Cosmetic Dermatology

Transcript

AMMONIO LATTATO "ATTIVATO"
LA RISPOSTA DERMATOLOGICA
ALLE IPERCHERATOSI
Ipercheratosi intrinseca
Ipercheratosi estrinseca
"ACTIVATED" AMMONIUM LACTATE
THE RIGHT REPLY TO HYPERKERATOSIS
Emulsione - Ammonio Lattato 14%
Emulsione - Ammonio Lattato 8%
Ammonio Lattato 7%
Oli i lineari e ramificati - Ammonio Lattato 5%
Moao" U'ìt
localmente 2 volte al dì.
A NEW MAVICEUTICAL®
Derrnatol
'ically tested
• Effective for initial and maintenance therapy (1•2 •3>
Compatible with all the drugs and cosmetics
Formulateci to treat mild-to-moderate inflammatory acne,
indispensable for patients with sensitive skin
CLI NICAL RESUL TS<
12 3
• • '
ACTIVITY CARRIEO OUT BY KERATOTAL ACNE ON THE
LINOLEIC ACIO ANO SQUALANE CONTENTS OF SURFACE
LIPIDS IN SUBJECTS AFFECTEO BY ACNE JUVENILIS
REDUCTION OF SURFACE LIPIOS OURING THE TREATMENT
WITH KERATOTAL ACNE
o....---"•_ 30_ _'--•<_o_oos_wtt.g--'-_...._
_ _ _ __
n • 30
9
4 ,0
3,5
z 6 0 + - - - -- - -- -
~ 3,0
0
~ sot--- - - -
~
!ila:
~ 2,0
=>
40+ - - - -
2,5
~ 1,5
~ 30
8z 1,0
20
~
0,5
10
10
15
gloml
l• untreated
20
•Treated
25
10
Httlmant
30
I
l• squalene • Llnolelc Acld
::) Decreases the Squalene content of
acne affected skin
12
I
::) Reduces excess lipids
EFAITG
z
o
90 + - - - - - - -_,__;___ _ __
~
85
~
8 0 + - - - - - -_,__
J:
z
i:
00
_
_
7 5 + - - - - ---oo--- - - - - 70 +-- - - -- -- -- - - - -
WEEK1
::) Significantly reduces EFA/TG ratio
_ __
WEEK2
::) lncreases skin hydration by 97%
Please see a brief summary of prescribing information on next page --)
BRIEF SUMMARY
KERATOrALACNE"'
THE GENTLE ANTIACNE
TREATMENT WITH
NO-DRUG CONTENT
DESCRIPTION
Keratotal Acne is a special fat-free lamellar
phosphatidylcholine emulsion developed
for the treatment of acne. lt is delivered in a
special phospholipidic-vehicle linoleic acid
rich which contains glicolic acid and salicilic
acid partially neutralized by a special
patented blend of aminoacids
INDICATIONS
Keratotal Acne is indicated for the
treatment of acne. Absolutely necessary as
a cosmetic substitute or support in presummer and summer periods, when
treatment with conventional keratolitic
agents (benzoi! peroxide, retinoic acid,
ecc.) is not recommended. Penetrates
pores to eliminate excess sebum, most
acne blemishes, acne pimples, blackheads
and whiteheads in a short period treatment.
lts continously use helps to prevent the
development of new acne efflorescences
For more information
cali to:
Mavi Sud Sri
V.le dell'Industria 1
04011 Aprilia (Lt)
ltaly
Tel. +39.6.92.86.261
Fax +39.6.92.81.523
E-Mail:[email protected]
URL=http://www.colosseum.it/st81/mavi
ADVERSE REACTIONS
In the first days of application transient
effect such as stinging or itching may be
observed
HOWTOUSE
Twice a day. Before applications cleanse
the skin thoroughly; if stinging occurs,
reduce application to once a day for the first
ten days of treatment
REFERENCES:
1,2 - Data on file Mavi Sud
- M. Ghiczy, H.P. Nissen, H. Biltz (1996) The treatment of Acne Vulgaris by phosphatidilcholine from
Soybeans, with a high conteni of linoleic acid. J. Appl. Cosmetol. 14, 137-145
3
A NEWMAVICEUTICAL®
'•
'
Lip protective with Glycoaminoacids<·>
INDICATIONS
Cosmetic adjuvant in all the forms
of cheilitis and lips dryness caused
by:
Such as
• Retinoids
• • • Cheilitis or chapped lips
• UV rays
• • • Actinic cheilitis (acute and chronic)
•Wind
• • • Allergie cheilitis
• Weather
••• Exfoliative cheilitis
• Environmental pollutants ••• Angular cheilitis
HOW TO USE
Use day and night as a regular lipstick
(o) partially neutralized by a special patented blend of aminoacids
Please see a brief summary of prescribing information on next page
__..,
L
BRIEF SUMMARY
KERATOrAl.LABBRAM
Lip protective with
Glycoaminoacids<->
IN ALL THE DISORDERS OF THE
MUCOCUTANEOUS INTEGUMENT OF
THE LIPS
DESCRIPTION
Keratotal Labbra is a fasta c ti n g, uncoloured
treatment to protect the lips
from premature ageing and
skin cancer due to UV rays.
lt helps to keeps the lips very
moist and well protected
from the dryness caused by
UV, wind, weather and
environment.
INDICATIONS
In all forms of dryness
caused by the use of
retinoids or other drugs, or
by environmental pollutants.
To avoid the premature
lips ageing caused by
UV activity.
ADVERSE REACTIONS
No adverse reactions to the
use of this product are
known.
Far more information call to:
Mavi Sud Sri
V.le dell'Industria 104011 Aprilia (Lt) ltaly
Tel. +39.6.92.86.261
Fax +39.6.92.81.523
E-Mail:mavi @colosseum.it
URL=http://www.colosseum.it/st81 /mavi
(•)partially neutralized by a special
patented blend of aminoacids
HOWTOUSE
Apply as a regular lipstick.
Keratotal Labbra is intended
for round-the-clock use.
DERMATOLOGIA COSMETOLOGICA
A cura di P. Morganti e L. Muscardin
Ed. International Ediemme
Indice 1° Volume
Sezione I Considerazioni Generali
1 Cenni storici
2 La bellezza della figura umana
Sezione Il Fisiologia e Biologia della cute
3
4
5
6
7
Sviluppo della pelle
La struttura della cute
Biochimica e Fisiologia dell'epidermide
Biologia del tessuto connettivo
Sistema Vascolare ed innervazione della cute
Sezione lli La Cute come organo di assorbimento
8 Nozioni basilari sulla permeabilità e sul1'assorbimento
9 Membrane e assorbimento
10 Metabolismo della cute e degli annessi cutanei
Sezione IV Chimica e Chimico-Fisica dei preparati topici
11 Materie prime e principi attivi di uso cosmetologico
12 Emulsioni ed emulsionanti
13 Tensioattivi di uso cosmetico
14 Gli antiossidanti e i fenomeni ossidativi dci grassi
15 Antimicrobici e preservanti cutanei
16 La prorumazione dei cosmetici
17 Chimica e tossicologia dei coloranti
18 Prodotti cosmetici in aerosol
Sezione IX Annessi cutanei e dermocosmesi
30 Ghlandole sudoripare e sebacee
31 Deodoranti e antisudore
32 Struttura e proprietà dei capelli
33 P,etersione, protezione e normalizzazione dei capelli e del cuoio
capelluto
34 Cosmetici decorativi ad effetto duraturo
35Le unghle
36 Prodotti decorativi ad effetto temporaneo superficiale
Indice 3° Volume
Sezione X Seborrea e dermocosmesi
37 Caratteristiche chimico-fisiche e funzioni fisiologiche dcl sebo
38 Produzione e modificazioni del sebo nel sano e nel seborroico
39 Influenza dei trattamenti cosmetologici sui lipidi di superfice del
viso e del capillizio
40 Attività ormonale e ghlandole sebacee
41 Il problema terapeutico dell'acne
42 Possibilità terapeutiche nella seborrea
Sezione XI Melanogenesi e dermocosmesi
43 Il sistema pigmentario
44 Filtri solari, pigmentanti diretti e depigmentanti
Sezione XIl Mucose orali e dermocosmesi
45 La salute della bocca e dei denti
46 Profilassi ed igiene dei denti e della bocca
47 Preparazioni cosmetiche per la cavità orale
Sezione XIIl Prodotti speciali
48 Omeopatia e cosmetici
49 Solmioni per lenti a contatto
50 Cosmetici ipoallergenici
51 Cosmesi su basi naturali
Indice 2° Volume
Sezione V Trattamenti dermocosmetici del viso e del corpo
19 Detersione, protezione e norma1izzazione della pelle
20 La cosmesi per l'uomo
21 Cosmetici per bambini
22 Preparati per il bagno
23 Maschere e peeling
24 I Depilanti
Sezione VI La cute senile
25 Invecchiamento cutaneo
26 Il trattamento della cute senile
Sezione VIl Cosmetici e Psiche
27 Aspetti psicosomatici e somatopsichici in
dermatologia cosmetologica
Sezione VIlI I danni cutanei
28 Patologia cutanea da cosmetici su base immunologica
29 Danni da cosmetici
Sezione XIV Trattamenti estetici correttivi
52 Interventi correttivi di chlrurgia plastica
53 Laserterapia
54 Crioterapia
55 Principi di mesoterapia
56 Ionoforesi
57 [nterventi correttivi di •camouffiage•
Sezione XV Controlli dermotossicologici
58 Valutazione delle materie prime e dei cosmetici finiti
59 Controlli tossicologici delle materie prime e del prodotto finito
60 Cosmetognosia. Funzionalità ed efficacia dei prodotti cosmetici
Sezione XVI Problemi normativi e cli Marketing
61 Nozioni di marketing e di pubblicità
62 Grafica pubblicita.r ia: implicazioni psicologiche
63 Normative di legge sui cosmetici nei vari paesi del mondo
64 La responsabilità civile dei trattamenti cosmetici
65 Giudizio medico-legale dcl danno estetico
INFORMAZIONI PER L'ACQUISTO
Il pagamento di Lit. 120.000 (Centoventimila) per l'acquisto del 1° volume di Dermatologia Cosmetologica pub essere effettuato mediante assegni
di conto corrente o per contanti indirizzandoli a:
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c/c bancario n. 3184/51 Banca di Roma Ag. 1, Aprilia (LT)
O Prenoto fin da ora i volumi 2° e 3°
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Copie n ......................................... del Volume n. 1
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TIMBRO E FIRMA
SpeclftcarecondlzlonidipagamentoefomireN" Codice Fiscale se è richiesta fattura.
Cosmetic Dermatology
Series Editor: P. Morganti
Volume 2
Every day Problems in Dermatology:
The Cosmetic Connection
Editors: P. Morganti, F.J.G. Ebling
Every day Problems in Dermatology:
The Cosmetic Connection is the second addition to the Cosmetic Dermatology Series
This book is comprised of 41 previously unpublished papers dealing with research in various fields
of cosmetic dermatology. The main themes covered are: inter-relationship between drugs and
cosmetic in the skin; the efficacy of, and the raction to, cosmetics; cosmetics in sports and work;
cosmetics in relation to sexuality and pregnancy; and finally, the interconnection existing between
cosmetics and diet. By so comprehensively covering the science of cosmetics, this text is indispensable to those involved in research and development for the cosmetics, toiletries and pharmaceutical
industries. It will also be a great benefit to university and hospital pharmacists and health care professionals entrusted with any aspect of skin care.
CONTENTS (Main Chapters)
Psycological aspects of every day cosmetic dermatology (E. Panconesi)
Cosmetic, drugs and common skin disorder (W. Raab)
Percutaneous absorption and lipids of the elderly skin (J. Wepiene)
Mechanism of solar erythema (E. Quencez, P. Agache)
.
The skin plasticisation effect of a medium chain alpha-hydroxy acid and the use of potentiators (J.C. Hill,
R.J. White, M.D. Banat, E. Mignini)
Analytical problems of cosmetic evaluation resulting from EEC ltalian regulatory procedures (L. Gagliardi, A. Amato)
Kathon C.G.: risk of sensitization (A.C. De Groot)
Methods for evaluating initant - erythematogenic activity irr.cosmetics (A. Sertoli, S. Gimgini, C. Martinelli, M.C. Melli)
Socia! problems related to perspiration: the cosmetic connection (C. Jacobson)
Barriers creams (L.C. Parish)
Evaluation of a new skin barrier providing water and solvent protection (P. Morganti, S.D. Randazzo)
Cosmetology and sexuality in the history of gynaecology (G. Forleo, M. Fraticelli)
Metabolism of steroids in human skin (A. Lanzone, A.M. Fulghesu, F.P. Bellante, A. Caruso, S. Mancuso)
The stucture and permeability of the ora! mucosa (A. Jarret)
Ora! mucosa and dental care problems (E. Benagian)
Vitamins and minerai nutrition in the skin (B . Berra, S. Zoppi, S. Rapelli)
Good manufacturing and quality contro! practices in the cosmetic industry (F. Pocchiari)
Cosmetology and public health (L.Toti)
400 pages about - Hard-bound
Price: U.S. $ 90.00 I in ltaly L. 120.000
Trimestrale di Dermatologia Cosmetologica
Quarterly Review of Cosmetic Dermatology
E DITOR
P. MORGANTI
PhD.
SECRETARY GENERAL
INTERNATIONAL SOCIETY of COSMETIC DERMATOLOGY
Via Innocenzo Xl. 41 • 00165 Roma (ltaly) ·Fax +39-6-63.80.839
ASSOCI ATE EDITOR
S.D. RANDAZZO
M.D.
Professor of DERMATOLOGY
UNIVERSITY OF CATANIA
Via lacona. 7 -95124 Ca1ania (llaly) · Fax +39-95-7159894
ASS ISTANT EDITOR
M.B.JAMES
M.D.
