
stati classificati in conformità alle linee guida del Joint National Committee-6th report (JNC 6).11 L’influenza dei trattamenti sulla pressione arteriosa è stata esaminata valutando la
frequenza di modificazioni rilevanti della pressione arteriosa, confermate da misurazioni ripetute, verificatesi nel corso dello studio. Per uniformare l’analisi dei dati, sono state
considerate rilevanti le variazioni della pressione arteriosa che
comportavano, durante il trattamento con CsA, una delle
seguenti condizioni: sviluppo di ipertensione borderline
(130-139/85-89 mmHg) o di vera ipertensione (≥ 140-90
mmHg) in soggetti precedentemente normotesi; passaggio da
una forma di ipertensione borderline a un’ipertensione conclamata; in soggetti già ipertesi, incremento significativo
dei valori pressori, tale, cioè, da peggiorare lo stadio dell’ipertensione o da richiedere un adattamento della terapia
anti-ipertensiva.
Risultati
La popolazione esaminata era composta da 203 pazienti:
101 soggetti (61 di sesso maschile e 40 di sesso femminile)
con un’età media di 42,8 anni (range: 25-83 anni) nel gruppo A e 102 (57 di sesso maschile e 45 di sesso femminile) di
età compresa tra 27 e 85 anni (età media: 44,6) nel gruppo B.
Nell’arco del periodo di osservazione, 17 pazienti hanno
interrotto prematuramente il trattamento per ragioni di vario
tipo: motivi amministrativi (3 nel gruppo A, 4 nel gruppo
B), eventi avversi (4 nel gruppo A, 2 nel gruppo A) o inefficacia (1 nel gruppo A, 3 nel gruppo B). Soltanto 55 pazienti sono stati sottoposti a valutazione clinica dopo un mese; pertanto, l’esiguo numero di casi valutabili in questa visita
Ottobre 2005
opzionale non ha consentito l’inserimento nell’analisi a fini
statistici.
Le condizioni generali di base dei pazienti reclutati nei 2
gruppi erano simili per tipo e frequenza, eccezion fatta per l’ipertensione arteriosa che risultava invece più frequente nei
pazienti del gruppo B (Tabella I). Dei soggetti ipertesi, 7 nel
gruppo A e 10 nel gruppo B assumevano farmaci anti-ipertensivi. La distribuzione dei valori del PASI tra i 2 gruppi di
trattamento risultava alquanto dissimile, con valori significativamente più alti nei pazienti candidati al regime di terapia continua (30,12±12,74 nel gruppo A versus 24,71±11,44
nel gruppo B, P=0,000). Molti pazienti (il 72% nel gruppo A
e il 68% nel gruppo B) riferivano la presenza di prurito di
variabile intensità; anche questo sintomo risultava più marcato
nei soggetti del gruppo A rispetto a quelli del gruppo B
(1,92±1,15 versus 1,58±1,04, P=0,057). Entrambi gli schemi
terapeutici hanno indotto un significativo miglioramento
(P<0,05) del PASI e del prurito a ogni visita (Tabella II). Una
differenza statisticamente significativa dei valori di PASI tra
i 2 gruppi si è mantenuta nel corso dello studio, mentre si è
persa la significatività statistica per il punteggio del prurito tra
i 2 gruppi sia al 4° mese sia al 6° mese. Una riduzione del PASI
di almeno il 50% rispetto al valore di partenza (PASI50) è stata raggiunta da un maggior numero di pazienti in terapia continua al secondo mese (60% versus 43%) e, nonostante una
tendenza a favore del trattamento continuo in termini di efficacia, le differenze di frequenza del PASI50 tra i 2 regimi
terapeutici si sono gradualmente attenuate con il prosieguo della terapia (Figura 1). Infatti, dopo 6 mesi, una riduzione del
PASI ≥ 75% è stata ottenuta nell’84% trattati in maniera continua e nel 78% dei casi in terapia intermittente.
Non sono state osservate alterazioni clinicamente rilevanti dei parametri di laboratorio, a parte il riscontro di leucocitosi, tra l’altro transitoria, in un paziente del gruppo A.
La frequenza di eventi avversi è stata pari al 31% nel gruppo A e 12% nel gruppo B (Tabella III). Ad eccezione dei
pochi casi che hanno richiesto l’interruzione precoce del
trattamento, nella restante parte dei casi gli eventi avversi sono
stati di entità lieve o moderata, transitori o, comunque, agevolmente controllabili. Nel gruppo A, rispetto al gruppo B,
si sono notati una frequenza lievemente aumentata di disturbi gastrointestinali e, soprattutto, un più frequente riscontro di variazioni rilevanti dei valori pressori (21% versus
7%).
Discussione e conclusioni
L’utilità di CsA nel trattamento di malattie cutanee infiammatorie croniche è ben documentata e confermata da numerosi studi clinici. La maggiore limitazione all’uso di CsA è
il rischio di tossicità, che risulta aumentato in maniera proporzionale alla durata della terapia. Queste considerazioni
generiche sulla sicurezza appaiono ancora più importanti
quando si riferiscono a una condizione cronica, quale la psoriasi, che non mette a repentaglio la vita dei pazienti e che può
colpire soggetti in buone condizioni generali.
Vol. 140 - N. 5
VENA
Numerosi dati indicano che gli eventi avversi indotti da
CsA, in particolar modo quelli a carico del rene, sono dosee tempo-dipendenti 12. La nefrotossicità della CsA è reversibile dopo cicli relativamente brevi; trattamenti prolungati
aumentano il rischio di evoluzione verso un progressivo danno strutturale renale con riduzione stabile della filtrazione glomerulare 13, 14. Per questo motivo, si consiglia di evitare una
somministrazione continua di durata superiore ai 2 anni 9, 12.
Vari studi hanno dimostrato l’efficacia nella psoriasi grave del
trattamento con CsA per un periodo massimo di 12 settimane 15-18. Nella pratica clinica, vengono utilizzati cicli
intermittenti di breve durata, in genere fino a 6 mesi 9, 12,
che consentono il recupero di una normale funzionalità renale 8. Un altro effetto collaterale frequentemente correlato a
CsA è l’ipertensione arteriosa. In pazienti con psoriasi sottoposti a cicli di breve durata con CsA alla dose di 5
mg/kg/die, si evidenzia un significativo aumento della pressione arteriosa con una riduzione lieve e transitoria, non
significativa, del filtrato glomerurale 19.
In realtà, il ricorso a cicli di terapia intermittente con CsA
nella psoriasi è stato finora attuato attraverso la somministrazione continua (giornaliera) per periodi limitati.
In questa esperienza, si sono valutati gli effetti di un nuovo schema terapeutico intermittente consistente nella somministrazione di CsA per 4 giorni consecutivi seguiti da 3
giorni di sospensione a settimana. I risultati indicano che
questo schema causa un significativo miglioramento delle
manifestazioni psoriasiche (PASI) e del prurito e suggeriscono
alcune considerazioni. In primo luogo, se si confrontano i 2
gruppi di trattamento, le caratteristiche basali della psoriasi
non erano uniformi, essendoci stata una tendenza verso l’inserimento di forme più severe nel gruppo candidato alla terapia continua. Allo stesso tempo, questo gruppo includeva
un numero decisamente inferiore di pazienti con valori pressori anomali. Ciò indica che, come talora può accadere negli
studi in aperto, l’inserimento dei pazienti in 1 dei 2 gruppi di
trattamento non sia stato attuato in maniera propriamente
random e che lo schema intermittente sia stato involontariamente preferito per pazienti con forme meno gravi di psoriasi e con condizioni generali meno favorevoli. La distribuzione sbilanciata dei valori basali del PASI e la conseguente disomogeneità delle 2 popolazioni non permettono,
pertanto, un confronto diretto dell’efficacia dei 2 tipi di trattamento. Va, tuttavia, notato che l’intensità del prurito, più elevata nel gruppo A nella visita basale e dopo 2 mesi, è divenuta simile nei 2 gruppi di pazienti alle visite successive
(P>0,05).