PROGRAM DIRECTOR
INTERNATIONAL SOCIETY of COSMETIC DERMATOLOGY
JAMES CLI NIC
Su ile 1076 Tannery Lane Camden, Maine 04843 USA· Fax 001-407-9972137
SECRETARY EDITOR
M. PASCOLI
Via Innocenzo Xl, 41 -001 65 Roma (ltaly) ·Fax +39-6-92.81.523
EDITOR I AL ADVISORY
BOARO
P. AGACHE
G. BELLOMONTE
W.F. BERGFELD
B. BERRA
R.CAPUTO
O. CARLESIMO
D.CERIMELE
E. CH IACCHIERIN I
i.COTTE
M.A. DINA
G. FABR!ZI
A. FIDANZA
D. GRAFNETTER
J.A.GRAHAM
L. GAGLIARDI
B. GUARNIERI
A.J.JOUHAR
F.H. KEMPER
A.M. KLIGMAN
N. LOPRIENO
S. MADDIN
G. PUGLISI
C.L. MENEGHIN I
t L. MUSCARDIN
N. ORENTREICH
E. PANCONESI
R. PAOLETTI
\V.E. PARISH
L. PUGLISI
W. RAAB
G. RABBIOSI
A.REBORA
V. RIZZA
G. SALVATORE
A. SANNA
P. SANTOIANNI
H. SCHAEFER
t F. SERRI
A. SERTOLI
A. STAMMATI
I. TADDEI
H. TRONNIER
V. VALKOVIC
MD. Prof. of Denmat. Cenire Hosp. Regional de Besançon (F)
CChem. Prof. of Chem., Food Depan lsl. Sup. Sanità • Rome (I)
MD, FACP Cleveland Clinic Ohio (USA)
DSc. Prof. of Biol. Chem. Univ. of Milano (I)
MD. Prof. and Chairman. Depan of Dem1a1. Univ. of Milano (I)
MD.• Prof. and Chairman, Depan. of Denmal. Univ. of Rome (I)
MD. Prof. and Chainman, Depan. of Derma!. Catholic Univ. of Rome (I)
CChem, Prof. and Chairman, Dcpan. Techn. of Commerce Univ. of Romc (I)
DSc. Prof. of Cosmel. !PIL Lyon (F)
MD. Prof. and Chairman, Dcpart. of Phatol. Anat. Catholic Univ. of Rome (I)
MD, Ass. Prof. of Pacdriaiic Dennatologist. Catholic Univ. of Rome ( I)
DSc, Prof. and Chairman. Depan. of Physiol. Univ. of Rome (I)
PhD, lnst. far Clinica! and Exp. Medicine Prague (CS)
B.Sc, PhD. Dep1. Denmatology Univ. of Pennsylvania (USA)
Chainman. Depart. of Phanm. Chem. lsl. Sup. Sanità of Rome (I)
MD. Prof. and Chainnan, Depart. of Dcnnal. Un iv. of Messina (I)
M.B.MRSC Beaconsfield (GB)
MD. Emcritus Prof., Phannacology & Toxicology. Univ. Munster (D)
MD. PhD, Prof. of Denma1ol. Univ. of Pennsylvania Philadelphia (USA)
DSc, Prof. of Genetica Univ. of Pisa (I)
MD, ERCP Clin. Prof. Denmatol. Div. Denmal. Univ. BR. Columbia. Vancouver (C)
CChcm. Dcpart. of Pharmacol. and Tox. Univ. of Catania (l)
MD, Prof. and Chainman, Depart. of Denmal. Univ. of Bari (I)
MD. Emeritus Prof. of Dennal. Cenlre Hosp. Rcgional IDI Romc (I)
MD, Clin. Prof. of Denmat, New York (USA)
MD. Prof. and Chainman. Depan. of Denmat. Univ. of Firenze ( I)
MD, Prof. and Chairman, Depart. of Phannacol. and Tox. Univ. of Milano (l)
MA, PhD. BVSc. Head or Environmental Safety Division. Unilevcr Rescarch Schan brook (GB)
DSc. Prof. of Phanmacognosy Univ. of Milano (I)
MD, Prof. and Chairman, Depan. of Denmal. Univ. of Wien (A)
MD. Prof. and Chainman, Dcpan. of Denmat. Univ. of Pavia (I)
MD. Prof. and Chainman. Depart. of Denmal. Univ. of Genova (I)
PhD. Prof. of Biol. Chem. Univ. of Catania ( I)
CChem, De pan. of Tox icol. Jst. Sup. Sanità of Rome (I)
MD, Prof. and Chainman, Depan. of Microbio!. Catholic Univ. of Rome (I)
MD, Prof. and Chainman, Depan. of Denmat. Univ. of Napoli (I)
Ph.D., Prof. and Scientific Director L'Oreal, Paris (F)
MD. Emeri1us Prof.. Depart. of Denmal. Catholic Univ. of Rome (I)
MD. Assoc. Prof. of Allergie and Occupational Dcrmat. Univ. of Firenze (I)
DSC. Depan. ofToxicol. lnst. Sup. Sanità of Rome (I)
B.Sc., Prof. and Chainnan. Depart. of Pharmacol.Scicnze Univ. of Siena (I)
MD, Emcrilus Prof., Dermatology,. Univ. Winen-Herdecke (D)
Ph.D. Prof. of Physic Ruder Boskovic lnSI. of Zagreb (CRO)
GENERAL INFORMATION
The JOURNAL OF APPLIED COSMETOLOGY is an intemational joumal devoted to publisching originai
papers, reviews and other materiai which represent a useful contribution to research on the skin and on cosmetics.
It is aimed at cosmetic chemists, dermatologists, microbiologists, pharmacists, experimental biologists, toxicologists, plastic surgeons, and ali other scientists working on products which will come into contaci with the
skin and its appendages.
The Joumal is publisched quarterly in English. It is distributed to cosmetic chemists, dermatologists, plastic
surgeons, medicai and pharmaceutical schools, medicai libraries, selected hospitals and research institutions
throught the world, and by subscription to any other interested individuals or organizations. Statements and
opinions expressed are persona! to the respective contributors and are not necessaril y endorsed by the
Editor(s), Advisers, Publishers of Distributors of this Joumal.
COPYRIGHT
Submitted materiai must be the originai work of the autor(s) and must not have been submitted for publication
elsewhere.
By submitting a manuscript, the authors agree that the copyright for their articles is transferred to the publi sher
if and when the article is accepted for publication. None of the conteni of this publication may be reproduced
in whole or in part, translated, stored in a retrieval system, or transmitted or distributed in any form or by any
means (electronic, mechanical, photocopy, recording or otherwise) without the prior written permission of the
Publishers.
Sections of Journal
The following sections will be features of the Joumal:
Originai Laboratory Studies: descriptions of originai investigative laboratory research in cosmetics and related areas.
Special Reports: Items of special interest to the readers, including reports on meetings, societies, legislation, etc.
Generai Articles: scienti fic articles of generai interest to our readers will be considered for publication. These
articles should be concemed with newer developments in such related fields as dermatology, biology, toxicology, etc.
Short Communications: the lenght should not exceed 5 typewritten pages with not more than 3 fi gures
included. Headings ("Materials", "Discussion", etc.) as well as Summaries are to be omitted. If accepted, these
submission will appear in print in a very short time.
Letter to the Editor: comments on Joumal articles are invited as well as brief contributions on any aspects of
cosmetic science. Letters may include figures, and/or references, but brevity is necessary.
Guest Editorials: concise, authoritative, substantiated commentary on specific topics of contemporary interest.
Book Reviews: book and monographs (domestic and foreign) will be reviewed depending on their interest and
value to subscribers. Send materiai for review to the Editor, Dr. P. Morganti. No such materiai will be returned.
Address:
all papers should be submitted to:
Dr. P. Morganti
INTERNATIONAL EDIEMME
Via Innocenzo Xl, 41
00165 Rome - Italy
Tel. 0039/6/393.78.788
Fax. 0039/6/63.80.839
INFORMATION FOR AUTHORS
Papers must be submitted in English. Authors whose mother tongue is not English should arrange for their
manuscripts to be written in proper English prior to submission.
Procedure of Submission of Manuscripts: submit three copies of both the manuscript and ali illustrative
materiai to the above address.
Organization of the Manuscript: investigative studies should be organized as follow: title, abstract page,
introduction, materiai and methods, results, discussion, acknowledgments, references, legend for figures,
tables. Ali pages shou ld be numered consecutively starting with the abstract. The entire manuscript is to be
typewritten, double-spaced, and with 3 cm margins.
Trade names must be capitalized: the common name for compounds may be used if the formai chemical name
as established by intemational convention is given after the first use. Any abbreviations other than those which
are generally accepted must be defined. In the text, references to dual authors will use both sumames throughout. For multiple authors, use the sumames of ali authors at the first reference and only the first author followed by "et al." thereafter. Please mark in the margin of the manuscript the desired position of the figu res and
tables. To allow faster publication only set of proofs will be fumisched to the author including the figures and
tables in their final position.
Title page: list the title, name(s) and degree(s) of author(s), department(s) and institution(s) at which the work
was done, city, state, and postai code. Any preliminary report or abstract of the work should be referred to as a
footnote to the title.
Summary: each paper must be headed by an English language title of not over 70 characters (including spaces) suitable for use as a running head and must also be proceded by an English su mmary not exceeding 300
words typed double-spaced. The summary will include statements of the problem, method of study, results,
and conclusions. Since this summary will be used by astracting journals, it must be self-explanatory a'nd
should not inlcude abbreviations, footnotes, and references.
Footnotes: shou ld be listed consecutively at the bottom of the page on which they fai!, designated by the fo llowing symbols in order *, +, +,§,Il,**, etc.
Key Words: key words for computerised storage and retrieval of information should be incorporated in the
summary.
References: the references have to be abbreviated as listed in the Index Medicus. The style of the references
must conform to the examples given below:
I) Robbins CR, Kellych ( 1970) Aminoacid composition of human hair. Text Res J 40:891 -896
2) Strehler BL (1977) Time, cells and aging 2nd edn. Academic Press, New York
3) Ebling FJ, Rook ( 1972) Ciclic activity of the follicle. In: Textbook of dermatology 11, Blackwell, Oxford, p.
1567-1573.
Illustrations: figures should be numbered consecutively using Arabic numerals Tables should be numbered
consecutively, using Roman numerals. All photographs should be black and white, glossy and unmounted. The
number and size of illustration should be restricted to the minimum needed to clarify the text. Authors requiring extra space for illustrations will be charge accordingly. This is also the case for color illustrations. Ali
figures, photographs, graphs, or diagrams should be submitted on separate sheets.
Animai Experiments: descriptions of animai experiments should include full details of the types of animai
used (inbred, etc.) and the conditions under which they were kept (standard diet, etc.)
Trade Names: ali common cosmetic ingredients should be referred to by their generic names, as indicated in
the latest edition of CTFA Cosmetic Ingredient Dictionary, and the Europ~an Pharrnacopeia. Ifa materials is
not listed, then the trademarked name can be used, with the chemical composition given in footnotes.
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Trimestrale di Dermatologia Cosmetologica
Contents
Originai Laboratory Studies
1
An in vitro selection of new cosmetic active compounds: from screening
tests on monolayered fibroblast culture to efficiency study on 3-D Dermal
Equivalent.
C. A ug ustin . V. Frei. E. Perrier. A. Huc and O. Damour
13
Demonstration of the anti-wrinkle efficace of a cosmetic product.
Correlation between clinica! observations and instrument methods.
J.G. Camarasa, P. Anthoine, M.J. Tribo Boixareu. E. Serra Ba ldrich and L. Aubert
21
Effect of phosphatidylcholine linoleic acid-rich and glycolic acid in acne
vulgaris.
P. Morganti. S.D. Randazzo. A. Giardina, C. Bruno. M. Vincenti and L. Tiberi
33
Role of topical glycolic acid and phosphatidylcholine linoleic acid-rich in
the pathogenesis of acne. linoleic acid versus squalene.
P. Morganti, A. Agostini, C. Bruno and G. Fabrizi
XIX
Book Review
Dermatologie surgery
Principles and practice
Edited by Roenigk & Roenigk's
J Appl. Cosmetol. 15. I- I I (January-March 1997)
AN IN VITRO SELECTION OF NEW COSMETIC
ACTIVE COMPOUNDS: FROM SCREENING
TESTS ON MONOLAYERED FIBROBLAST CULTURE
TO EFFICIENCY STUDY ON 3-D DERMAL
EQUIVALENT
Corinne AUGUSTIN", Valérie FREI ••, Eric PERRIER ••, Alain HUC •• and Odile DAMOUR*.
• Laboratoire des Substituts Cutanés (CNRS-URA 1341). Hòpital Edouard Herriot.
3 Piace d' Arsonval. 69437 Lyon. FRANCE.
•• Coletica. 32 rue Saint Jean de Dieu. 69007 Lyon. FRANCE
Received: January 15. 1997
Key words: Fibroblast; Derma/ Equiva/ent: Efficiency; Cosmetic: In vitro.
Synopsis
The requirement for documentation of effic iency w ith technical data in support of claims attributed
to cosmetic products has been recently introduced by the European Comm unity directi ve concerning cosmetics. The use of monolayered fibroblast c ultu re e nables the fas t acquisition of information, however, this model is oversimplified compared with the in vivo architecture where the cells
are di spersed in the Extracellular Matrix (ECM) network. Consequenlly, the use of 3-D Derma!
Equivale nt model incl uding a collagen-GAGs-chitosan matrix, major components of ECM, popul ated by fibroblasts is a bette r way of studying the effects of new molecules on fibroblast metabolism
and o n ECM synthesis. We present here an in vi tro selection of promising new active compounds
produced by biotechnology. Four biopeptides were preselected after screening the effects of 200
biopeptides on the stimulation of celi proliferation on monolayered fibroblast c ultures. Then, these
fo ur biopeptides were tested on monolayered fibroblast c ulture, both fo r glycosaminoglycan and
protein synthesis. T he most effecti ve one, Milk derived biope ptide, was used for an efficiency study
using a Derma! Equivalent (DE) mode l. T hi s Milk biopeptide was tested on DE by systemic applicatio n ( l.25 % (v/v) in cul ture medium) for 8 days. Then, tota! protein, collagen, g lycosaminoglycan a nd elastin synthesis were measured respectively by radioactive proline incorporation, e lectrophores is followed by densitometry and colo rimetric assays. These in vitro techniques have Iead
to the selection of an active cosmetic compound which could have interesting properties, due to the
significant activation of glycosaminoglycan neosynthesis it is able to induce, for the treatment of
aged and dehydrated sk in.
Riassunto
L'esigenza di una documentazione che attesti l'efficacia dei prodotti cosmetici, insieme a dati tecn ici c he ne supporti no la validità è stata recentemente introdotta dalla dire ttiva della Comuni tà Europea sui cosmetici. L'uso di colture monostrato di fibroblasti permette la rapida acquisizione di informazioni. Tuttavia questo modello è eccessivamente semplificato se paragonato all'archittettura in
An 1n vtfro select1on of new cosmet1c act1ve compounds from screening test on 3-0 Derma/ Equ1valent
vivo, dove le cellule sono sparse nella rete della Matrice Extracellulare (ECM). Di conseguenza,
l ' uso del modello 3-D Dermal Equivalent, c he incl ude una matrice collagene-GAG-chitosano quale
maggiore componente dell 'ECM, popolata da fibroblasti, è un modo migliore per stud iare gli effetti
d i nuove molecole sul metabolismo dei fibroblasti e sulla sintesi della ECM. Viene presentata qui
una selezione in vitro di promettenti nuovi principi attivi prodotti dalla biotecnologia. Sono stati
preselezionati quattro biopeptidi dopo aver analizzato gli effetti di 200 biopeptidi sulla stimolazione
della proliferazione cellulare su colture monostrato di fibroblasti. Questi quattro biopeptidi sono stati poi testati su una coltura monostrato di fibroblasti, sia per la sintesi dei glicosaminoglicani che per
la s intesi proteica. Il più efficace, un biopeptide derivato dal latte, è stato usato per uno studio
sull'efficacia uti lizzando un modello Dermal Equi valent (DE). Questo biopeptide da latte è stato testato sulla DE attraverso applicazioni sistematiche ( 1.25% (v/v) in un medium culturale) per 8 giorni. Il totale proteico, il collagene, i glicosam inoglicani e la sintesi di elasticità sono state poi misurate rispettivamente attraverso incorporazione di prolina radioattiva, elettroforesi seguita da saggi di
densitometria e colorimetria. Queste tecniche in vitro hanno portato alla selezione di un principio attivo che la neosintesi dei glicosaminoglicani è capace di indurre, per il trattamento della pelle invecchiata e di sidrata.
2
C Augustm. V Fre1. E Pemer, A Huc and O Damour
INTRODUCTION
minoglycans-chitosan populated by normai human fibroblasts which neosynthesise an organised extracellular matrix, with striated collagen
fibers surrounding the cells ( 11 , 12, 13).
As biotechnologies allow the production of a
large number of molecules, the most promising
of the m must be preselected prior to evaluation
in in vivo cosmetology tri als.
We present he re in vitro techniques used forselecting new acti ve cosme tic compounds. Four
biopeptides were preselected after screening the
effects of 200 biopeptides on the stimulation of
celi proliferation on monolayered fibroblas t c ultu res. This paper reports the resu lts of tests
using these fo ur biopeptides on both for g lycosam inoglycan a nd prote in synthesis in monolayered fibroblast cultures. The most effective,
Mil k derived biopeptide, was used for an efficiency study using a Dermal Equivale nt (DE)
model.
The increasing interest in cosmetics in recen t
years has led to considerable development of
new technologies. The formula tion of a new cosmetic product involves complex technologies
such as rheology, su rface-active c hemistry,
e mulsion science and biotechnology.
Accord ing to the 6th amendment and following
uptades of the E uropean Community directive
on cosmetic products (93/35/EEC), the manufacture rs of marketed cosmetic formu lat ions
must keep information regarding: a) the assessment of the safety to human health, b) existing
data on undesirable health effects resulting from
the use of the product, and c) proof of the effect
claimed fo r the cosmetic product. This implies
the need to identify and codify experimental tests that are able to de monstrate biological cosmetic activity.
In vitro studies carried out in order to demonMATERIALS ANO METHODS
strate the biological effects of an active cosmetic compound are usually pe1formed on monolayered cultures. Even if today normai human
Tested Biopeptides
Fo ur biopeptides iss ue fro m biotechno logies
fibroblasts, keratinocytes, melanocytes and enwere obtained by fermentation of several prodothelial cells can be maintained in monolayered culture, these conventional models are overteins (Wheat, M il k, Soya) by different micro-ors impl ified and do not fu ll y reproduce the in
ganisms (Coletica, France). Various hydrolysavivo situation because the cells are not in their
tes were obtained according to the micro-orgaphysiological three-dimensional environment,
nism used for the ferme ntation: Wheat I , Milk
I, Soya I and Soya 2.
in which each celi are subjected to celi-celi and
cell-matrix interactions.
Test procedure on
The developme nt of three-d imensional models
Monolayered Fibroblast Culture
allows us to assess these kinds of interactions
but also allows topical applications of the tested
Celi proliferatio n: norma i hu man fibrob lasts
(I O 000 cells/cm 2) were seeded in I 2-wells pi ate
formulation, mimicing the standard cond itions
of use of the finis hed product. Today, a number
(Costar, USA) and were c ultured with culture
of such three-dimensional models have been demedi um containi ng Dul becco's Modified Eave loped, a nd are ab le to reproduce in v itro
g le's Medium DMEM (Life technologie, Franeithe r a multilayered epidermis cultured on vace) supple mented with 2% foe tal calf serum
rious substrates (I, 2, 3), a derma I equivalent (4,
(Boehringer, Mannaheim), 50 µ g/ml streptomy5) or a reconstructed skin (6,7,8). The Dermal
cin, 2 mM L -Glutami n, 100 Ul/ml penicili n
Equivalent (9) developed by our laboratory for
(Biomérieux, France) supplemented with 1.25
pharmaco-toxicological appl ications includes a
% (v/v) of each biopeptide. Contro! fibroblast
Dermal Matrix ( I O) made of collagen-glycosacultures were done in the same conditions in the
3
An m vitro select1on of new cosmet1c octive compounds: tram screening test on 3-0 Derma/ Eqwvolent
absence of biopeptides. After 2 to 4 days of culture, fibroblasts (n=3 wells) were detached by
incubation with trypsine-EDTA (Sigma, USA)
and counted using a Coulter Counter (Coultronics, USA). Tota! proteins assay (14): fibroblasts (23 000 cells/cm 2) were cultured in 75 cm2
flasks for 7 days with a culture medium containing 10% foetal calf serum (FCS) . After this
proliferation step, the cells were cultured for 7
additional days with 5% FCS culture medium
supplemented by 1.25 % (v/v) of each biopeptide except for the contro!. Then, the culture medium was removed, the fibroblast layers rinced
with PBS (Sigma, USA) and harvested by scraping. The cells were lysed with l % Tryton X
100 prior to Micro BCA method to determine
the amount of proteins in the suspension. Glyco saminoglycans assay ( 15): the fibrobla sts
were cultured in monolayer for 14 days as described above. In the collected culture medium,
the g lycosaminoglycans were extracted by ethanolic precipitation, separated by electrophoresis
on cellulose acetate gel in a O. I M pH 5 barium
buffer solution and stained with GAGs-specific
alcian blue. The GAGs were identified by comparison with standards (Hya luronic Acid, Chondro"itin-4-Sulphate, Derm atane and Heparane
Sulphate) and quantified by integrating peak
surfaces. The assay was done in triplicate.
Test procedure on Derma/
Equivalent
Human fo reskin fibroblasts (200 000 cells/cm2)
were seeded into freeze dried Derma! Matrix
made of 72% bovine collagen types I and III,
8% ovine chondro"itin-4-sulphate and 20% chitosan. DEs were cultured in DMEM supplemented with 10% neonata! calf serum, 25 mg/I gentamycin, 100 000 UI/l penicillin, 1 mg/I amphotericin B and 40 mmol/l L-glutamine and 50
mg/I ascorbic acid (Sigma, USA) for 3 weeks
and the medium was changed twice a week.
Mature DEs were cultured for 8 days in a culture medium containing 1.25% (v/v) of selected
biopeptide, except for contro! DEs. At the end
4
of the systemic application period, synthesised
GAGs , tota! proteins and collagenic proteins
and elastin were assayed on collected and freezed culture media and into DEs. Histological
contro!: three DEs were fi xed in Boin 's solution and embedded in paraffin ; 5 µ m sections
were stained with hematoxylin-phloxin-saffron
and examined under a ZEISS IM 35 microscope. MTI Test (16): DEs were incubated with 2
ml of a solution of I mg MTI/ml of PBS. The
reduced MTI dye was extracted with 4 ml of
HCl 0.04N acidified isopropanol (Merk, USA)
and mechanical agitation at room temperature
for 30 minutes. A I 00 µI sample of each extraction was placed in a 96 wells-plate and the absorbance was read at 550 nm on a plate reader
(DYNATECH MR 4000) with 100 µI isopropanol as a blank. Tota! and collagenic proteins assay (17): briefly, on the 7th day of biopeptide
culture, 12 DEs were cultured with culture medium supplemented with 5 µCi/ml of (5-3H)proline for 24 hours. Half of them were used for
determining the tota! incorporateci rad ioactivity
using a B scintillation counter Packard Tri-carb
2 100 TR. The other half (n=6 DEs) was used in
order to measure the radioactivity incorporated
in the non-collageni c proteins, after a specific
degradation of collagen by collagenase (Advance Biofacture, USA). Substracting one from the
others gave the amount of radioactivity incorporated in collagen expressed in radioactivity units
(dis integrations per minutes (dpm) per DE).
Glycosaminoglycans assays: the quantity of glycosaminoglycans synthesised after 5 d ays of
treatment was assayed as described above in the
collected culture medium. Elastin assay ( 18):
the soluble fraction of elastin was assayed using
Fastin elastin kit (B iocolor, Ireland) in the DE
culture medium, sampled on the last day of biopeptide application (n=6 fractions).
C. Augustin. V Frei. E. Perrier. A. Huc and O. Damour.
RESULTS
The study of new biopeptides on the fibroblast
metabolism was divided in 3 steps: (1) Preselection screening of 200 biopeptides on fibroblasts
proliferation cultured in monolayer (2) Evaluation of 4 preselected biopeptides on GAGs and
tota! proteins synthesis by fibroblasts cultured
in monolayer (3) Efficiency study of the most
effective biopeptide on three-dimensional Derma) Equivalent including measurements of the
synthesised tota) proteins, collagen, GAGs and
elastin.
ration rate versus the contro! culture (Student's
test, p<0.01). This effect is more significant after 2 days culture than after 4 days culture as
shown by the respective activation percentages:
81% and 36% for Wheat 1, 76% and 34% fo r
Milk 1, 64% and 50% for Soya I and, 50% and
36 % for Soya 2.
The synthesised glycosaminoglycans released in
the culture medium by fibroblasts treated with
biopeptides versus contro] were measured by
electrophoresis followed by densitometry. The
60
O
Evaluation of 4 biopeptides on
Monolayered Fibroblast Culture
Proliferation rate of fibroblasts cultured in monolayer in the presence of various biopeptides
added to the culture medium (1.25% v/v) was
determinated by celi counting after 2 and 4 days
culture (n=3) and are presented in Fig.1 a.
]
e:
8
]
~
e
"
Hyaluronic Acid
Chondro"1lin·4·Sulphotc
50
•o
Q)
-=
~
"
~
~
30
e:
.Q
o
·~
.:t.
20
o
O
2 doyscultvre
•
4 doys culture
a.I!
10
T
1500 -
T
T
T
Whool
I
Soyo
I
Soyc
2
~
~
E IOCX> -
T
z"
Fig. I b. Synthesised G/ycosaminog/ycans evaluated by
electrophoresis of the culture medium of biopeptidetreated monolayered fibrob/ast culture versus contro/
and expressed as activation % versus contro/ (n=3) .
J
500 -
o- Unlrooled
Contro!
Whool
I
Milk
I
Soyo
I
Soyc
2
Fig. I a. Proliferation Rate of fibroblasts cultured in monolayer according to the different biopeptides added in
the culture medium ( 1.25% v /v) determinated by celi
count after 2 and 4 days culture (n=3).
For ali the tested biopeptides, we observe a significant enhancement of the fibroblast prolife-
results, presented in Fig. l b, were expressed as
percentages of activation versus untreated contrai for each glycosaminoglycan detected in the
culture medium: H yaluro nic Acid (HA) and
Chondro'ltin-4-Sulphate (C4S) . The amounts of
GAGs measured in ali the biopeptide-treated
culture media are significantly increased versus
the untreated contro! (Student's test, p<O.O I ).
Milk l biopeptide is the most effective substance tested regarding the activation percentages of
both HA (39%) and C4S (53%).
The synthesised total proteins were evaluated
5
An 1n vitro select1on of new cosmet1c octive compounds from screening test on 3-0 Derma/ Equivolent
using the Micro BCA method performed on
biopeptide-treated monolayered fibroblast cultures and untreated controls, and are illustrated
in Fig. l c. We observe that ali the tested biopeptides stimulate the tota! proteins synthesis;
however, only Mil k I and Soya 2 biopeptides
induce a significant activation, respectively of
7% and 20%, compared with the untreated contro! (Student's test, p<0.01 ).
ee:
25
]
20
(")
!"I Student s
1
8
test (p<0.01)
~e
:>
Ql
-=
15
~
Fig. 2 Histological contro/ of a mature Derma/ Equivalent
~
e
(X 320)
a
o
(F) Fibroblast (ECM) Extrace//ular Matrix (P) Pore of Dermal Matrix
10
:~
~
o
5
i)'1
Wheot
Soyo
Soyo
I
I
2
Fig. I c. Synthesised Tota/ protenis evaluated after Micro
BCA m ethod pertormed on biopeptide-treated mono/ayered fibrobla st culture versus contro/ and expressed
as activation % versus contro/.
Efficiency study on ThreeDimensional Derma/ Equivalent
(Fig. le)
After the selection of Milk I biopeptide (Hydrakine®, Coletica, France) according to the activation of GAGs synthesis, we wanted to confirrn and validate these resul ts using a 3-D DE
model.
Histological contro! of the Derrnal Equivalent
used for this study is illustrated in Fig.2. Fibroblasts (F) have migrated, proliferated and invaded the porous structure (P) of the Derma) Matrix. We observe a production of neosynthesised
human Extracellular Matrix (ECM) surrounding
each fibroblast.
6
The cellular viability correlated with the number of cells was evaluated by a MTT test performed on control un treated DEs and on Milk I
biopeptide-treated DEs (n=6). The control DEs
absorbances were no t s ignifi cantly differe nt
from those of Milk l biopeptide- treated DEs
(Student's test, p<0,0 1), that means th at there is
the same number of cells in control and biopeptide-treated DEs.
The glycosaminoglycans released into the culture medium were evaluated by electrophoresis
and densitometry of the culture media of biopeptide-treated D Es and contro! DEs (n=6) .
These results expressed as percentages of activ ation ve rs us th e contrai are prese nted in
F ig.3 a. H yaluronic Acid and Chondro'itin-4Sulphate are detected in the culture media of
both treated and contro) DEs. Mil k I biopeptide
induces a significant increase of HA (114 %)
and of C4S (54 %) versus control DEs culture
media (Student's test,p<0.01).
Tota! and Collagenic Proteins synthesised by
the fibroblasts into the DEs were evaluated after
(5-3 H)-proline incorporation and specific degradation by collagenase in biopeptide-treated DEs
C. Augustin, V. Frei, E. Perrier. A. Huc and O. Damour.
15
O
Contro! DE culture medium
•
M;lk l lreoled DE cullure medium
spective activation percentages of 9.5 % and 97
% versus control DEs (Student's test, p<0.05).
Elastin synthesis was evaluated using an Elisa
kit, in biopeptide-treated DEs culture medium,
and contro! DEs culture medium (n=6) and is il1ustrated in Fig.3c. A significant increase of
64% in elastin synthesis is observed in the presence of the biopeptide (S tudent's test, p<0.01).
150
E'
:::>
'i5
Q)
E
Hyoluronic Acid
Chondro'ilin·4·Sulphate
~
.2
:;
100
V
Fig. 3o. Synthesised Glycosominoglycons evoluated by
e/ectrophoresis of the culture medium of biopeptidetreoted DEs versus contro/ and expressed as octivotion %
versus contro/ (n=6).
:i
8
1
.:::::.
O'>
2:
2
e
50
e
·s
u::;
20000
UJ
o
--....
2
15000
Contro/ DE
Milk l treoted
culture medium
culture medium
:::>
.Ee
Fig. 3c. Synthesised Elastin evoluoted by colorimetry in
biopeptide-treated DEs culture medium ond contro/ DEs
culture medium (n=6) .