Per quanto riguarda la tollerabilità, i dati cumulativi evidenziano che lo schema intermittente sembra essere più vantaggioso. In particolare, a fronte di una maggiore inclusione
di pazienti ipertesi, nel gruppo B, modificazioni importanti
della pressione arteriosa si sono verificate con una frequenza nettamente inferiore. Pertanto, la sospensione infrasettimanale potrebbe migliorare il profilo di tollerabilità di CsA.
Esistono poche segnalazioni relative all’uso di schemi
basati sulla somministrazione intermittente infrasettimanale di CsA nella psoriasi. In uno studio in aperto 20, la CsA,
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5 mg/kg/die, è stata utilizzata di continuo per 4 settimane e,
nella successiva fase di mantenimento, è stata somministrata, allo stesso dosaggio, ogni 4 giorni. In uno studio randomizzato 21, la CsA è stata usata 3 volte a settimana per 12 settimane, dopo una terapia continua d’attacco di 12 settimane.
Goodman et al. 22 hanno studiato gli effetti di 3 dosi settimanali di CsA, assunte a distanza di 12 h l’una dall’altra, per
10 settimane. Il suddetto schema era stato proposto in considerazione del fatto che il ciclo cellulare dei cheratinociti psoriasici è di 36 h; tuttavia, con questo schema venivano osservati importanti effetti collaterali a causa degli alti dosaggi utilizzati (fino a 10 mg/kg).
In conclusione, i risultati ottenuti in questo studio pilota
suggeriscono che un nuovo schema di terapia intermittente
infrasettimanale con CsA (4 mg/kg/die per 4 giorni consecutivi a settimana) è efficace e ben tollerato in pazienti con
psoriasi severa. Sono, tuttavia, necessari studi randomizzati per confermare questi risultati preliminari e, eventualmente, valutazioni in doppio cieco per confrontare l’efficacia e la tollerabilità di questo schema con quello tradizionale continuo.
Riassunto
Obiettivo. Lo scopo di questo studio pilota in aperto è
stato valutare l’efficacia e la tollerabilità di un nuovo schema di terapia intermittente con ciclosporina A (CsA) nella
psoriasi severa.
Metodi. Pazienti adulti con psoriasi a placche grave (PASI
≥ 12) hanno ricevuto 2 diversi schemi terapeutici con CsA alla
dose di 4 mg/kg/die, entrambi della durata di 6 mesi: il pri-
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mo schema si basava sulla somministrazione continua di
CsA (gruppo A), il secondo consisteva nell’uso intermittente di CsA per 4 giorni consecutivi a settimana (gruppo B). I
pazienti sono stati visitati prima della terapia e dopo 2, 4 e 6
mesi; a ogni visita, la gravità delle manifestazioni cliniche era
determinata tramite calcolo del PASI e si valutava l’intensità
del prurito mediante una scala semiquantitativa con punteggio da 0 a 4.
Risultati. I pazienti arruolati sono stati in totale 203: 101
nel gruppo A e 102 nel gruppo B. Il valore basale del PASI
era significativamente maggiore nel gruppo A rispetto al
gruppo B (30,12±12,74 versus 24,71±11,44, P=0,000),
così come l’intensità del prurito (1,92±1,15 versus
1,58±1,04, P=0.057). Allo stesso tempo, la presenza concomitante di ipertensione arteriosa, comunque controllata,
era più frequente tra i pazienti arruolati nel gruppo B (47
soggetti) rispetto a quelli inseriti nel gruppo A (25 pazienti). Entrambi gli schemi terapeutici hanno indotto un significativo miglioramento del PASI e del prurito (P<0,05).
Eventi avversi si sono verificati nel 31% dei pazienti sottoposti a terapia continua e nel 12% di quelli trattati con lo
schema intermittente; in 4 pazienti del gruppo A e in 2 del
gruppo B si è resa necessaria l’interruzione precoce del
trattamento per problemi di tollerabilità. Il rilievo di valori pressori anomali è stato più frequente nel gruppo A (21%
versus 7%).
Conclusioni. I risultati preliminari suggeriscono che questo nuovo schema di terapia intermittente con CsA è efficace nella psoriasi severa e apparentemente meglio tollerato della tradizionale terapia continua.
Parole chiave: Psoriasi, diagnosi - Ciclosporina A - Psoriasi, terapia.
Ottobre 2005
SPECIAL ARTICLES
Psychopatological status and coping strategies
in psoriasic patients:
objectives and methodology of the Psoriasis:
SurveY for the Control of Anxiety
and dEpression (PSYCHAE) study
A. FINZI 1, M. D. COLOMBO 2, A. CAPUTO 3, L. ANDREASSI 4, S. CHIMENTI 5, G. VENA 6,
L. SIMONI 7, S. SGARBI 7, A. GIANNETTI 8 for the PSYCHAE study group*
Psoriasis: SurveY for the Control of Anxiety and dEpression
(PSYCHAE) is a multicentric, observational, prospective study
for evaluation of the psychological impact and strategies for
coping with psoriasis in Italian patients. A cohort of 1 580
patients was enrolled in 39 University Dermatological Departments in Italy. A total of 3 follow-up visits were programmed
after enrollment. Psychological status was evaluated using specific psychometric scales (general health questionnaire [GHQ12], brief symptoms inventory [BSI], and Brief-Cope). GHQ12 was chosen due to its easy use and rapid clinical assessment
of general psychological status. The BSI scale provides a structured and more in-depth analysis of psychological symptoms.
Brief-Cope permits the analysis of cognitive stress regarding
psoriasis. On the basis of the methodology chosen and the large number of patients enrolled, it is expected that the study
will provide a realistic assessment of the psychopathological
Ospedale Maggiore, University of Milan, Milan, Italy
2Novartis Pharma, Milan, Italy
3Department of Psychiatry 34, Milan, Italy
Le Scotte Hospital, University of Siena, Siena, Italy
Tor Vergata Hospital, Rome, Italy
Ospedale Consorziale, University of Bari, Bari
7MediData Studi e Ricerche, Modena, Italy
Hospital University of Modena, Modena, Italy
*) Participating centers in the PSYCHAE study (order based according to the number of patients enrolled): S. Chimenti, L. Bianchi, Tor Vergata Hospital, University of Rome; C. Veller, M. Gallo, University of Padua; P. Santoianni, A. Baldo, Federico II Hospital, University of Naples; L. Andreassi, L. Flori, Le Scotte Hospital, University of Siena; P. Calzavara Pinton, C. Zane, Spedali Civili, University of Brescia; G. De Panfilis, S. Di Nuzzo, University of Parma; A. Finzi, A. Cattaneo, University of Milan; A. R. Virgili, E. Altieri, S. Anna Hospital, University of Ferrara; V. Rocco, A. Lo Schiavo, Federico II Hospital, University of Naples; G.
Trevisan, F. Kokelj, Cattinara Hospital, University of Trieste; A. Tulli, G. Andreassi, SS. Annunziata Hospital, University of Chieti; R. Caputo, A. Locatelli, Ospedale Maggiore, University of Milan; C. Varotti, F. Bardazzi, S. Orsola Malpighi Hospital, University of Bologna; A. Giannetti, M. Coppini, University of Modena
and Reggio Emilia; L. Marchesi, A. Reseghetti, Ospedali Riuniti of Bergamo, University of Milan; P. Fabbri, L. Amato, University of Florence; G. Micali, F.
Lacarrubba, University of Catania; S. P. Cannavò, C. Manfrè, G. Martino Hospital, University of Messina; K. Peris, R. Cavallaro, S. Salvatore Hospital, University of L’Aquila; M. Pippione, E. Soro, S. Giovanni Battista Hospital, University of Turin; A. Barba, D. Schena, Ospedale Civile Maggiore, University of Verona;
G. Angelini, G. A. Vena, Ospedale Consorziale, University of Bari; D. Cerimele, M. A. Montesu, University of Sassari; M. G. Bernengo, A. Bonvicino, S. Giovanni
Battista Hospital, University of Turin; P. Biggio, M. Zucca, S. Giovanni di Dio Hospital of Cagliari; P. Amerio, A. Garcovich, Sacro Cuore Cattolica University of
Rome; M. Aricò, M. R. Buongiorno, University of Palermo; P. Baracchini, G. Giuliano, University of Pisa; G. Leigheb, E. Zavattaro, University of Piemonte
Orientale; A. Lodi, P. Rossigni, University of Milan; F. Cantuccio, F. Loconsole, Ospedale Consorziale, University of Bari; P. Lisi, S. Simonetti, University of Perugia; G. Panasiti, G. Gallo, Sant'Andrea Hospital, La Sapienza University of Rome; V. De Francesco, F. Favot, Gemona Hospital, University of Udine; F. Drago, E.
Repetto, University of Genova; B. Giannotti, E. M. Difonzo, University of Florence; S. Calmieri, G. Campione, Umberto I Hospital, La Sapienza University of Rome;
A. Offidani, O. Simonetti, Umberto I Hospital, University of Ancona; M. Papini, P. Cecchetti, Santa Maria Hospital of Terni, University of Perugia.
This study was supported by an educational grant from Novartis Italia.
Received: June 14, 2005. Accepted for publication: September 29, 2005.
Address reprint requests to: Prof. A. Finzi, Istituto di Dermatologia, Ospedale Policlinico Maggiore, Università degli Studi di Milano, Via Pace 9,
20122 Milano. E-mail: [email protected]
Vol. 140 - N. 5
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FINZI
PSYCHOPATOLOGICAL ASSESSMENT IN PSORIASIC PATIENTS: METHODOLOGY OF THE PSYCHAE SURVEY
status of patients with psoriasis in Italy. The rationale behind
the choice of the methodology utilized for obtaining objective
results is discussed.
KEY WORDS: Psoriasis - Psychopathology - Epidemiology.
choice of dermatological therapy was decided by the
individual physician and no evaluations were made
concerning therapeutic efficacy.
Study objectives
P
laque psoriasis is one of the most common cutaneous pathologies with an estimated incidence of
1-2% in industrialized countries 1 with concomitant
and potentially grave psychological impact. Recent
studies 2 have indicated that psoriasis can lead to significant loss of self-esteem and psychological confidence. The negative psychological consequences of
psoriasis as evaluated by the Salford psoriasis index
(SPI) and hospital anxiety and depression scale
(HADS) scales gives rise to depression in at least 43%
of patients.3 One relevant aspect that should be underlined is that the impact on the psychological status
does not correlate with severity of disease,4, 5 at least
as evaluated by the PASI index, demonstrating the fact
that even slight to moderate symptoms can have a psychological consequences.
Clinical studies have shown that a greater severity of
disease correlates with a lower compliance of topical
pharmacological treatments.6 Nonetheless, independently of the body surface affected, 40% of patients
with psoriasis are not satisfied with therapeutic strategies.2, 7 Moreover, 32% of these patients believe that therapeutic strategies adopted are not aggressive enough.
This suggests that the type of therapy should consider,
in addition to objective clinical evaluation, eventual
psychological repercussions that the disease may bring.8
Psoriasis: SurveY for the Control of Anxiety and
dEpression (PSYCHAE) is a cooperative, observational, longitudinal study involving the Società Italiana di DErmatologia medica, chirurgica, estetica e di
MAlattie Sessualmente Trasmesse (SIDEMAST)
group comprising 39 University dermatological clinics with the objective of evaluating any correlations
between severity of disease and mental status of
patients with psoriasis. After enrollment of a total of
1 580 patients, the scope of the present report is to
describe the methodology utilized in this study.
Methods
PSYCHAE is a multicentric, observational, longitudinal study comprising 39 University dermatological clinics with ample geographic coverage in Italy. The
584
The objectives of the transversal phase were:
1) Evaluation of the psychological status of patients
with psoriasis using psychometric instruments.
2) Evaluation of the severity of disease.
3) Determination of any correlation between psychological status and severity of disease.
4) Evaluation of eventual relationships between character traits of the curing physician and the patient
management strategies adopted.
The longitudinal phase consisted in 3 follow-up
visits at 3, 6, and 12 months after enrollment and included the following objectives:
1) Evaluation of the incidence of psychopathologies.
2) Evaluation of the principal outcomes of prescribed dermatological therapy (compliance with treatment, tolerance, therapy effectiveness including reevaluation of psychopathological status).
Characteristics of patients enrolled
Both male and female patients aged from 18 to 65
diagnosed with psoriasis (either previously or at the
enrollment visit) were consecutively enrolled among
those who presented to participating specialist centers between April and December 2002. Subjects affected by chronic/degenerative disease, by an obvious
inability to understand the questionnaires, with diagnosed psychological disorders, or those taking major
antipsychotic drugs were excluded from the study.
Instruments for patients assessment
Clinical report forms (CRF) and a predefined set of
questionnaires were used for collection of data. The
CRF was anonymous and contained standardized portions for clinical and family history, diagnosis, and
therapeutic strategy. The severity of psoriasis was carried out by evaluation of the body surface area (BSA)
according to the rules of 9 area assessment (RoNAA)
method,9 which provides simple objective results. The
PSYCHAE study had the intention of observing routine clinical practice using valid and applicable instru-
Ottobre 2005
ments, even when not considered as part of the objectives of the present study. Psychological status was
evaluated using 3 different questionnaires compiled
by the patient during the 4 visits (basal and follow-up
visits at 3, 6, and 12 months).
GENERAL HEALTHH QUESTIONNAIRE-12 ITEMS
This questionnaire was compiled by each patient before each of the 4 clinical visits. General health questionnaire-12 items (GHQ-12) is useful for revealing the presence of generic psychological symptoms that are often
associated with more significant psychological disturbances 10 and makes reference to the general health status during the previous weeks to the exam. A total GHQ12 score value of 3 or higher is commonly used as a cutoff identification of psychological sufferance.11-13 According to Piccinelli et al.,14 this threshold value is a good
compromise between sensitivity and specificity.