~
Q)
o..
"'oe
10000
·e
]'
DISCUSSION
e
·;;;
i:S
5000
Totol protcins
Collogen
Fig. 3b. Synthesised Toto/ ond Co/logenic Proteins evo/uoted ofter (5-3H)-proline incorporotion ond specific degradotion by collagenase in biopeptide-treoted DEs versus contro/ DEs (n=6).
versus contro( DEs (n=6). The results are expressed as percentages of activation versus untreated control and are illustrated in Fig.3b.
Milk I biopeptide has a significant activation
effect on total protein synthesis and especially
on collagenic proteins synthesis as shown by re-
Manufactu rers will increas ingly have to take
into account a recent directive from the European Community which requ ires that the claimed properties of the cosmetic products be supported by technical data and documentation demonstrating efficacy. In vitro techniques will
play an increasing role in the development of
active cosmetic compounds, because they represent an interesting alternative to tests on animals
or humans. On the other hand, biotechnology
now allows the production of numerous molecules that could have important effects in cosmeto logy. However, these new mo lec ul es
should be selected using a well defined strategy.
7
An in vitro setection of new cosmetic octive compounds: from screening test on 3-D Derma/ Equivolent
In this paper, we present the in vitro techniques
used for selecting biopeptides, produced by biotechnology, which are able to stimulate neosynthesis of ECM components, and especially
GAGs. As a matter of fact, the decrease in concentration of those molecules during ageing is
spec ially linked with dehydratation of aged
skin. A stimulation of their neosynthesis might
be an interesting way of reducing some ageing
effets on ski n structure and properties as the
polyanionic nature of these macromolecules are
responsible for skin turgescence due to their capacity to retain water ( 19).
The choice of the two in vitro models, monolayered fibroblast cultu re and Derma! Equivalent was motivated by the crucial role of fibroblasts in cutaneous ageing mechanism and
dehydrated skin phenomena. Briefly, cutaneous
ageing is characterized by a decrease in the
synthesis of proteins, such as collagens and proteoglycans. Also, a spontaneous and progressive
degradation of the elastic fibers take piace (20)
and a low cellularity develops (21). Consequently, the most visible changes occuring in ageing
skin are in the dermis where the destruction of
the relationship between fibroblasts and the interstitial matrix occurs (22). Fibroblasts play a
pivotal role in the morphogenesis and dynamic
remodelling of the dermis including the synthesis of ECM components and specific enzymes
involved in ECM degradation. Consequently research on skin ageing should focus on fibroblasts, whose role consists in maintaining the matrix structure and fonctionality.
The model used for screening should give rapid
results using relatively easy and inexpensive
analytical techniques. This is the reason why
monolayered fibroblast culture was used for a
preselection of the most promising biopeptides
among about 200 products of initial interest
(Coletica, France) using the criteria of the cellular proliferation rate. Four of these biopeptides
were able to give very interesting results on fibroblast renewal, when c ultured in monolayer.
In this paper, we have caffied out investigations
8
on these four selected biopeptides by checking
if this proliferation stimulation effect is accompanied by an increase of the synthesis of extracellular matrix components such as proteins and
glycosaminoglycans. At this stage, two biopeptides appears to have potential cosmetic properties: Soya 2 biopeptide for its effect on proteins
synthesis stimulation which is the subject of
another study (23), and Milk 1 biopeptide fo r its
significant activation of GAGs synthesis (39%
increase of HA and 53% C4S). The latter was
selected for further efficiency study. The aims
of such study are to comfirm and to improve on
the previous results using more sophisticated
analytical methods for measuring dy n amic
synthesis of ECM components in a DE, where
fibroblasts are in a more physiological environment. In this three-dimensional model, the fibroblasts are non proliferative and surrounded
by their own human neosynthesised extracellular matri x, with a very si mi lar organization to
that in normai dermis (I 2). After treatment of
DEs by Milk I biopeptide, no variation on the
celi number using the MTT viability test was
found versus contro!, and we conclude that the
increase in ECM proteins and GAGs are due to
a real activation of sy nthesis . The collagen
synthesis was significantly increased (97 %) by
Milk I biopeptide treatment. The effect of Milk
I biopeptide on GAGs synthesis was confirmed
as we obtai ned an increase of 114 % of Hyaluronic Acid and 54 % of Chondroi:tin-4-Sulphate
synthesis compared with the contro! DE. Moreover, the presence of the Milk l biopeptide in
the culture medium induced an elastin synthesis
activation of 64%. Elastin is a macromolecule
characterized by its high physical and chemical
strength g iving suppleness and plasticity to the
skin. During derma! ageing, there is a spontaneous and progressive degradation of the elastic
fibers. This phenomenon is attributed to a reduced synthesis of elastin molecules and also to a
concomitant increase in the fiber degradation
susceptibility by proteases (24).
Cutaneous ageing is an insidious and progressi-
C. Augustin. V Frei E Pemer. A Huc and O. Damour.
ve degenerative process, inevitable in course
and predictable in outcome. However, cosmetic
active compounds can slow down the normai
and photoinduced ageing phenomena and can
improve extemal aspects of aged skin. Indeed,
modem cosmetology proposes various active
compounds such as alpha-hydroxy acids, ceramides or actives extracted from seaweed or
plants with different mechanisms of action. Today, biotechnologies open a way to the discovery of a wide range of powerful and innovative
cosmetic active compounds like the Milk I biopeptide studied in this paper. New processes based on fermentation by different micro-organisms characterized by their highly developed
enzyme systems, give rise to radically different
peptides than those obtained with conventional
chemical method. The biopeptides produced in
this way are highly specific and are structural
analogues of celi mediators like cytokines. They
might be recognised by biological receptors and
can induce biological effects. Moreover, the
biopeptides selected for this study are very
small molecules that penetrate deeper in the
skin than macromolecules such as those are present in conventional cosmetic filmogene substances. Consequently, Milk I biopeptide is a
radically innovative cosmetic moisturizing
agent, because it induces the endogenous restauration of the moisturizing potential of the skin
by stimulating the neosynthesis of glycosaminoglycans.
study and prove the efficiency of new cosmetic
molecules.
ACKNOWLEDGEMENTS
This work was supported by Coletica, DRET
n° 95060, CNRS and Hospices Civils de Lyon.
Thanks to Jim Torbet for reading th is
manuscript.
CONCLUSION
Modem cosmetology requires the detailed testing of efficacy together with the use of new
and origina] active compounds. Biotechnologies
are very effective in the production of innovative active compounds. However, the wide range
of products need to be evaluated with fast and
accurate methods allowing the selection of the
most promising molecules. Consequently, the
use of in vitro models are going to be increased
in the future to ensure the safety (25) and to
9
An in vitro selection of new cosmetic oct1ve compounds: from screening test on 3-0 Derma/ Equivolent
REFERENCES
1) Tinois E, Tollier J, Gaucherand M, Dumas H, Tardy M, Thivollet J (1991) In vivo and post
transplantation differentiation of keratinocytes growth on the human type IV collagen film of
bilayered derma) substitute. Exp.Cell Res. 191: 310-319.
2) Cannon L, Neal J, Kubilus J, Klausner M (1993) New epidermal model for derma) irritancy
testing. Symposium "Current trends in in vitro Skin Toxicology and Eye Irritancy testing".
Ottawa, Canada.
3) Rosdy M, Clauss C (1990) Terminal epidermal differentiation of human keratinocytes grown
chemically defined medium on inert filter substrates at the air-liquide interface.
J. lnvest. Dermatol. 409-414.
4) Naughton K, Jacob L, Naughton A (1989) A physiological skin model for in vitro toxicity
studies. In "in vitro toxicology: mechanisms and new technology".
Goldberg A. Ed. New York, Mary Liebert lnc. 183-189.
5) Bell E, lvarson B, Merrill C (1979) Production of a tissue-like structure by contraction of
collagen lattices by human fibroblasts of different proliferative potential in vitro.
Celi Biol. 76: 1274-1278.
6) Laska D, Poulsen R, Horn J, Meador V, Hoover D (1992) An evaluation of TESTSKIN : an
alternative derma! irritation model. In Vitro Toxicol. 5: 177-189.
7) Saintigny G, Bonnard M, Damour O, Collombel C (1993) Reconstruction of an epidermis
on a chitosan cross-Iinked collagen-GAG lattice: effects of fibroblasts.
ActaDerm. Venereo!. 73: 175-180.
8) Shahabeddin L, Berthod F, Damour O, Collombel C (1990) Characterisation of skin
reconstructed on a chitosan-cross-linked collagen glycosaminoglycan matri x.
Skin Pharmacol. 3: 107-114.
9) Augustin C, Damour O (1995) Development of a kit for predicting cutaneous toxicity in vitro
using 3-D derma) equivalent : Phase 1 Reproducibility of derma) equivalent.
J. Cellular Engineering. 1: 58-62.
10) Collombel C, Damour O, Gagnieu C, Marichy C, Poinsignon F (1987) Biomaterials of
collagen, chitosan and glycosaminoglycans; process for preparing them and their application
in human medecine. French patent 8708252, 1987.
European patent 884101948, 1988. US patent PCT/FR/8800303, 1989.
11) Berthod F, Damour O, Collombel C (1993) Collagen synthesis by fibroblasts cultured within
a collagen sponge. Biomaterials. 14: 749-754.
12) Berthod F, Sahuc F, Hayek D, Damour O, Collombel C (1996) Deposition of collagen fibril
bundles by long-term culture of fibroblasts in a collagen sponge. J. Biom. Mat. Res. 32:
87-94.
13) Sahuc F, Nakazawa K, Berthod F, Collombel C, Damour O (1995)
Mesenchymal-epidermal interactions regulate gene expression of type VII collagen and kalinin
in keratinocytes and dermal-epidermal junction formation in a skin equivalent model.
Wound repair and Regenerartion. 93-102.
14) Smith K, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto
E, Groeke N, Olson B, Klenk D (1985) Measurement of protein using bicinchoninic acid.
Anal. Biochem. 150: 76-85.
15) Cappelletti R, Del Rosso M, Chiarugi VP (1979) A new electrophoretic method for the
10
C. Augustm. V Frei. E. Perner, A Huc ond O. Domour.
16)
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19)
20)
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23)
24)
25)
complete separation of ali known animai glycosaminoglycans in a monodimensional run.
Anal. Biochem. 99: 3 11 -3 15.
Mossman T (1983) Rapid colorimetric assay for cellular growth and survival: applicati on to
proliferation and cytotoxicity assay. l. lmmunol. Meth. 65: 55-63.
Diegelman RF, Peterkofsky B (1972) Collage n biosynthesis during connective tissue
development in chick embryo. Deve/op Biol. 28: 443-453.
Winkelman J, Spicer S (1962) Staining with tetraphenylporphinesulfonate in vivo.
Stain technology. 37: 303-305.
Tovard C (1987) Biochemistry of Hyaluronan. Acta Otolaryngology. 442: 7-24.
Montagna W, Carlisle K (1990) Structural changes in aging skin.
81: f. Dermatol. 122: 6 1-72.
West M (1994) The cellular and molecular biology of skin aging.
Arc/1. Dermato/. 130: 87-95.
Pierragi M, Julian M, Bouissou H (1984) Fibroblasts changes in c utaneous ageing.
Virchows Arch. 402: 275-287.
Frei V, Augustin C, Perrier E, Orly I, Damour O, Huc A (1997) Activation of fibroblasts
into equivalent dermis: a way to select proved activities from promising biopeptides.
lnt. J. Cosm. Sci. In press.
Gilchrest A (1984) Aging. J. Am. Acad. Dermatol. 11: 995-997.
Augustin C, Collombel C, Damour O (1997) Use of in vitro dermal equivalent and skin
equi valent kits for evaluating cutaneous toxicity of cosmetic products.
In vitro Toxico/ogy.10: 2 1-29.
11
J. Appl. Cosmetol. 15, 13-20 (January-March 1997)
DEMONSTRATION OF THE ANTl-WRINKLE
EFFICACY OF A COSMETIC PRODUCT.
CORRELATION BETWEEN CLINICAL
OBSERVATIONS ANO INSTRUMENT METHODS.
J,G. Camarasa•, P. Anthoine 0 , M.J. Tribo Boixareu•, E. Serra Baldrich* and L. Aubert 0 •
• Catedra de Dermatologia. Universitat Autonoma de Barcelona.
Hospital del Mar. Passeig Maritim, 25-29.
08003 Barcelona. SPAIN
0
BIOTHERM
Avenue du Prince Héréditaire Albert, MC 98000, MONACO.
Received: December 23, 1996
Key words: anti-wrinkle, photoaging, cosmetic.
Synopsis
Demonstration of the anti -wrinkle efficacy of a cosmetic product was carried out in 41 women presenting with photoagei ng of facial skin. The subjects applied the product to the whole of the face,
twice a day, for 8 weeks. Evaluation of the state of the skin and cutaneous re lief was caJTied out
before the start of treatment and after 2, 4 and 8 weeks of treatment using a clinical score method,
together with instrume nt measurements: replicas of the area around the eye and Image Analysis.
Evaluation by the subjects was recorded. Skin tolerability was also veri fied.
The clinica! observations showed a significant and progressive improvement in cutaneous rel ief as
well as a significant increase in the firmness and cosmetic qualities of the skin during treatment
These results were confirmed by Image Analysis of the replicas: reduction in the number and depth
of the wrinkles, as well as evaluation of product efficacy by the treated subjects.
Riassunto
La dimostrazione dell 'attività anti-rughe di un prodotto cosmetico è stata svolta su 41 donne che
presentano fotoinvecchiamento della pelle del viso. I soggetti hanno applicato per 8 setti mane il prodotto sull'intera superficie de l volto, due volte al giorno. La valutazione dello stato della pelle e del
rilievo cutaneo è stata svolta sia prima dell ' inizio del trattamento c he dopo 2, 4 e 8 settimane di trattamento utilizzando un metodo di punteggio clinico, insie me a misure strumentali: riproduzioni
dell ' area intorno agli occhi e Im magine Ana litica Computerizzata. Le valutazioni dei soggetti sono
state registrate. É stata anche verifica la tolleranza del la pelle. Le osservazioni cliniche hanno dimostrato un significativo e progressivo miglioramento del rilievo cutaneo, insieme ad un aumento significativo del tono e delle qualità cosmetiche della pelle durante il trattamento. Questi risultati sono
stati confermati dalla Immagine Analitica Computerizzata delle riproduzioni: riduzione nel numero e
nella profondità delle rughe, insieme alla valutazione dell 'efficacia de l prodotto data dai soggetti trattati.
13
Demonstrat1on of the ant1-wrinkle eff1cacv of a cosmet1c product Correlat1on between .
INTRODUCTION
Cutaneous ageing is the result of two distinct
processes: intrinsic or chronological ageing and
photoageing which is induced by repeated exposure to UV radiation from the sun ( I, 2, 3). In
exposed areas, particularly on the face, numerous cutaneous alterations are fou nd. In the epidermis, an effect on division (4) and differentiation of keratinocytes leads to cutaneous dryness
and a loss of elasticity in the Stratum Corneum.