The choice of this questionnaire was based on several different factors. First and foremost was the necessity of providing a valid screening tool to the specialist regarding the psychological consequences of psoriasis that was also simple for the patient (low number
of items, easy to understand). Secondly, it gave the
curing physician an initial psychopathological status
of the patient.
Brief symptoms inventory
The brief symptoms inventory (BSI) has been used
in several studies for various pathologies to measure
psychological response and distress to physical illness and disability.15 Therefore, in order to provide a
structured and detailed analysis of psychological sufferance of patients with psoriasis, it was decided to
use the BSI at each visit in order to compare the results
with those obtained with the GHQ-12, evaluating the
actual differences in the detection of cases with psychological sufferance in a dermatological setting. The
scale is composed of 53 items in 9 different symptomatic areas including somatization, obsession-compulsion, interpersonal sensitivity, depression, anxiety,
hostility, phobic anxiety, paranoid ideation, and psychoticism and takes into account possible disturbances
that may have been experienced by the patient during
the past 7 days.16 Different modes exist for evaluation
of the BSI according to 4 global indexes (total BSI
index, global severity index [GSI], positive symptom
distress index [PSDI], positive symptoms total
Vol. 140 - N. 5
FINZI
[PST]).17 The simplest index (total BSI index) is the
sum of the points of the 53 items. Dimension scores are
calculated by summing the values for the items included in that dimension and dividing by the number of
items endorsed in that dimension. The GSI is calculated
using the sums for the 9 symptoms dimensions plus the
4 additional items not included in any of the dimension
scores, and dividing by the total number of items to
which the individual responded. It is also possible to
calculate the PST that sums the items with a positive
response or PSDI that sums the values of the items
receiving non-zero responses divided by the PST, this
index provides information about the average level of
distress the respondent experiences. The GSI score is
the most sensitive indicator of the respondents distress
level and combines information about the number of
symptoms present together with their severity. Lastly,
the T-score is the normalized GSI index: a threshold
value of 63 is used as a cut-off in order to define any
eventual psychological sufferance (in the USA).
BRIEF-COPE
One major goal of the PSYCHAE study was to evaluate the therapeutic management of patients with psoriasis. It is of fundamental importance for the physician to understand how the patient copes with psoriasis.
In a cognitive-relational light, coping may be defined as
the set of cognitive and behavioral aspects used to deal
with specific external and internal needs that are viewed
as imposed or as superior to the resources available to
the subject.18 The Brief-Cope questionnaire was adopted for these purposes in an abbreviated version consisting of 28 items in 14 subject areas, each composed of
2 items 19 self-distraction, active coping, denial, substance use, use of emotional support, use of instrumental
support, behavioural disengagement, venting, positive
reframing, planning, humor, acceptance, religion and
self-blame. Some authors have indicated that the awareness of coping strategies adopted by the patient is a key
aspect during the physician-patient interview that might
influence successive therapeutic strategies.20 For the
present study, it was evident that the physician-patient
relationship was not insignificant, not only for evaluation of coping strategies adopted by the patient, but also
to provide the possibility for detection of a relation
between therapeutic strategies adopted by the physician and his character traits. Based on this supposition,
it was decided that the investigator should also compile the Brief-Cope questionnaire during the initial visit.
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FINZI
1 800
1 600
1580
1353
Numero pazienti
1 400
1213
1 200
948
1 000
800
N=815 (52%)
600
400
200
0
Basal
3 (follow-up)
6 (follow-up) 12 (follow-up)
Months
Figure 1.—Number of patients included in the study at basal, 3, 6, and 12
months.
Figure 2.—Percentage of patients included in the study at all 3 and followup visits compared to the number of patients enrolled at the basal visit.
Data management and statistical analysis
prevalence and incidence were considered in the study
population in patients classified as cases with psychiatric sufferance evaluating the status of severity of
psoriasis at the initial visit with the clinical evolution
of the BSA involved. All data were treated according
to the guidelines contained in the respective manuals
for the individual questionnaires.
In order to standardize data collection as far as possible, each center appointed a physician who was in
charge of the individual center’s participation (Investigator). This person oversaw patient enrollment, carried out visits according to established study criteria,
compiled CRFs, and distributed questionnaires according to the study protocol. The Investigator also collaborated with the data elaboration group at MediData for quality control of all data. The CRF were distributed in 2 copies in order to allow one copy to be sent
to MediData Studi e Ricerche, Modena and another
copy to be archived at each center for eventual control
and verification as needed. All data were kept in an
anonymous manner, in full respect of current normatives. Data were periodically sent to MediData and
inserted in a single database containing the entire
patient cohort and periodically subjected to appropriate quality control in order to reveal the presence of
illegible data, inconsistencies, and missing values.
The control procedures were logged in a specific report
that was sent after data lock at predefined study phases to each participating center in order to reveal potential problems. Any data corrections were therefore
carried out using predefined procedures.
Statistical analyses were carried using the SAS statistical software package (SAS Institute). Analysis of
CRFs was carried out according to the methodology
recommended by the Advisory Board of the SIDEMAST group and was principally aimed at the evaluation of the project objectives. Concerning the scales and questionnaires employed in the study, both the
586
Current study status
A cohort of 1 580 patients suitable for evaluation was
enrolled (Figure 1). Of this large number of cases,
data are available relative for 1 459 GHQ-12 (92.3%),
1 299 BSI (82.2%), and 1 108 Brief-Cope (70.1%)
questionnaires. At present, the study has been concluded with the third follow-up visit at 12 months. A
total of 815 patients finished the study per protocol
having completed all 3 follow-up visits (Figure 2).
The compliance regarding the compilation of the questionnaires was 73% for the GHQ-12 and 55% for the
BSI, considering only those patients who finished per
protocol (Figure 3).
Conclusions
On the basis of the large number of patients enrolled and because of the high number of questionnaires compiled, it is concluded that the study can be
expected to provide a realistic assessment of the psychopathological status of patients with psoriasis in
Italy. In particular, due to the psychometric scale cho-
Ottobre 2005
100
Compliance (%)
80
60
40
20
0
Basal
Follow-up
GHQ-12
BSI
Figure 3.—Compliance with compilation of the GHQ-12 and BSI questionnaires at all 3 follow-up visits.
sen, we obtained a high compliance frequency at the
basal visit that was sustained to acceptable levels after
3 follow-up visits, confirming the validity of the study
and the possibility to obtain sound data.
The PSYCHAE study is complementary to previous
studies wherein other authors have indicated weak
points, especially regarding the diverse strategies adopted by patients with psoriasis, in contrast to what observed in other chronic pathologies.5 The compilation of
the Brief-Cope questionnaire by the curing physician
renders the study unique and for the first time will
permit a scientific evaluation of the possible relationship between character traits of the curing dermatologist and therapeutic strategies adopted, with respect to
the severity of disease and/or psychological status.
References
1. Rea JN, Newhouse ML, Halil T. Skin disease in Lambeth. A community study of prevalence and use of medical care. Br J Prev Soc Med
1976;30:107-14.
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2. Williamson D, Gonzalez M, Finlay AY. The effect of hair loss on
quality of life. J Eur Acad Dermatol Venereol 2000;15:137-9.
3. Richards HL, Fortune DG, Griffiths CE, Main CJ. The contribution
of perceptions of stigmatisation to disability in patients with psoriasis. J Phycosom Res 2001;50:11-5.
4. Kirby B, Richards HL, Woo P, Hindle E, Main CJ, Griffiths CE. Physical and psychologic measures are necessary to assess overall psoriasis
severity. J Am Acad Dermatol 2001;45:72-6.
5. Fortune DG, Richards HL, Main CJ, Griffiths CEM. Patients’
strategies for coping with psoriasis. Clin Exp Dermatol 2002;27:
177-84.
6. Zaghloul SS, Goodfield MJ. Objective assessment of compliance
with psoriasis treatment. Arch Dermatol 2004;140:408-14.
7. Stern RS, Nijsten T, Feldman SR, Margolis DJ, Rolstad T. Psoriasis
is common, carries a substantial burden even when not extensive, and
is associated with widespread treatment dissatisfaction. J Investig
Dermatol Symp Proc 2004;9:136-9.
8. Jonckheere P, Bourlond A, Grazian N. The bifocal approach to psoriasis. G Ital Dermatol Venerol 2000;135:139-45.
9. Colangelo PM, Welch DW, Rich DS, Jeffrey LP. Two methods for
estimating body surface area in adult amputees. Am J Hosp Pharm
1984;41:2650-5.
10. Goldberg DP. The detection of minor psychiatric illness by questionnaire. Oxford: Oxford University Press; 1972.
11. Bellantuono C, Fiorio R, Zanotelli R, Tansella M. Psychiatric screening in general practice in Italy. A validity study of the GHQ (General Health Questionnaire). Soc Psychiatry 1987;22:113-7.
12. Lattanzi M, Galvan U, Rizzetto A, Gavioli I, Zimmermann-Tansella
C. Estimating psychiatric morbidity in the community. Standardization of the Italian versions of GHQ and CIS. Soc Psychiatry Psychiatr Epidemiol 1988;23:267-72.
13. Picardi A, Abeni D, Renzi C, Braga M, Puddu P, Pasquini P. Increased psychiatric morbidity in female outpatients with skin lesions on
visible parts of the body. Acta Derm Venereol 2001;81:410-4.
14. Piccinelli M, Bisoffi G, Bon MG, Cunico L, Tansella M. Validity and
test-retest reliability of the Italian version of the 12-item General
Health Questionnaire in general practice: a comparison between three
scoring methods. Compr Psychiatry 1993;34:198-205.
15. Piersma H, Reame W, Boes J. The Brief Symptom Inventory as an outcome measure for adult psychiatric inpatients. J Clin Psychol
1994;50:555-63.
16. Derogatis LR, Melisaratos N. The Brief Symptom Inventory: an introductory report. Psychol Med 1983;13:595-605.
17. Derogatis LR. Brief Symptom Inventory. Baltimore, MD: Clinical
Psychometric Research; 1975.
18. Lazarus RS. Progress on a cognitive-motivational-relational theory of
emotion. Am Psychol 1991;46:819-34.
19. Carver CS. You want to measure coping but your protocol is too long:
consider the brief cope. Int J Behav Med 1997;4:92-100.
20. Griffiths CEM, Richards HL. Psychological influences in psoriasis.
Clin Exp Dermatol 2001;26:338-42.
Impatto psicopatologico e strategie di coping nella psoriasi.
Obiettivi e metodologia dello studio Psoriasis:
SurveY for the Control of Anxiety and dEpression (PSYCHAE)
L
a psoriasi a placche rappresenta una delle più comuni forme di patologia cutanea. Si calcola che nei Paesi industrializzati la psoriasi si presenti con un’incidenza variabile
dall’1% al 2% 1. La psoriasi presenta un considerevole impat-
Vol. 140 - N. 5
to sullo stato psicologico del paziente con conseguenze anche
molto gravi. Recenti studi 2 mostrano come la psoriasi sia causa di una perdita significativa dell’autostima e della sicurezza psicologica nella vita di relazione. Le conseguenze
587
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psicologiche negative della psoriasi, valutate tramite scale
quali il salford psoriasis index (SPI) e l’hospital anxiety and
depression scale (HADS), possono portare all’instaurarsi di
processi di depressione che possono colpire fino al 43% dei
pazienti 3. Un aspetto rilevante da sottolineare, dimostrato da
alcuni studi4, 5, è che l’impatto sul vissuto psicologico del
paziente può non correlarsi con la gravità oggettiva della
malattia, misurata con l’indice PASI, a dimostrazione del
fatto che anche un interessamento di entità lieve-moderata può
avere conseguenze psicologiche molto pesanti.
Studi clinici sperimentali indicano che, maggiore è il grado di disabilità provata dal paziente psoriasico, minore è la
sua compliance al trattamento farmacologico topico 6. Tuttavia, indipendentemente dall’interessamento della superficie corporea, il 40% dei pazienti psoriasici non si ritiene soddisfatto delle cure del medico curante 2, 7. Inoltre, il 32% di
questi pazienti ritiene troppo poco aggressiva la strategia
terapeutica adottata dal proprio medico. Questi dati suggeriscono come la scelta del tipo di terapia antipsoriasica
dovrebbe tenere conto, oltre che delle valutazioni cliniche
obiettive, anche delle ripercussioni psicologiche che la malattia provoca su ciascun paziente secondo un approccio di trattamento bifocale 8.
Lo studio Psoriasis: SurveY for the Control di Anxiety
and dEpression (PSYCHAE) è uno studio cooperativo, di
tipo osservazionale longitudinale, promosso dalla Società
Italiana di DErmatologia medica, chirurgica, estetica e di
MAlattie Sessualmente Trasmesse (SIDEMAST) in 39 cliniche dermatologiche universitarie, con l’obiettivo di valutare la correlazione tra la gravità della malattia e lo stato
mentale del paziente.
Il numero di pazienti arruolati alla visita basale è pari a
1 580. Questo lavoro intende descrivere i metodi e gli strumenti utilizzati nello studio.
Metodi
PSYCHAE è uno studio osservazionale multicentrico,
longitudinale condotto in 39 centri universitari italiani di
Dermatologia, con ampia copertura geografica del territorio
nazionale. Non sono state previste valutazioni di efficacia terapeutica. È stata data libera e assoluta facoltà a ogni Centro
partecipante di scegliere in piena autonomia la terapia dermatologica ritenuta più idonea.
Obiettivi dello studio
Gli obiettivi della fase trasversale dello studio sono:
1) Valutazione dello stato psicologico del paziente psoriasico tramite l’utilizzo di appositi strumenti psicometrici.
2) Valutazione della gravità clinica della malattia.
3) Verifica della correlazione tra stato psicologico e gravità della malattia.
4) Valutazione dell’eventuale relazione tra tratto caratteriale del medico e strategia di gestione del paziente.
588
Lo studio, tramite la valutazione del paziente a 3, 6 e 12
mesi dall’inizio dell’osservazione, intende, inoltre, perseguire i seguenti obiettivi (fase longitudinale):
1) Valutazione dell’incidenza delle psicopatologie.
2) Valutazione dei principali outcome della terapia dermatologica prescritta (compliance al trattamento, tollerabilità, esito, inclusa rivalutazione del quadro psicopatologico).