In the dermis, a di sorganisation of the fibre
network can be observed with accumulation of
abnormal elastic fibres (5) and degeneration of
collagen (6) which is seen as the appearance of
deep wrinkles and loss of firmness, suppleness
and elasticity of the skin.
Vitreoscilla fi/iformis, a bacterium obtained
from sulphur thermal springs, has been cultivated in vitro by biotechnology (7); recent studies
have shown that an extract of Vitreoscilla filiformis was abie to stimulate the proliferation
of human keratinocytes in vitro (C.M. Lapière
et al, unpublished observations). Its activity has
also been demonstrated in human fibroblast c ultures which it stimulates to proliferate and produce ILIP (J.A. Grimaud et al, unpublished observations), and in cultures of human macrophages where it induces cellular activation and production of ILI b (V. Bayer et al, unpublished observations). In the complex mechanisms of derma! repair, ILI induces proliferation of fibroblasts, probably by the intermediary of the autrocrine production of PDGF (8) which in turn, indirect ly stimulates the production of collagen
(9). ILI p also acts on regulation of elastin expression (I 0) and in the epidermis, it induces
proliferation of keratinocytes ( 11 ).
The properties of Vitreoscilla filiformis observed in vitro suggest a potential in vivo action on
the restructuration of cutaneous tissue and thus
improvement in the relief and state of the skin.
In to order to verify this hypothesis, an extract
of Vitreoscilla filiformis was introduced into a
cosmetic product and its efficacy was evaluated
14
over 8 weeks of treatment in a group of women
presenting c utaneous photoageing of the face.
The evolution of cutaneous relief and the state
of the skin was determined by clinica! observations (scoring method) together with methods of
instrumental analysis of the skin surface (replicas and Image Ana lysis) and by a subjective
evaluation (evaluation of the efficacy by the
subjects in the study).
MATERIAL ANO METHODS
1 - Protocol
An open, non-randomised study was carried out in
subjects presenting facial cutaneous photoageing.
2 - Subjects in the study
43 female volunteers in good health, of Caucas ian origin, aged between 35 and 58 years
(mean age: 44 years) participated in this study.
The subjects had a normai skin and presented
moderate to severe facial photodamage corresponding to degrees 3 to 5 on the photonumeric
scale defined by Larni er et al (12). Ali the
subjects gave their writte n informed consent in
conformity with the ethics of cosmetic experimentation.
3 - Produci
The product studied was an oil in water emulsion containing I% Vitreoscilla filiformis extract together with traditional cosmetic active
ingredients.
4 - Treatment
- Method of product application
The subjects had to apply the product to the
whole of the face, moming and even ing, for 8
weeks. No other cosmetic product was allowed
during the course of the study with the exception of cleansing products.
- Conduct of the study
This study including four contro! visits: before
J.G Comoroso. P. Antho1ne M J Tnbo 801xoreu. E Serro Boldnch ond L Aubert
the beginning of the treatment (TO), after 2
weeks (T 2S), after 4 weeks (T4S) and after 8
weeks of treatment (T8S) . Evaluation of the
state of the skin and cutaneous relief was carried out at each visit A questionnaire concerning the efficacy and cosmetic aspects of the
product was given to the subjects after 4 and 8
weeks of treatment.
5 - Evaluation methods
The subjects had to judge the efficacy of the
product on cutaneous relief (satisfactory or unsatisfactory results obtained), as well as its activity on firmness, softness and hydration of the
skin, according to a scale of O to 4 for each criterion (0: unsatisfactory; 4: satisfactory).
6 - Tolerability
Skin tolerabi lity was evaluated by the investigator after 2, 4 and 8 weeks of treatment.
- Clinica/ observafions: scoring mefhod
7 - Statistica/ Analysis
(13)
A two-tailed Student 's t test on paired series
was used to analyse the differences between the
values obtained before treatment and after 2, 4
and 8 weeks of treatment (clinica! scores and
Image Analysis). The differences were considered to be significant when p < 0.05. The correlation coefficient r and its leve! of significance p
were calculated using linear regression analysis
in order to determine the correlation between
the results recorded by the scoring method and
those obtained by Image Analysis.
Evaluati on of the state of the facial skin was
carried out using clinica! scores defined on four
areas of the face: forehead, crow 's feet area,
cheeks and medio-facial reg ion. The criteria studied were the following: cutaneous relief, suppleness, firmness, hydration, complexion. Each
criterion was evaluated indi vidually by the investigator using a scale of O to 1O (0: negative value for the criterion, IO: positive value for the
criterion). Tue mean of the scores obtained for
the four zones was calculated for each criterion.
- lnsfrumenfal mefhods: replicas and
lmage Analysis
Silfio replicas of the cutaneo us surface (1 4)
were made fro m the chosen zones and delineated precisely in the area around the eye (crow's
fee t area). These replicas were photographed
and analysed qualitatively by o bserving macrophotographs and quantitati vely by Image
Analysis according to the technique described
by Corcuff et al (1 5, 16), using a video-camera
(Cohu) together with a microcompu ter using
Image Analysis software (Quantrides, Monaderm). This technique enabled evolution in the
two parameters characterising relief to be evaluated: the number of wrinkles per unit surface
area (u.s.) and their depth (mm).
- Subjecf evaluafion
The efficacy of the product was evaluated by
the subject after 4 and 8 weeks of treatment.
RESULTS
1 - Early Discontinuations
Two subjects did not present for the contro! visit
at 2 weeks; these subjects were excluded from
the study. No o the r observations concernin g
them were taken into account during analysis of
the res ults. The res ults th erefore include 4 1
subjects who finished the study.
2 - Evolution in cutaneous re/ief
and state of the skin during
treatment
- 2. 1 Clinica/ observafions
The treatment induced significant and progressive improvement in cutaneous relief.
The mean of the scores obtained for ali subjects,
at each contro! visit, fo r each parameter studied,
is given in Table 1. The variations, expressed as
15
Demonstration of the anti-wnnkle eff1cacy of a cosmet1c product Correlat1on between .
Table I
CLINICAL SCORES
(mean +/-standard deviation of the mean, % of evolution
with respect to time TO and p determined using Student's t test)
TO: Before starting the treatment; T2S: After 2 weeks of treatment;
T4S: After 4 weeks of treatment; TSS: After 8 weeks of treatment
CRITERIA
TO
T2S
T4S
T8S
CUTANEOUS
RELIEF
% evolution
p
4.5+/-0.3
5.0+/-0.3
5.8+/-0.3
6.8+/-0.2
+10.2%
< 0.05
+29.0%
< 0.05
+49.4%
< 0.05
FIRMNESS
% evolution
p
5.6+/-0.3
6.4+/-0.3
+ 13.0%
< 0.05
7.0+/-0.3
+23 .5%
<0.05
7.6+/-0.3
+34.6%
< 0.05
SUPPLENESS
% evolution
p
5.2+/-0.3
5.8+/-0.3
+ 11.4%
< 0.05
6.5+/-0.3
+25.2%
<0.05
7.l+/-0.3
+36.5%
< 0.05
HYDRATION
% evolution
p
5.6+/-0.3
6.4+/-0.3
+13.0%
< 0.05
7.0+/-0.3
+23.5%
< 0.05
7.6+/-0.3
+34.6%
< 0.05
COMPLEXION
% evolution
p
5.l+/-0.2
5.9+/-0.2
+15.0%
< 0.05
6.7+/-0.2
+30.7%
<0.05
7.5+/-0.2
+47.3%
<0.05
a percentage with respect to TO (before treatment) as well as the results of the statistica!
comparison with respect to TO, are also given
in the same table. An improvement in cutaneous relief of 1O, 29 and 49% respecti ve! y
after 2, 4 and 8 weeks of treatment was observed.
In the same way, the firm ness of the skin increased significant ly during treatment. The
use of the product also progressively and significantly improved the cosmetic qualities of
the skin: suppleness, hydration, complexion.
16
• 2.2 Qualitative and quantitative
analysis of the rep/icas
The observation of replicas by macrophotographs taken of ali subjects enable a qualitative
visual observation in the progressive improvement of cutaneous relief during treatment to be
made. For example, the macrophotographs of
replicas carried out on two subjects are given in
Figure I (a and b).
The quantitative evolution in cutaneous relief
was determined by Image Analysis of the replicas. Table 2 presents the evolution in the num-
J.G Camarasa. P. Anthome. M.J. Tr1bo 801xareu. E Serra Baldrich and L Aubert
TO
T2S
T4S
T8S
T4S
Figure I o : Visuolisotion of repllcos from crow's feet
areo corried o ut on o womon 50 yeors of oge
T8S
Figure lb: Visuolisotion of rep/icos from crow's feet
areo corried out on o womon 58 yeors of oge
FIGURE I:
Mocrophotogrophs of replicos corried out on two subjects (Fino/ enlorgement: x 10)
Figure lo: subject n° I - Figure Jb: subject n ° 2
TO: Before the start of treotment: T2S: After 2 weeks of treotment; T4S: After 4 weeks of treotment: TBS: After 8 weeks
of treotment;
Table Il
QUANTITATIVE ANALYSIS OF REPLICAS: NUMBER OF WRINKLES PER UNIT
SURFACE AREA AND DEPTH OF WRINKLES IN µM
(mean +/-standard deviation of the mean, % evolution with respect to time TO
and p determi ned by Student's t test).
TO: Before starting the treatment; T2S: After 2 weeks of treatment;
T4S: After 4 weeks of treatment; T8S: After 8 weeks of treatment
TO
T2S
T4S
T8S
NUMBEROF
WRINKLES
per u.s.
% evolution
p
92.9+/- J.8
85.4+/-l .9
81.8+/-2.2
74.6+/-2.6
-8.1%%
< 0.05
-12.0%
< 0.05
-20.0%
< 0.05
DEPTHOF
116.9+/-7 .9
98.l+/-6.7
79.5+/-6. I
6 l.0+/-5.6
-16.1 %%
< 0.05
-32.0%
<0.05
-47.8%
< 0.05
WRINKLES (µm)
% evolution
p
17
Demonstrat1on ot fhe ant1-wnnkle eff1cacy of a cosmet1c produci Correlat1on between .
linear equotion: - 23.67X + 219.70
r--0.99
lineor equolion: · 7.41X + 124.64
p = 0.01
r = · O. 98
150
p s 0.02
100
TO
TO
125
•
Il
:;;
-~-
o1
-=a.
100
JiI
75
~
:ij
80
•
T8S
•
70
T8S
•
5
~
T4S
-B
T4S
50
4
V
'O-§
•
~
~
T2S
•
~~
T2S
•
•
90
6
60
7
4
Scores: cutoneaus relief
Figure 2a: Correlafion between scores (cutaneous re/ief)
and depth of wrinkles.
5
6
7
Scores: cvtoneous relie(
Figure 2b: Correlation between scores (cutaneous relief)
and number of wrinkles.
Figure 2: Correlation between scores and the results obteined by lmage Analysls.
TO: Before the start of treatment; T2S: After 2 weeks of treatment: T4S: After 4 weeks of treatment: TBS: After 8 weeks of
treatment;
ber and depth of the wrinkles fo r ali subjects.
The number of wrinkles was reduced in a significant fashion duri ng the treatment (-8, -12 and 20% after 2, 4 and 8 weeks of treatment respectively) as well as the depth of the wrinkles (- 16,
-32 and -47%).
- 2.3 Correlation between the clinica/
scores and resulfs obtained with lmage
Analysis
F ig ure 2a represe nts correlati on over time
between the mean of the clinical scores (cutaneous relief) and mean of the v·a lues corresponding to the depth of the wrinkles.
The correlation coefficient r = -0.99 is significant at the 0.01 level.
T here also ex is ts a s ig ni fica nt co rre lati o n
between the clinica! scores and the number of
wrinkles per unit surface area (Figure 2b):
r = -0.98, p = 0.02.
- 2.4 Subjective evaluation
A nalysis of the qu es tionn aires given to the
subjects confi rms the efficacy of the product
18
with respect to improvement in cutaneous relief:
in term s of w ri nk le im proveme nt , 92% of
subj ects j udged the result obtained to be satisfactory from the four th week of treatment
onwards. The subjects also noted a good activity of the product on the firmness, softness and
hydration of the skin.
3 - Tolerability
No undesirable cutaneous reaction was observed during use of the product: tolerability was
excellent.
DISCUSSI ON
This study showed the efficacy of the produci
studied based on the most characteristic signs of
photoageing of the face: wrinkles and loss of
fi rmness. The clinical observations showed an
improvement in these criteria and the cosmetic
qualities of the skin during treatment. T hey
were confirmed by the res ults obtained after
quantitative an alysis of replicas: reduction in
the number and depth of wrinkles. The evalua-
J.G. Camarasa. P. Anthoine. M J. Tribo 801xareu. E Serra Baldrich and L Aubert.
tion of the subjects also corroborates the observations made.
The correlation between the clinica! observations and the results recorded using instrumental
methods demonstrates the reliability and value
of the clinica! score method when attributed by
a dermatologist who is expert in the evaluation
of cutaneous relief.
lnsofar as activity of the product is concemed,
ali the results confirm the basic hypothesis, i.e.
that the activity of Vitreoscilla filiforma measured in vitro on cutaneous cells has been verified
in vivo: cellular activation induced by the bacterial extract can induce an increase in renewal
and cellular cohesion in vivo in the epidermis as
well as an increase in density of connective tissue, leading to an improvement in cutaneous relief and the state of the skin.
19
Demonstration of the anti-wrinkle efficacy of o cosmetic product. Correlation between ..
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«Br. J. ofDermatology» 130: 167-173.
13) Costa C., Rilliet A., Nicolet M., Saurat J. H. (1989): Scoring Atopic Dermatitis: the
simpler, the better? «Acta Dermatovener. Stockholm» 69: 41-45.
14) Makki S., Barbenel J /C., Agache P. (1979): A quantitative methodfor the assessment of the
microtopography of lluman skin. «Acta Dermatovener. Stockholm» 59: 285-29 l.
15) Corcuff P., de Rigai J., Lévèque J.L. (1982): lmage analysis of the cutaneous microre/ief,
«Bioengineering and the skin» 4 (1): 16-3 1.
16) Corcuff P., Chatenay F., Lévèque J.L. (1984): A fully automated system to study skin
surface pattems, «lnt. J. Cosmet. Sci.» 6: 167-176,
20
J. Appl. Cosmetol. 15. 21-32 (January-March 1997)
EFFECT OF PHOSPHATIDYLCHOLINE LINOLEIC
ACID-RICH ANO GLYCOLIC ACID IN ACNE
VULGARIS
P.Morgonti '*, S.D.Rondazzo2, A. Giardino', C. Bruno', M.Vincenti' ond LTiberi '
' MAVI SUD S.r.l. Research and Development Aprilia CLT) - ltaly.