Caratteristiche dei pazienti arruolati
Sono stati inclusi nello studio i pazienti, di entrambi i sessi, che, consecutivamente e spontaneamente, si sono presentati all’osservazione dello specialista di ogni singolo centro nel periodo compreso tra aprile e dicembre 2002. Sono stati arruolati tutti i pazienti di età compresa tra i 18 e i 65 anni
e una diagnosi (pregressa o concomitante all’arruolamento) di psoriasi. Sono stati esclusi pazienti affetti da gravi
malattie cronico-degenerative e/o caratterizzati da un’evidente incapacità di comprendere i questionari, pazienti che
avessero una pregressa diagnosi psichiatrica certa oppure
facessero uso di antipsicotici maggiori.
Strumenti per la valutazione dei pazienti
Gli strumenti utilizzati per la raccolta dei dati consistono
in una scheda raccolta dati (clinical report forms, CRF) e in
un set predefinito di questionari. La CRF, realizzata in forma cartacea, è strutturata al fine di raccogliere, in forma
anonima e standardizzata, elementi anamnestici, clinici, diagnostici e terapeutici.
La valutazione del grado di severità della psoriasi è stata
effettuata tramite la valutazione della superficie corporea
(body surface area, BSA) secondo il metodo rules di nines
area assessment (RoNAA) 9, uno strumento semplice di
valutazione oggettiva. L’intento dello studio PSYCHAE è, in
effetti, osservare la routine clinica quotidiana, attraverso
strumenti validi e applicabili anche al di fuori del contesto dello studio stesso.
Lo stato psicologico del paziente è stato valutato attraverso 3 differenti strumenti autosomministrati al paziente
in occasione delle 4 visite previste (basale e follow-up a 3,
6 e 12 mesi).
GENERAL HEALTH QUESTIONNAIRE-12 ITEM
Questo questionario è stato somministrato al paziente prima di ognuna delle 4 visite. Il general health questionnaire
12 items (GHQ-12) è utile per rilevare la presenza di generici sintomi psicologici che risultano spesso associati a disturbi psichiatrici più importanti 10. Fa riferimento allo stato di
salute generale del paziente durante le ultime settimane. Il
valore di soglia comunemente utilizzato per l’identificazione di una probabile presenza di qualche forma di sofferenza
psichiatrica corrisponde a uno score totale del GHQ-12 uguale o superiore a 3 11-13. Secondo Piccinelli et al. 14, un valore soglia pari a 3 risulta un buon compromesso tra sensibilità e specificità nell’identificazione dei casi.
Ottobre 2005
La scelta di utilizzare tale tipo di strumento è dettata da
diversi importanti fattori. In primo luogo, era necessario fornire agli specialisti uno strumento di screening delle comorbidità psicologiche nella psoriasi validato e di semplice utilizzo
per il paziente (basso numero di item, facile comprensione).
In secondo luogo, il medico poteva utilizzarlo per una prima
valutazione dello stato psicopatologico del paziente.
BRIEF SYMPTOMS INVENTORY
La brief symptoms inventory (BSI) è utilizzata in numerosi studi e per diverse patologie come strumento volto a
valutare l’evoluzione di una malattia e le conseguenze psicologiche clinicamente rilevanti 15. Pertanto, al fine di fornire un’analisi maggiormente strutturata e approfondita della sofferenza psichiatrica dei pazienti psoriasici arruolati
nello studio PSYCHAE, si è deciso di somministrare la BSI
a ogni visita al fine di poter poi confrontare i risultati con
quanto rilevato dal GHQ-12, valutandone le effettive differenze nella rilevazione dei casi con sofferenze psichiatriche
in ambito dermatologico. La scala, che è autosomministrata dal paziente, risulta composta da 53 item che costituiscono 9 dimensioni sintomatologiche: somatizzazione, ossessività, sensibilità interpersonale, depressione, ansia, ostilità,
ansia fobica, paranoia, psicoticismo. Fa riferimento a possibili
disturbi di cui possa aver sofferto il paziente durante l’ultima settimana, compreso il giorno in cui viene somministrata 16. Esistono diversi modi per valutare il questionario BSI
secondo 4 indici globali (indice totale BSI, global severity
index [GSI], positive symptom distress index [PSDI], positive symptom total [PST]) 17. L’indice più semplice (indice
totale BSI) è la somma dei punteggi dei 53 item. Se tale
somma è calcolata, invece, per dominio, si ottiene uno punteggio specifico per ognuna delle dimensioni della scala. Un
altro indice, detto indice globale di stress (global stress index,
GSI), prevede di rapportare tale somma al numero totale dei
53 item che compongono il questionario. Il GSI è calcolato
sommando le 9 dimensioni sintomatologiche più i 4 item
addizionali non inclusi in nessun punteggio delle dimensioni e dividendo per il totale degli item a cui ha risposto l’individuo. Inoltre è possibile calcolare anche l’indice dei sintomi positivi (PST) come somma degli item con risposta
positiva. Il GSI score è l’indicatore più sensibile della sofferenza psichiatrica, in quanto combina informazioni circa
il numero di sintomi presenti e la gravità del sintomo. Infine il T-score è il GSI normalizzato: il valore soglia di 63 è utilizzato come cut-off per definire un’eventuale sofferenza
psicologica (nella popolazione statunitense).
BRIEF-COPE
Lo studio PSYCHAE ha tra i suoi obiettivi anche la valutazione della gestione terapeutica del paziente psoriasico.
Nella pratica clinica, risulta di fondamentale importanza per
il medico capire in quale modo un paziente affronta la psoriasi, ovvero quali sono le strategie di coping adottate. Il
coping, in un’ottica cognitivo-relazionale, può essere definito
come l’insieme degli sforzi cognitivi e comportamentali per
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FINZI
far fronte a specifiche esigenze esterne e interne che sono vissute come imposizioni o come superiori alle risorse del soggetto 18. Il questionario adottato per tali valutazioni è il BriefCope, una versione ridotta del Cope, costituita da 28 item articolati in 14 scale ciascuna composta da 2 item 19: ristrutturazione positiva, distogliere l’attenzione, espressione, uso
del supporto strumentale, affrontare operativamente, negazione, religione, umorismo, disimpegno comportamentale,
uso del supporto emotivo, uso di sostanze, accettazione, pianificazione e autoaccusa. Da alcuni Autori 20 viene evidenziato che la conoscenza delle strategie di coping adottate
dal paziente è un aspetto chiave dell’intervista medicopaziente che dovrebbe influenzare la scelta di gestione terapeutica successiva. Nell’ambito dello studio PSYCHAE,
volto alla valorizzazione del rapporto medico paziente, è
parso di interesse non trascurabile valutare non solo il coping
del paziente ma anche quello del medico e rilevare, se esiste,
un legame tra le modalità adottate di gestione del paziente e
il suo tratto caratteriale. Basandosi su tali presupposti, si è
deciso di somministrare anche agli investigatori coinvolti il
Brief-Cope durante la visita iniziale.
Elaborazione dei dati e analisi statistica
Al fine di standardizzare il più possibile la rilevazione dei
dati, ogni centro ha incaricato un medico per la conduzione
dello studio. Tale medico arruola i pazienti, effettua le visite secondo i criteri definiti nel protocollo di studio, compila le schede raccolta dati e cura la somministrazione dei questionari secondo quanto definito nel manuale operativo. Infine, collabora con il Centro Elaborazione Dati MediData per
il controllo qualità dei dati raccolti.
Le CRF sono predisposte in forma copiativa per consentire l’invio di una copia al Centro Elaborazione Dati (MediData Studi e Ricerche, Modena) e al contempo mantenere l’originale presso il centro, permettendo successive verifiche e
controlli. Tutti i dati vengono trattati in forma anonima, nel
rispetto della normativa di legge. I dati inviati periodicamente al Centro Elaborazione Dati MediData vengono
immessi nel data base nazionale della casistica aggregata e
periodicamente sottoposti a procedure di controllo qualità
(validazione) per l’individuazione di dati illeggibili, incongruenze e dati mancanti. Tali controlli risultano in una specifica reportistica che viene inviata a chiusura di predefinite fasi dello studio a ciascun centro partecipante affinché
provveda alla risoluzione dei problemi evidenziati. Le correzioni dei dati immessi nel database vengono, pertanto,
effettuate esclusivamente seguendo le indicazioni scritte pervenute dai centri.
Le analisi statistiche sono effettuate tramite il pacchetto statistico SAS (SAS Institute). L’analisi dei dati della scheda clinica è stata effettuata secondo le modalità suggerite dall’Advisory Board della SIDEMAST e, comunque, indirizzando la valutazione agli obiettivi del progetto. Per quanto
riguarda le scale e i questionari utilizzati nello studio, sono
state valutate prevalenza e incidenza, nella popolazione studiata, dei pazienti classificati come «casi con sofferenze psi-
589
FINZI
chiatriche» valutandone al contempo lo stato di severità al
basale della psoriasi e la sua evoluzione clinica nei termini
di BSA coinvolta. I dati sono stati trattati secondo le indicazioni contenute nei relativi manuali d’uso dei questionari.
re. Permetterà, infatti, di tracciare per la prima volta in modo
sistematico e scientifico attraverso una scala validata un profilo delle caratteristiche caratteriali dei medici dermatologi
e di metterlo in relazione, se possibile, con la strategia di
gestione del paziente rispetto alla gravità della malattia e/o
alla sofferenza psicologica.
Lo stato dello studio
La coorte di pazienti arruolati e valutabili al basale è di 1
580 (Figura 1). Su tale ampia casistica sono disponibili i
dati relativi a 1 459 questionari GHQ-12 (92,3%); 1 299
BSI (82,2%) e 1 108 questionari Brief-Cope (70,1%). Attualmente lo studio si è concluso con la terza visita di follow up
a 12 mesi. I pazienti valutabili per tutte le tre visite di follow
up sono risultati pari a 815 (Figura 2). La compliance alla
compilazione dei questionari, considerando come valutabili solo quelli compilati per intero e disponibili per uno stesso paziente a tutte e tre le visite di follow up, è risultata pari
al 73% per il GHQ-12 e pari al 55% per il BSI (Figura 3).
Conclusioni
Sulla base della numerosità del campione effettivamente
arruolato e della compliance alla compilazione dei questionari, è possibile prevedere con ragionevole sicurezza la possibilità di ottenere dallo studio PSYCHAE informazioni
valide ai fini di rappresentare la realtà italiana per quanto
concerne gli obiettivi dello studio. In particolare, grazie
all’utilizzo delle scale psicometriche scelte, è stata ottenuta
un’ottima percentuale di compilazione al basale che si è
mantenuta entro livelli accettabili anche alle 3 visite di follow up, rafforzando i propositi iniziali di mantenere un buon
compromesso tra fattibilità dello studio e informazione scientifica rilevabile dalle stesse.
Lo studio PSYCHAE si inserisce in un panorama scientifico dove già da altri Autori 5 viene evidenziata la carenza
di dati soprattutto in merito alle diverse strategie adottate
dai pazienti psoriasici nei confronti della propria patologia,
al contrario di quanto accade per altre malattie croniche. La
compilazione del Brief-Cope da parte dei medici, riteniamo che contribuisca a rendere lo studio unico nel suo gene-
590
Riassunto
Lo studio Psoriasis: SurveY for the Control of Anxiety
and dEpression (PSYCHAE) è uno studio osservazionale,
multicentrico prospettico, il cui scopo è valutare l’impatto psicopatologico e le strategie di coping in una coorte di pazienti italiani affetti da psoriasi.
Una coorte di 1 580 pazienti valutabili è stata arruolata in
39 Cliniche Universitarie di Dermatologia su tutto il territorio
italiano. Per ogni paziente arruolato al basale erano previste
3 visite di follow-up a 3, 6 e 12 mesi dalla data di arruolamento. Lo stato psicopatologico dei pazienti osservati è stato misurato attraverso l’utilizzo di specifiche scale psicometriche (general health questionnaire [GHQ-12], brief
symptoms inventory [BSI] e Brief-Cope). Il GHQ-12 è stato scelto perché rileva la presenza di generici sintomi psicologici con solo 12 item ed è molto comodo da utilizzare permettendo una visione clinica immediata. La scala BSI è in
grado di fornire un’analisi maggiormente strutturata e
approfondita della sofferenza psichiatrica. Il Brief-Cope permette di analizzare gli sforzi cognitivi e comportamentali
del paziente di fronte al problema «psoriasi» e del medico di
fronte al paziente inteso olisticamente.
Sulla base delle scelte metodologiche qui esposte, oltre
naturalmente alla numerosità campionaria raggiunta, è possibile prevedere con ragionevole sicurezza la possibilità di
ottenere dallo studio PSYCHAE informazioni valide ai fini
di rappresentare la realtà italiana per quanto concerne lo stato psicopatologico dei pazienti affetti da psoriasi. In questo
lavoro vengono esplicitati il motivo di certe scelte metodologiche e gli strumenti necessari per ottenere l’obiettivo desiderato.
PAROLE CHIAVE: Psoriasi - Stato psicopatologico - Epidemiologia.
Ottobre 2005
LETTERS TO THE EDITOR
rythema nodosum is a cutaneous reactive process
due to a hypersensitivity immunologic reaction to
a variety of antigens and may occur in association
with several systemic diseases or drug therapies, or it
may be idiopathic.1-5
We report the case of a 77-year-old man presenting
with multiple erythematous papules, some of them
with a central tense, clear, unilocular vesicles and others with turbid pustules surrounded by a red areola. The
lesions were most numerous on the trunk and on the
face. Vesicles were also present in the mouth, especially on the palate, with multiple small ulcers. Skin
eruption appeared after one day of high fever and
malaise. Varicella was diagnosed and a treatment with
aciclovir was performed. Past medical history was
negative for this infection.
Painful skin eruptions appeared on the legs one week
Figure 1.—Red cutaneous nodule of erythema nodosum on the leg.
Figure 2.—Multiple papules and dry crusts on the trunk following varicella.
Erythema nodosum following varicella
P. ROSINA, M. R. ZAMPERETTI,
F. S. D’ONGHIA, C. CHIEREGATO
Section of Dermatology and Venereology,
Department of Biomedical and Surgical Sciences,
University of Verona, Verona, Italy
Dear Editor,
E
Vol. 140 - N. 5
591
ROSINA
ERYTHEMA NODOSUM FOLLOWING VARICELLA
later. Tender erythematous and painful nodules were
present on the extensor surface of the legs. The border was poorly defined and the lesions varied from 13 cm (Figure 1). The eruptive phase began with fever,
generalized aching and legs’ arthralgia. Dry crusts and
some papules with shallow, pink depression were present in the area affected by varicella (Figure 2).