• Dept. of Dermatology. Dermatologists Training School. Il University of Naples. ltaly
2
Dept. of Dermatology, University of Catania - ltaly
' Head Dept. of Dermatology. Umberto I Hospital, Siracusa - ltaly
• Physiology lnstitute. University of Urbino - ltaly
Received: December IO, 7996
Key words: Acne, phosphatidylcholine, linoleic acid, skin lipids, skin hydration, glycolic acid,
3C System~.
Synopsis
Severa! studies have highlighted as the concentration of linoleic acid in the sebum and in the
epiderm al lipids seems to be strictly connected to the onset of different pathologies, and among
those of acne.
lt has been found also that the wax ester content of the sebum and of the epidermal acylceramides containg linoleic ac id seems to be inversely proportional to the sebum production.
It has been proven that the acylceramides of the comedones and the skin su rface of acne patients
contain much less linoleic acid than the acylceramides from the skin surface of contro! subjects
(6% versus 45% in the normai physiological state).
Other studies seem to suggest that a localized increase of squalene and oleic acid and contemporary reduction in levels of linoleate esterified to ceramide I should be considered as a causative
factor in comedogenesis and acne.
The present study was conducted on 40 vo luntar patients in order to determine whether the supposed overproduction of oleic acid, squalene and sebum, of an acne-affected skin could be influenced by topica! application of cosmetic emu lsions containing as active ingredients an high
concentration of esterified linoleic acid together with glycolic acid buffered by a special mixture
of aminoacids.
A li the treated patients showed a significant decrease of the free fatty acids/triglycerides ratio
together with decrease of the skin surface I ipids.
From the other hand the obtained clinica! resu lts show that the contemporary use in the same
phospholipidic emulsion of g lycol ic acid partially buffered with aminoacids, of salicilic acid
and chlorexidine digluconate, can effective ly relieve the severity of acne ever since the first
weeks of treatment.
Given the positive results obtained we believe that this cream can be considered as a new cosmeceutical and a useful aid for the normai acne therapies.
21
Effect of phosphot1dylcholme lmole1c oc1d-nch ond glycoilc OCld m acne vulgons
Riassunto
Diversi studi hanno messo in luce come la concentrazione di acido linoleico nel sebo e nei lipidi
dell'epidermide sembri essere strettamente correlata all'insorgere di diverse patologie, tra cui l'acne.
È stato anche scoperto che il contenuto del sebo e delle acilcerammidi epidermiche contenenti acido
linoleico sembrano essere inversamente proporzionali alla produzione del sebo.
È stato provato che le acilcerammidi dei comedoni e la superficie cutanea dei pazienti affetti da acne
contengono molto meno acido linoleico delle acilcerammidi della superficie cutanea dei soggetti
controllati (6% contro il 45% del normale stato fisiologico).
Il presente studio é stato condotto su 40 volontari con lo scopo di determinare se la supposta sovraproduzione di acido oleico, squalene e sebo di una pelle affetta da acne potesse essere influenzata da
applicazioni topiche di emulsioni cosmetiche contenenti come principi attivi un 'alta concentrazione
di acido linoleico esterificato insieme ad acido glicolico tamponato da una speciale miscela di aminoacidi.
Tutti i pazienti trattati hanno mostrato una riduzione sign ificati va del rapporto acidi grassi liberi/trigliceridi, insieme ad una diminuzione dei lipidi cutanei di superficie.
D'altro canto i risultati clinici ottenuti dimostrano che l'uso contemporaneo nella stessa emulsione
fosfolipidica di acido glicolico parzialmente tamponato con aminoacidi, di acido salicilico e di clorexidina digluconato possono migliorare efficacemente la gravità dell 'acne già dalle prime settimane di trattamento.
Dati i positivi risultati ottenuti riteniamo che questa crema possa essere considerata, quale nuovo cosmeceutico, di utile ausilio nelle normali terapie dell'acne.
22
PMorgant1, S D.Randozzo, A Giardina, C Bruno, M Vincenti and L T1ben
INTRODUCTION
Severa! studies have highlighted as the concentration of linoleic acid in the sebum and in the
epidermaJ lipids seems to be strictly connected
to the onset of different pathologies, and among
those of acne,
As it is well-known linole ic acid seems to represent a fundamental e le me nt of ceramide l considered the main repository of essential fatty acids
in the stratum corneum, although it is also esterified to the corneocyte envelope and N-acylated to sphingosine in ceramide 2 ( l-3).
It has been found also that the wax ester content
of the sebum and of the epiderm al acylceramides containg linoleic acid seems to be inversely
proportional to the sebum production (4).
It has been proven that the acy lceramides of the
comedones and the skin surface of acne patients
contain muc h less linoleic acid than the acylceram ides from the sk in surface of co ntro ]
subjects (6% versus 45% in the normai physiological state)( 1,5). Other studies seem to suggest that a loca lized increase of squalene and oleic
acid and conte mporary red uc tions in levels of linoleate esterified to ceramide l should be conside red as a causative facto r in comedogenesis
and acne ( l ,6-8).
The present study was conducted to determ ine
whether the supposed overproduction of oleic
ac id, sq ualene and sebum, of an acne-affected
skin could be influenced by topica! application
of cosmetic e mulsions containing as active ingredients an high concentration of esterified linoleic acid together with g lycolic acid buffered
by a special m ixture of am inoacids (9, 10).
In order to define the activi ty of these emulsions, fo urthy voluntar patients suffe ring from
acne vulgari s were controlled wi th regard to the
tota! quantity of sebum and free fatty acids/triglycerides ratio, and to the tota! numbe r of inflammatory lesions.
Were also controlled the supe rficial skin-lipids
and the skin hydration.These parameters surely
are not normai at the leve! of acne-affected skin.
MATERIALS ANO METHODS
Materials
Vehicle: soybean liposome containing 10% lecithin fraction with 80% phosphatidylchol ine linoleic acid-rich (cream B)
Active ingredients: vehicle + glycol ic acid
buffered to pH 4 .5 by a special blend of aminoacids - chlorexidine dig luconate and salicylic
acid (cream A).
Even if the same veh icle (7) should be considered as an active ingredi e nt having proven of
be ing capable of increasing the presence of linoleic acid to detriment of oleic acid and squale ne, as active ing redients have been utilized
glycolic acid, aminoacid-buffered and salicylic
ac id for their well-k nown keratolytic activi ty
( 13, 14) . Clorexydine digluconate to red uce
excessive presence of propionibacterium acne
and of ali the surface aerobic flora (15).
With this particular formulat ion we intended to
reduce the keratinized Stratum Corneum excess
through the use of salicylic and glycolic ac id;
to diminish the sebum a nd fatty acids producti on through the use of linoleic and buffered
glyco lic acid; to decrease both the aerobic and
the anaerobic flora by usi ng the clorexydine digluconate. The used soybean vehicle is capable
of penetrating through the pilosebaceous follicles dragg ing with itself the used active ingred ients.
Patients
Forthy healthy vo luntee rs of both sexes (20
women, 20 men) wi th an average age of 16±3
years with a mild to moderate acne vulgaris ,
partecipated in this study after providing informed consent. The nature of the study was explained to them in full.
A li patients were required to have a Cunliffe
score of at least 1.0 but less than 4 (11 ).
Exclusion criteria included patie nts with more
tha n fi ve nodules and cysts and patients who
had used topica! antibiotics, retinoids or ben-
23
Effect of phosphat1dylcholine linoleic aCJd-nch and glyco/1c acid in acne vulgans
zoyl peroxide in the past 14 days, systemic antibiotics in the past 30 days, systemic retinoids
in the past 2 years, any other topica! acne treatments including medicated soaps, creams or
make up in the past 7 days, topica! corticosteroids in the past 14 days or systemic corticosteroids in the past 12 weeks.
Test substance
Each patient was supplied with two tubes labelled as cream A and cream B together with a
cleansing cream (Mavigen" Idroschiuma).
Procedure
This was a 12-week, randomized, double-blind,
vehicle-controlled study. The subjects were instructed to apply the test-creams on their face
twice a day for three months, and they were not
allowed to use any other skin care product during the study. Each subject was used as his or
her own contro!; the test creams (A and B)
being applied on a randomized basis, on the right or on the left area of the face.
Moreover they were instructed to apply the
same cream always to the designed site after
washing, first thing in the morning and just
before retiring in the evening. Subjects were
also instructed that only the cleansing cream
supplied to each at the beginning of the study
should be used to cleanse the test area.
Other instructions included that the patients
use no other acne treatment during the study
and not to apply the creams the day of evaluations or wash their face 4 hours before evaluations.
Clinica/ evaluation
Clinica! examinations were performed on the
first day (baseline), and at 2, 4, 6, 8, 1O and 12
week (end of the treatment). Clinica! evaluations included indi viduai lesion counts of open
and closed comedones, papules, and pustules,
and were quantified by separately calculating
them by means of a transparent millimeter grid
of cellophane, according to Morganti et al (16).
24
Biophysical non-invasive
measurements
Measurements were performed, on the 1st day
(baseline), after 2, 4, 6, 8, 1O and 12 weeks,
(end of the treatment), by means of the computerized 3C System (Dermotech, Rome, Italy)
( 17). This instrument measures the surface skin
lipids having absorbed them by a special frosted plastic fo il. The determinations we re
always carried out on fo ur sities of right or left
areas (forehead, cheek, chin and nose) before
valuating the patients for the calculation of inflammatory lesions . To achieve an higher degree of assurance ali evaluations were performed after a 30 minutes acclimatization period
in a room at 21 °C to 22°C and 45% to 50 humidity, even if the 3C System automatically adjusts environmental conditions to 22°C and 50%
relative humidity.
Measurement equipment
Skin surface lipids. The skin surface lipid levels were measured with the 3C System" (Dermotech S.r.l., Rome, Italy) (I 7). Determination
is based on photometric measurement of light
transmission through a skin surface imprint obtained applying to the designed skin area a frosted plastic foil. lt allows adherence of skin lipids in a I cm2 area. The obtained readings are
automatically converted into (µg/cm 2).
Free fatty acids/friglycerides
ratio
The special and protected plastic foil was applied on the four areas with gentle pressure by
20 strokes of a gloved finger and carefully removed. Stratum corneum lipids were extracted
from the frosted plastic foil using chloroform:
methanol (2: I ) for 2 hours at room temperature, the solvent was dried under nitrogen and
the lipids were redissolved in chloroform. Lipid fractions were chromatographycally separated on 0.25mm-thick layers of silica gel into
their individuai lipid classes and were successively separated by plastic foi l contaminants,
P.Morganti. S.O.Randazzo. A. Giardina. C. Bruno. M. Vincenti and L. Tiberi
using solid phase extraction colums, according
to Cav ina et al. (18). All lipid fractions, redissolved in chloroform, were stored at -20°C unti! required.
The isolated triglycerides and free fatty acids
were then quantified separately in order to determine the free fatty acids/triglycerides ratio.
sions imp roved as follows: A treatment (A
cream - active) 33% on average if compared
with the basic values;
B treatment (B cream - vehicle) about 17%.
After three months of treatment improvements
were noticed on ali the lesions counts with the
A cream (active) and they resulted very high,
equa! to about 80% (Tab. I and Fig. 1).
Skin hydration
The hydration of the horny layer was assessed
by measuring electrical capacitance of the skin
surface by means of the 3C System"' ( 17).
When the probe is applied to the skin (recording time 0.5 s), the capacitance is displayed
digi tally in arbitrary 3C units. The results are
expressed as mean values of the measurements
performed on fo ur different ri ght or left sites
(cheek, forehead, chin and nose ).
Statistica/ analysis
Student's test was used in evaluation of ali the
data before and after the treatment period. Ali
the analyses were produced using the SAS statistica! package, version 5. 18 (SAS Institute
Inc., Cary, N.C.). Probabilities less than 0.05
were considered significant.
RESULTS
Clinica/ evaluation
Of the 40 patients enrolled 36 patients (90%)
completed the study. Four patients (3 active
treated, I vehicle-treated) dropped from the
study for the following reasons: two for skin
irritation (active-treated), due probably to glycolic acid, and two for persona! reasons. The
mean results of the clinica! parameters evaluated are shown in Table I and Fig. l.
Both the treatments, cream A and cream B,
had significantly reduced ali the acne Jesion
counts, even if cream A (active) has proven
more effective than cream B (vehicle) and having a q uicker activity. In fac t after the first
month of treatment al i the inflammatory le-
FIG 1
The vehicle also, however, has proven to be, as
it was expected, hig hly effective (46 %)
towards ali the checked parameters. This fact
has further proven the activity carried out by
the high concentration of the linoleic acid in
the liposomal emulsion.
25
Effect of phosphat1dylcholine linoleic acid-flch and glycol1c ac1d in acne vulgaris
Table I
MEAN INFIAMMATORY C OUNTS IN MILD AC NE TREATED BY
PHOSPHATIDYLC HOLINE-C REAM GLYCOLIC AC ID ENRICHED
n=36
AC NE LESIONS
COUNTS
Base/ine
Week4
Week8
Week 12
change
22
27
15 (55%)
20
12(35%)
17
-65%
-50%
33
38
25
32
20
26
-53%
-39%
5
6
3
4
l
3
-86%
-57%
20
29
15
20
12
18
-67%
-5 1%
Reduction
Open Comedones
Baseline
Active (cream A)
Vehicle (cream B)
34.3
Closed Comedones
Baseline
Active (cream A)
Vehicle (cream B)
43
Pustoles
Baseline
Acti ve (cream A)
Vehicle (cream B)
7
Papules
Baseli ne
Active (cream A)
Vehicle (cream B)
37
Ali p values are significant (p<0.0 1) as to groups and highly {p<0.005) significant as baseline values.
Skin surface lipids
Ali the patients showed at baseline an higher
content of surface lipids (casual level) ( 175 ±
50 µ g/cm 2) .
At week 2 the va lues a lready dec reased o f
a bo ut 7 0 % for both th e ve hi c le and ac ti ve
cream. As seen in Fig. 2 and Tab. II both the
ve hi c le (B cream ) and th e ac ti ve cream (A
cream) are able to reduce the skin lipids casual
level since the first month of treatment (- 70%)
proving a remarkable topic effectiveness.
26
These val ues seem to prove what stated by
G hyczy et al (7): phos pholipidic e muls ions,
when rich in linole ic acid, are able to reach the
cells of the sebaceous g land inducing them to
reduce the sebum secretion.
The high percentage of linoleic acid, entering
into competion wit h the o leic acid , due to a
mec hani s m yet un k now n , w o ul d ac t as a
" brake", red ucing its excessive presence also
at level of the surface lipidic film .
P.Morganti. S.D.Randazzo. A. Giardina. C. Bruno. M. Vincenti and L. Tiberi
Table Il
RIDUCTION OF SURFACE SKIN LIPIDS BY
A PHOS PHATIDYLCH OLINE-C REAM GLYCOLIC AC ID ENRIC HE D
(µg/c m 2) n=36 t=22°C RH=50 %
Weeks
o
Active
baseline
Vehicle
baseline
8
195±50
2
138±33
4
110±24
6
94±24
80±1 8
10
78±21
12
59±16
187±48
134±35
121 ±3 1
107±27
95±25
85±22
7 1±18
Ali p values are not signi ficant as to groups and highly significant (p<0.005) as to baseline values.