He was otherwise well, exception made for mild
hypertension in treatment with calcium antagonist
(amlodipine) for 10 years.
A biopsy was performed on a nodule of the leg. Histology showed a septal panniculitis with slight superficial and deep perivascular inflammatory lymphocytic infiltrate. Erythema nodosum was diagnosed.
Throat culture, intradermal skin tests to rule out
tuberculosis, complete blood count, antistreptolysin
titer, renal and liver function tests, urinary analysis
and stool examination were normal. Only ESR was
increased (67 mm/h). A chest X-ray was negative.
Serologic test for varicella-zoster virus infection was
positive for IgM antibodies.
The patient was given only symptomatic relief by
nonsteroidal anti-inflammatory drugs, cool wet compresses, elevation and bed rest. The color changed
in the second week from bright red to bluish. This disappeared within 3 weeks without scarring or atrophy.
In northern and western Europe and the USA the
most common cause of erythema nodosum is streptococcal infection in children and young adult, whereas other infectious processes, drugs systemic diseases
(sarcoidosis, malignant diseases etc.), pregnancy,
autoimmune disorders and inflammatory diseases of
the bowel are the most commonly associated disorder in adults.1-5
Viral causes were rarely reported. Among them
milkers’ nodes, infectious mononucleosis and hepatitis B virus should be mentioned.1 Also some cases of
upper respiratory tract infections without microbiological or serological evidence of streptococcal
pharyngitis could be suspected as viral.5 Our patient
showed the second described association of erythema
nodosum with varicella. Tay reported the first case of
a 16-year-old female who developed a typical erythema nodosum 2 weeks after the onset of varicella.5
In our patient it was also unusual to contract chickenpox in old age but there is a temporal relationship
between infection and clinical manifestations of erythema nodosum. Other known causes of this panniculitis were excluded.
592
References
1. Bondi EE, Margolis DJ, Lazarus GS. Erythema nodosum. In: Freeberg
IM, Eisen AZ, Wolff K, Austen KF; Goldsmith; Katz S et al. editors.
Fitzpatrick’s Dermatology in General Medicine. 5th ed. New York:
McGraw-Hill; 1999. p.1284-6.
2. Hassink RI, Bianchetti MG, Laux-End R. Conditions currently associated with erythema nodosum in Swiss children. Eur J Pediatr
1997;156:851-3.
3. Puavilai S, Charuwichitratanna S, Rajatanavin N. Etiology of erythema nodosum. J Med Assoc Thai 1995;78:72-5.
4. Psychos DN, Voulgari PV, Skopouli FN, Drosos AA, Moutsopoulos
HM. Erythema nodosum: the underlying conditions. Clin Rheumatol
2000;19: 212-6.
5. Tay YK. Erythema nodosum in Singapore. Clin Exp Dermatol
2000;25:377-80.
Address reprint requests to: P. Rosina, MD, Sezione di Dermatologia
e Venereologia, Piazzale Stefani 1, 37126 Verona (Italy).
Eritema nodoso secondario a varicella
Egregio Direttore,
L
’eritema nodoso è un processo reattivo cutaneo dovuto
a una reazione di ipersensibilità immunologica verso
vari antigeni associati a patologie sistemiche, infezioni o
farmaci, oppure di natura idiopatica 1-5.
In questo lavoro viene presentato il caso di un paziente di
sesso maschile di 77 anni, giunto alla nostra osservazione per
la comparsa di papule eritematose multiple, alcune delle
quali presentavano al centro vescicole uniloculari, tese, chiare e altre con pustole a contenuto torbido circondate da un’areola eritematosa. Le lesioni erano numerose a livello del
tronco e del volto; anche la mucosa del cavo orale, specialmente del palato, presentava vescicole con multiple piccole
ulcerazioni. L’eruzione cutanea compariva dopo un giorno di
febbre elevata e malessere. Veniva posta diagnosi di varicella e veniva eseguito un trattatamento con aciclovir. L’anamnesi patologica remota risultava negativa per questa infezione.
Una settimana dopo l’infezione compariva un’eruzione
cutanea a livello della regione estensoria delle gambe rappresentata da noduli eritematosi dolenti con margini scarsamente definiti e con dimensioni variabili da 1 a 3 cm di diametro (Figura 1). La fase eruttiva iniziava con febbre, dolore generalizzato e artralgia agli arti inferiori. Le aree affette
da varicella presentavano lesioni crostose e alcune papule con
una lieve depressione rosea (Figura 2).
Il paziente non presentava altre patologie rilevanti tranne
un’ipertensione di grado lieve in trattamento con calcio antagonisti (amlodipina) da circa 10 anni.
Veniva eseguita una biopsia di un nodulo della gamba e l’esame istologico evidenziava una panniculite settale con scar-
Ottobre 2005
ERYTHEMA NODOSUM FOLLOWING VARICELLA
so infiltrato infiammatorio linfocitario perivascolare sia
superficiale sia profondo caratteristico dell’eritema nodoso
così diagnosticato.
Gli esami bioumorali routinari eseguibili nel caso di eritema nodoso e, in particolare, tampone colturale faringeo, test
intradermico per la tubercolosi, esame emocromocitometrico completo, titolo anti-streptolisinico, test di funzionalità epatica e renale, analisi delle urine e delle feci, risultavano nella norma. Solamente la VES era aumentata (67 mm/h). La
radiografia del torace era negativa. L’esame sierologico per
l’infezione da varicella-zoster virus risultava positivo per gli
anticorpi IgM.
Il paziente veniva sottoposto solamente a trattamento sintomatico con anti-infiammatori non steroidei, con impacchi
umidi freddi e riposo a letto.
Nella seconda settimana il colore delle lesioni nodulari
cambiava da rosso vivo a bluastro e le lesioni si risolvevano
dopo 3 settimane senza lasciare esiti cicatriziali o atrofici.
Nel Nord e Sud Europa e negli USA la più comune causa di eritema nodoso è rappresentata dall’infezione strepto-
Vol. 140 - N. 5
ROSINA
coccica per l’infanzia e per i giovani adulti, mentre altri processi infettivi, farmaci, patologie sistemiche (sarcoidosi,
neoplasie etc.), disordini immunitari e malattie infiammatorie
intestinali rappresentano le più comuni cause associate, per
gli adulti 1-5.
L’eziologia virale viene riportata raramente e, a questo
riguardo, si può ricordare l’infezione del nodulo dei mungitori, della mononucleosi e dell’epatite B1. In alcuni casi di
infezioni delle alte vie respiratorie, in cui non si dimostra nè
microbiologicamente nè sierologicamente una faringite streptococcica, può essere sospettata un’eziologia virale 5. Il
nostro paziente è il secondo caso descritto di eritema nodoso associato a varicella. Tay riporta il primo caso di una
paziente di 16 anni che ha manifestato l’eritema nodoso 2 settimane dopo l’insorgenza di varicella 5.
Nel nostro paziente, risulta anche insolito il fatto che abbia
contratto la varicella in età avanzata, esiste, comunque, una
relazione temporale tra infezione e le manifestazioni cliniche dell’eritema nodoso. Altre cause conosciute di questa
panniculite sono state escluse.
593

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