REDUCTION OF SURFACE SKIN LIPIDS BY A PHOSPHATIDYLCHOLINECREAM GLYCOLIC ACIDS ENRICHED
cn
225
210
195
180
165
150
135
120
105
90
(J
75
60
N
E
~
~
cn
ea..
...J
z
~
...J
<
IJ..
RH=50%
6
8
45
a:::
w
30
15
cn
o
a..
::>
n=36 t=22 °C
o
2
4
10
12
WEEKS
ID Active
• Vehicle
All p values are not significant as to groups and highly significant {p<0.005) as to baseline values
Fig2
27
Effect of phosphatidylcholine linole1c acid-r1ch and glycolic acid in acne vulgaris
Table lii
EFFECT OF TOPICAL APPLICATION OF PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON FREE FATTY ACIDS/TRIGLYCERIDES RAT IO
n=36 t=22°C RH=50%
Product
number of
patients
Weeks
0.96
4
0.84
6
088
10
36
2
0.84
8
Vehicle
(cream B)
Active
(cream A)
0.82
0.80
12
0.85
36
0.95
0.56
0.45
0.36
0.32
0.31
0.26
o
Ali p values are highl y significant both as to groups (p<0.005) and to baseline values.
EFFECT OF TOPICAL APPLICATION OF A PHOSPHATIDYLCHOLINE-CREAM
GLYCOLYC ACID ENRICHED ON FREE FATTY ACIDS / TRIGLYCERIDES RATIO
n=36 t=22 °C RH=50%
0,9
0,8
o
0,7
...
0,6
:;::::;
11:1
...J
(!)
I-
0,5
LL
LL
0,4
0,3
0,2
0,1
o
o
2
4
6
WEEKS
8
10
IDVEHICLE (CREAM B) •ACTIVE (CREAM A) I
All p values are highly significant (p<0.005) both as to groups and to baseline values
Fig3
28
12
PMorganti, S.D.Randazzo. A Giardina. C. Bruno. M Vincenti and L.T1ben
Table IV
MEAN VALUES OF SKIN HYDRATION IN ACNE PATIENTS TREATED BY
A GLYCOLIC ACID ENRICHED PHOSPHATIDYLCHOLINE CREAM
n=36
t=22°C
RH=50 %
Weeks
Active
Ve hicle
o
2
4
6
8
78±11
74±9
ns±11
I LO±
123±14
128±16
L28±21
133±17
126±19
138±18
10
132±20
141±23
12
134±24
140±22
Ali p values are not significant as to groups and highly significant (p<0.005) as to baseline values.
MEAN VALUES OF SKIN HYDRATION IN ACNE PATIENTS TREATED BY A
PHOSPHATIDYLCHOLINE-CREAM GLYCOLIC ACID ENRICHED
n=36 t=22 °C
160
RH=50%
140
Ciì
:t::
e:
120
~
. ra
...
100
...
80
"':::>w
60
:::>
-:e
~
~
-
~
V
-
-
-
10
12
/
....I
~
o
('?
40
20
o
o
2
4
WEEKS 6
8
1-- ACTIVE (CREAM A) - - VEHICLE (CREAM B) I
All p values are not significant as to groups and highly significant (p<0.005) as to baseline values
Fig4
29
Effect of phosphatidylchoilne linoleic acid-rich and glycolic ac1d m acne vulgaris
The contemporary presence of the g lycol ic
acid would facilitate, moreover, the transcutaneous pene tration of phospholipids, improv ing
in this way the ir activity.
Free fatty Acids/Triglycerides
ratio
This particular phospholipidic emulsion has proven to be highly active also towards another parameter of the acne development, namely the presence of free fatty acids, that are the main cause
of the abnonnal inrrafollicular keratinization. Free
fatty acids have an inflammatory effect and become potential irritants, wh ich when released into
the dennis cause cysts to fonn.
As see n in Fig . 3 a nd Tab. III the free fatty
acids/triglycerides mean ratio decreases of 4 1%
just after two weeks of treatment, and further reduces up to about 7 1% after three months.
During this time period it seems that this activity
is perfonned only by the active cream (A cream)
for the probable antiseptic activity carried out by
the glycolic acid, and particularly by the salicilic
acid and the clorexidine digluconate. In fact, as it
has been proven e lsewhere, there is a significant
reduction of propionibacterium acnes colonies,
with a subsequent drop in the production both of
enzyme esterase and of free fatty acids ( 19).
Skin hydration
Ali the patients showed a significant increase in
moisture content and maintained these values unti! the end of the study. The results are shown in
Table IV and Fig. 4.
DISCUSSION
In acne, as known, defects in the keratinization of
the "epithelial" lining of the pilosebaceous follicle
block eagress of sebum, leading to "plugging".
Propionibacterium acnes, exerts lipolytic effect on
stagnant sebum, releases free fatty acids and causes inflammation when they escape from the pilosebaceous follicle.
In this study, as also suggested from Ghyczy et
30
al (7) the use of soybean derived phospatidylcholine seems to be able to nonnalize excess of
sebum.
Ali the treated patients showed a significant decrease of the free fatty acids/triglycerides ratio togethe r with decrease of the skin surface li pids
(Tab IIl and Fig. 3).
From the other hand the obtained clinica] results
show that the contemporary use in the same phospholi pid ic emulsion of glycolic acid partially
buffered with aminoacids, of salicilic acid and chlorexidine digluconate, can effectively relieve the
severity of acne ever since the first weeks of treatment. It has been noted an high reduction of a li
the inflammatory acne lesions such as small or
large solid bumps, pus pimples, " blackheads" and
whiteheads (Fig. I and Tab. I). The use of the glycolic acid, even if in rather high concentration
(I 0%) did not cause clear irritating forms or stinging activity.
Only two patie nts had to stop immediately the
treatme nt, consideri ng it not suitabl e for the ir
skin type.
The high bearableness of the used compound is
due, in our opinion, both to the strong presence of
phosphol ipids and to the special buffer we already
used to neutralize the aggressiveness of the glycolic acid (12).
Un like a li th e acne treatments utili zed, thi s
phospholipidic preparation not only reduces in
drasti c way the presence of lipids and acneic lesions, but rehydrates the skin since the first period of application, mantaining it e lastic (Fig . 2,
4 and Tab. II, IV).
Given the positi ve resu lts obtained we be lieve
that this cream can be considered as a new cosmeceutical and a usefu l aid for the nonna! acne therapi es.
Author address:
P. Morganti, PhD
Via Innocenzo Xl , 41
00165 Roma l taly
[email protected]
URL=http://www.colosseum.it/st 81 lmavi
P.Morganti, S.O.Randazzo, A. Giardina, C. Bruno, M. Vincenti and L. Tiberi
REFERENCES
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The cornposition of the ceramides frorn human Stratum Corneum and frorn cornedones,
J. lnvest. Dermato/., 84:410-412.
2) Wertz P.W., Madison K.C. and Downing D.T. (1989) Covalently bound lipids of human
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3) Colarow L.J. (1992) Quantitation of cerarnides with essential fatty acids rnoieties in the
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activity and irnproving activity of AHAs, J. Appl. Cosmetol., 14:79-91.
13) Van Scott E.V. and Yur J., (1974) Contro! of keratinization with alpha hydroxy acids and
related cornpounds - Topica) treatrnent of ichtyotic disorders, Arch Dermatol., 110:586.
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glycolic acid regirnen, Cosm. Dermatol., 5:32.
15) Wallhausser K.H. (1984), Praxis der Sterilization - Disinfection - Konservierung. 3, Auflage
Thierne, Stuttgart, p. 27.
16) Morganti P., Randazzo S.D., Bruno C. and Cardillo A. (1988), Ethyl lactate and benzoyl
peroxyde in acne vulgaris, J. Appl. Cosmetol., 6: 15-30.
17) Cardillo A. and Morganti P. (1994), Fast and non-invasive method for assessing skin hydration,
J. Appl. Cosmetol, 12: 11-16
18) Cavina et al.(1965), Ann. Ist. Super. Sanità, 1, 566
19) Morganti P., Agostini A., Bruno C. and G. Fabrizi, (1997), Role of topica! glycolic acid and
phosphatidylcholine linoleic acid-rich in the pathogenesis of acne. linoleic acid versus squalene.,
J. Appl. Cosmeto/. 15:33-41
31
J. Appl. Cosmetol 15. 33-47 (January-March 7997)
ROLE OF TOPICAL GLYCOLIC ACID ANO
PHOSPHATIDYLCHOLINE LINOLEIC ACID-RICH
IN THE PATHOGENESIS OF ACNE.
LINOLEIC ACID VERSUS SQUALENE
P. Morganti', A. Agostini2, C. Bruno' and G.Fabrizi'
' President/Director. Research & Development - Mavi Sud S.r.l. Aprilia CLD, ltaly
Department of Dermatology, Dermatologists Training School. Il University of Naples. ltaly
' Dermatological Hospital, University of Pisa, ltaly
' Physiology lnstitute. University of Urbino. ltaly
• oepartment of Dermatology. Catholic University of Rome, ltaly
Received: December 27, 1996
Key-words: Acne, Phosphatidy/choline, Linoleic acid, Squalene, Glycolic Acid, 3C System®.
Synopsis
Acne is a disease that commonly occurs during adolescence with the production of testosterone and
the activation of the sebaceous glands. Factors such as stress, the environment, drugs, greasy cosmetics and mechanical irritants may contribute to or aggravate this disease.
Many facts indicate that free fatty acids released in sebaceous follicles through the action of bacterial lipases on sebaceous triglycerides play an important role in the overall pathogenesis of acne.
Moreover, it has been shown that there is a significant decrease in the levels of linoleic acid in sebum of patiens with acne and a contemporary increase of squalene and oleic acid.
Supported by our recent data that demonstrate an anti-acne activity carried out by an emulsion based
on phosphatidylcholine and glycolic acid buffered through a special mixture of aminoacids we controlled TEWL, corinebacterium acnes colonies and the presence, at cutaneous level, of linoleic acid
and squalene on 20 patients with an average age of 18±2 with a mild to moderate acne.
The data obtained proved a remarkable decrease of squalene and a contemporary increase of linoleic
acid in the stratum comeum lipids, together with a considerable improvement of the skin 's look of
the patients.
Therefore it seems possible to assert that phospholipidic creams particularly rich in linoleic acid can
be used as new cosmeceutical means adjuvant in the acne juvenilis therapy.
Riassunto
L'acne è una malattia che si verifica di solito durante il periodo dell'adolescenza con la produzione
di testosterone e l'attivazione delle ghiandole sebacee. Fattori come lo stress, l'ambiente, i farmaci, i
cosmetici grassi e gli irritanti meccanici possono contribuire o aggravare la malattia.
Molti fattori indicano che gli acidi grassi liberi rilasciati nei follicoli sebacei attraverso l'azione di
lipasi batteriche sui trigliceridi sebacei giocano un ruolo importante nella patologia complessiva
33
Raie of topico/ glyco/ic ocid ond phosphotid1/coline /Jnoleic ocid-rich 1n the pothogenesis of acne
dell 'acne. Inoltre è stato provato che si verifica una diminuzione significativa nei livelli di acido lino leico nel sebo dei pazienti affetti da acne ed un contemporaneo aumento di squalene ed acido
oleico.
Sostenuti dai nostri recenti ri sultati che dimostrano un 'attività anti-acne svolta da un 'emulsione basata su fosfatidilcolina e acido g licolico tamponato da una speciale miscela di aminoacidi, abbiamo
controllato la TEWL, le colonie di corinebacterium acnes e la presenza, a livello cutaneo, di linoleico e squalene su 20 pazienti con una età media di 18±2 affetti da acne debole o media.
I risultati ottenuti hanno dimostrato un a considerevole diminuzione di squalene ed un contemporaneo aumento di acido linoleico nei lipidi dello strato corneo, insie me ad un miglioramento considerevole dell 'aspetto cutaneo dei pazienti.
Per questi motivi sembra possibile affermare che le creme fosfolipidiche particolarmente ricche di
acido linole ico possono essere utilizzate come nuovi mezzi cosme ti ci ad iu vanti nella te rapia
dell'acne juvenilis.
34
P Morganti. A. Agostirn. C. Bruno and G Fabriz1
INTRODUCTION
Acne is a di sease that commonly occurs during
adolescence with the production of testosterone
a nd the activation of the sebaceous glands. Factors suc h as stress, the e nviro nm en t, drugs,
greasy cosmetics and mechanical irritants may
contribute to or aggravate this disease (1-3).
A connection between acne and high rates of sebum secreti on is well recognized (4-5)
Many facts indicate that free fatty acids released
in sebaceous follicles through the action of bacterial lipases on sebaceous triglycerides play an
important rote in the overall pathogenesis of the
acne (5,6).
Since hyperkeratosis is part of the essential fatty
acid deficiency syndrome, it has been also seen
that a decrease in the amount of essential fatty
acids might be involved in irritating hyperkeratinization within the follicle (7,8).
It is known that in the condition of essenti al
fatty acid deficiency, essential fatty acids of the
n-6 fami ly are replaced by non-essential fatty
ac ids of the n-9 family.
Moreover, it has been shown that there is a signi ficant decrease in the levels of linole ic acid
in sebum of patiens with acne and a contemporary increase of squalene and oleic acid (9).
Supported by the results obtained by the work
of Ghyczy et al. (I O) and by our recent data (11)
we decided to study in detail the cutaneous activity carried out by an emulsion based on phosp ha t idy lc ho l ine and g lyco lic acid buffered
throug h a special mixture of ami noacids on acne
affected skin, through the evaluation of two defin ite parameters: corinebacterium acnes colonies and the presence at cutaneous leve) of linoleic acid and sq ualene.
We decided, moreover, to check also the possible variations that may be found in the Transepide rmal Water Loss (TEWL), since the reduced presence of essential fatty ac ids makes the
membrane structure more perrneable to water.
MATERIALS ANO METHODS
Materials
Vehicle: soybean liposome containing 10% lecithin fraction with 80% phosphatidylcholine linoleic acid-rich (cream B)
Active ingredients: vehicle + glycolic acid buffered to ph 4.5 by a special blend of a minoacids, chlorexidine digluconate and salicylic acid
(cream A).
Patients
Twenty patients ( I O fornaie, IO male) with an
average age of 18±2 with a mild to moderate
acne, partecipated in the study. Ali were volunteers and the nature of the study was explained to
them in full. As described elsewhere ( li) exclusion criteria included patients with more than 5
nodules and cysts and patients who had used topica! antibiotics, retinoids or benzoyl peroxide in
the past 14 days, systemic antibiotics in the past
30 days, systemic retinoids in the past 2 years,
any other topica) acne treatments including medicated soaps, creams o r make up in the past 7
days, topical corticosteroids in the past 14 days,
or systemic corticosteroids in the past 12 weeks.
Test procedure
The study was conducted as a 12-weeks double-blind paired comparison, with treatment assignements randomized, as described elsewhere
( 11 ). Each patient was supplied with two tu bes
containing the test creams (A and B) to apply on
the left or on the right area of the face on a ra ndomized basis for three months. They were also
instructed to apply always the same cream to
the designed sites after washing first thing in the
morning and just before retiring in the evening.
A mild non-irritant washing cream (Mav igene
Idroschiuma) was suppli ed by us to be used
throughtout the study. Other instructions included that the patients use no other acne treatment
during the study and not to apply the creams the
day of evaluations or wash their face 4 hours
before evalutions.
35
Rote of top1col glycolic ocid ond phosphotidilcolme linole1c ocid-rich in the pothogenesis of acne.
Biophysical non-invasive measurements
Measurements were performed on the lst day
(baseline) and after 2, 4, 6, 8, 10 and 12 weeks
(end of treatment). The medium value, as described elsewhere (1 1), was always carried out
on two different sities of right or left areas of
forehead and automatically evaluated by 3C System®(Dermotech, Rome, Italy) (12).
methodology (12).
The 3C System®probe consists of a cylindrical
open chamber measuring system, diameter 14
mm, height 10 mm and skin area 0.95 cm2, two
sensor units, containing thin capacitative film
transducer, are placed at 3 and 7 mm distance
from the skin surface. TEWL is calculated digitally in g/m2 h. The obtained results are shown
in Tab. I and Fig. I.
Transepidermal Water Loss (TEWL)
Squalene and Linoleic acid
Ali evaluations were performed after a 30-minute acclimatization period in a room at 22±2°C
and 50% humidity.
Water evaporating from the skin surface was
measured quantitatively with the 3C System®
Ali lipids extracted by 3C System"' methodology from both the half-forehead from all the
twenty patients were separately dampled and
collected samples from each half-forehead were
Table I
TEWL MEASUREMENTS OF ACNE AFFECTED PATIENTS TREATED
BY PHOSPHATIDYLCHOLINE-CREAM GLYCOLIC ACID ENRICHED
n=20 T=22°C RH=50%
Weeks
Vehicle (cream B)
Active (cream A)
o
22.3±7.2
24.5±8.I
2
20.4±6.8
18.6±7.6
4
15.2±7.0
12.0±3.5
6
14.8±6.5
11.2±2.7
8
12.1±4.8
11.5±3.2
10
11.8±3. l
11.6±2.9
12
11.7±3.5
11.8±2.5
Ali p values are not significant as to groups and highly significant (p<0.005) as to baseline values.
36
P Morgont1. A Agosf1nt, C Bruno ond G Fobnz1
TEWL MEASUREMETS OF ACNE AFFECTED PATIENTS TREATED BY
PHOSPHATIDILCHOLINE CREAM GLYCOLIC ACID-ENRICHED
n = 20 - t = 22 •e
30
25
.e
20
-
15
sw
10
~
........
N
E
O>
.....J
RH = 50%
~
~
'-"'
-
--------
I5
o
o
4
6
8
10
12
WEEKS
1--ACTIVE (Cream A)--VEHICLE (Cream B)I
All p value are not significant as to groups and highly significant (p<0.005) as to baseline value
Fig I
pooled. Collections from the right and the left
areas were analyzed separately. Ali solvents
used were chromatography grade. Tota) lipids
were exstracted from the 3C plastic foil (1 cm 2)
using ethyl ether methanol (2: I) for 3 h. at
room temperature according to Folch et al. (13).
The solvent was dried under nitrogen and the lipids were redissolved in chloroform (2: 1) according to Cavina et al. (14). Lipid fractions were
chromatographilly separated into their individuai lipid classes.
Fatty acids were then converted to their methyl
esters according to Stoffel et al. (1 5). Oleic and
linoleic acid methyl esters and squalene were
quantitatively identified by using internal standards (Sigma Chemical).
The obtained resuls are shown in Fig. 2 and
Tab.II.
Colonies of propionibacterium
acnes
The evaluation of P. acnes was carried out according to the method of Williamson and Kligman (16). Samples were taken at lst day and after 2, 4, 6, 8, I Oand 12 weeks of treatment.
Before taking the sample, both cheeks of the
subjects were cleaned by using a sterile gauze saturated with a 0.1 % sterile solution of Triton x100, followed by a further cleaning with sterile
distilled water and finally rubbed with a gauze
saturated with hexane for 30 seconds. Tue cleansed skin was protected with a porous sterile pla-
37
Rote of topica/ glycolic acid and phosphatidi/coline linoleic acid·rich in the pathogenesis of acne.
EFFECT OF PHOSPHATIDYLCHOLINE·CREAM GLYCOLIC ACID ENRICHED ON LINOLEIC
ACID AND SQUALENE CONCENTRATIONS OF ACNE-AFFECTED SKIN
n=20
e:
o
;
...ca
e:
Q)
CJ
e:
o
CJ
o~
4,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
2
4
6
8
10
12
weeks
IO Squalene •
Linoleic Acid
I
All p values are highly significant (p < 0.005) as to baseline values
Fig2
Table Il
TOPICAL APPLICATION EFFECT OF PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON LINOLEIC ACID AND SQUALENE
CONCENTRATIONS OF ACNE-AFFECTED SKIN.
n=20 T=22°C RH=50%
Weeks
Squalene
%concentration
Linoleic acid
% concentration
o
3.8±0.2
3.2±0. 1
2.7±0.3
2±0.l
1.2±0.1
1±0.1
0.9±0.l
1.2±0.1
2±0.l
2.9±0.2
3.2±0. l
3.4±0.2
3.7±0.1
3.9±0.2
2
4
6
8
IO
12
All p values are significant (p<0.05) both as to weeks and to baseline values.
38
P. Morganti, A. Agostini, C. Bruno and G.Fabrizi
TOPICAL APPLICATION EFFECT ON QUANTITATIVE P.ACNES COUNTS OF
PHOSPHATIDYLCHOLINE-CREAM GLYCOLIC ACID ENRICHED
60
n= 20 t=22 °C
RH=50 %
I/)
w
z
~
50
:E
::>
o::
w
I-
~
z
o
a:
o
o::
c..
u.
o
z
o
j::
(.)
::>
e
w
o::
~
VEHICLE (CREAM B)
WEEKS
4
ACTIVE (CREAM A)
8
4
12
8
12
All p values are highly significant (p< 0.005) as to baseline and groups
Fig3
Table lii
TOPICAL APPLICATI ON EFFECT OF A PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON QUANTITATIVE P. ACNES COUNTS
n=20
Product
Number
of subjects
Week
propionibacterium acnes (Log/cm2)
o
4
8
12
Vehicle (cream B)
IO
6.0842
5.7087
4.5219
3.5610
Active (cream A)
10
6.1533
4.2538
3.4636
2.9776
All p values are significant (p<0.01) as to groups and highly significant (p<0.005) as to baseline values.
39
Rote of topica/ glycolic acid and phosphatidilcoline linoleic acid-rich in the pathogenesis of acne.
stie gauze, so as to maintain normai evaporative
activity. After one hour a cylinder of sterile glass
(internal area 3.8 cm2) with hollow base was applied to the area. Into said cylinder 1 ml of sterile
solution of 0.1 % Triton x-100 in a pH 7.9 phosphate buffer was introduced. Having cleaned the
area with a teflon spatula for one minute, two
successive samples of liquid were taken. The
samples thus obtained were diluted a number of
times with a solution of 0.05% Triton x-100, immediately set in culture with a solution of O. I %
Tween 80 and incubated anaerobically for 7 days.
The colonies of P. acnes were determined according to Mc Finley et al (17). The obtained mean
results are shown in Table ill and Fig. 3.
Statistica/ analysis
Differences between means were calculated
using the Student's test. Statistica[ correlations
between percent values of squal ene and linoleic
acid and absolute tota[ amount recovery of lipids were calculated using the Pearson correlation coefficient (Statistica! Analysis System,
SAS, North Caroline, USA).
RESULTS ANO DISCUSSION
According to data obtained from Ghyczy et al
(I O), and as it can be seen in the fig. 2 and Tab.
Il, the squalene concentration decreases drastically since the second week of treatment, while
at the same time it can be noted a regular increase of the Iinoleic acid present in the stratum
corneum lipids. It seems that this activity can be
ascribed exclusively to the phospholipidic vehicle rich in linoleic acid.
The data obtained from the cutaneous areas
treated with the cream B (vehicle) compared
with the co rrespo nding areas of th e same
subject treated with the cream A (active), resulted almost similar and are therefore reported as
the average of a unique treatment (Fig. 2 and
Tab. li).
With regard to the presence of corinebacterium
acnes (Fig. 3 and Tab. III), the active cream
40
(cream A) resulted much more effective than
the vehicle (cream B). The vehicle also (cream
B) reduces the presence of corinebacterium acnes of about 50% after 12 weeks of treatment.
This unexpected result is to be ascribed probably to the antioxidant activity typical of the soya
phospholipids, that is Iikely to interfere with the
survival and the development of corineabacterium acnes.
According to what we verified (18) it has also to
be pointed out that by adding to the vehicle only
the glycolic acid buffered to pH 4.5 it was obtained a further decrease of the whole bacterial
charge, apart from the well-known bactericide
activity carried out by both the salicilic acid and
clorexidine. It has been verified experimentally
that the glycolic acid alone buffered wi th special mixtures of aminoacids increases of a
further about 30% the bacteriostatic activity typical of the used phosphatidylcholine.
Finally, as it was expected, it was possible to
verify a considerable decrease of TEWL in ali
patients treated by both the vehicle and the active cream (Fig. 1 and Tab. I).
Considering the res ults obtained by our previous study, the results obtained by Ghyczy et
al. (I O, l l) and the data obtained wi th this work
it seems possible to assert that phospholipidic
creams particularly rich in linoleic acid can be
used as new cosmeceutical means adj uvant in
the acne juvenilis therapy.
These emulsions have, moreover, proved to be
even more active when the right proportion of
glycolic acid, properly buffered with aminoacids, is added to them.
Author address:
P. Morganti, PhD
00165 Roma ltaly
[email protected]
URL=http://www.colosseum.it/st 81 Jmavi
P. Morganti, A Agostini, C. Bruno and G.Fabriz1
REFERENCES
1) Kligman AM (1974) An overview of acne J. l nvest. Dermatol. 62:268
2) Fortrom L (1980) The influence of sex hormones on acne Acta Derm. Venereol 89 (sup):27
3) Wu SF, Kinder BN, Trunnell T N and Fulton J E (1988) Role of anxiety and anger in acne
patients: a relationship with the severity of the disorer J. Am. Acad. Dermatol. 18:325-333
4) Pochi PE and Strauss JJ (1964) Sebum production, casual sebum levels, titratable acid ity of
sebum, and urinary fractional 17-ketosteroid excretion in males with acne.
J . l nvest. Dermatol. 43:383-388.
5) Kellum RE (1968) Acne vu lgaris: studies in pathogenesis: relative irri tancy of free fatty ac ids
from C2 to Cl 6 Arch. Dermato! 97 :722
6) Kellum RE (1979) Free fatty acids hypothesis In : Frank SB (ed) Acne: Update fo r the pratitioner
Yorke Medicai Books. N, pp 65-73
7) Williams ML (1991) Lipids in normai and pathological desquamation In: Advances in li pid
research voi 24 (Elias, Havel, Small Edtrs) Acad. Press, NY, p 237.
8) Dowining DT, Stewart ME, Wertz PW and Strauss JS (1986) Essential fatty acids and acne
J. Am. Acad. Dermato/. 14:22 1-25
9) Morello AM Dowining DT and STRauss J S (1976) Octadecadienoic acids in the skin surface
lipids of acne patients and norma! subjects. J. !nvest Dermatol 66: 319-323
1 O) Ghyczy M, Nissen HP and Biltz H (1986) The treatment of acne vulgaris by phosphatidylchol ine from soybeans, with an high conteni of lino leic acid J. App/. Cosmetol. 14: 137-145
11) Morganti P, Randazzo SD, Giardina A, Bruno C and Tiberi L (1997) Effect of phospatidylcholine linoleic-rich and glycolic acid in acne vu lgaris J. Appl. Cosmetol., 15, 21
12) Cardillo A and Morganti P. (1994) Fast and non-invas ive method for assess ing skin hydration.
J.A ppl. Cosmetol. 12: 11- 16
13) Folch J. et al.(1957), J. Biol. Chemistry, 226, 497
14) Cavina G. et al.(1965), Ann. lst. Super. Sanità, 1, 566
15) Stoffel A. et al.(1959), Ann. Chem. 31, 407
16) Williamson P, Kligman AM (1965) A new method for the quantitative investigation of
cutaneous bacteria J . !nvest. Dermatol. 45:498
17) McFinley K, Welster G, Leyder J (1965) Regional variations in cutaneous propionibacteria
J. App/. Env. Micro 45 :998
18) Morganti P. (1996), unpublished data
41
Bookreview
DERMATOLOGIC SURGERY
Principles and practice - Il Edition
Edited by Randall k. Roenigk & Henry H. Roenigk Jr.
1344 pages illustrateci, hard bound
ISBN#:0-8247-9503-2
u.s. $ 175.00
marcell dekker, lnc.
Cimarron road, Monticello N.Y. 12701
914-796-1919
It took long for o ur journal to review the second editon of this very interesting text of "Dermatologie Surgery" by Roeni gk & Roenigk's, as the board wanted it to be read by both dermatologists and
plastic surgeons, to whom it is essentially dedicated.
I personally devoted many hours to read this reall y complete text of clinica! diagnoses and updated
therapies, ranging from the detailed description and treatment of the always neglected wh ite popular
lesions called Milia, particularly frequent in the skin of Asian peoples, to the different techn iques
for the surgical management of skin tumours.
No topic of a dermo-surgical interest is left aside by the "directors" of this interesting text, written
by many experts of the academic world.
In fac t, it begins by describing the preparation of the skin before any treatment and the various surgical tools required, without omitting the important matter of the infotmed consent by the patient
who asks for the treament. For legai purposes, in fact, the patient must be informed of ali indicaiions
and contraindications of the proposed treatment, and of any alternative treatment options.
Two entire chapters are devoted to the techniques and problems of an effective locai anaesthesia
with reference to methodologies and chemical compounds, which will have to be carefully evaluated fo r the required quickness and duration of action, also considering any systemi c toxicity. A wide
space is also dedicated to ali the precautions and special proceeding the physician wi ll have to comply with before and after treatment to prevent any complications, including careful evaluation and
psychogical support for the patient before and soon after a surgical operation.
The book also describes the attitude to be adopted in the various emergencies which might occur during a surgical operation, ranging from fainting to convulsions and allergie reactions, up to a cardiac
arrest, although extreme ly rare in dermatologie office surgery. Two inte resting c hapters dea! with
wound healing and wound dressing, which very clearly describe the physiological and biochemical
aspect of wound fornrntion and healing, as well as ali methodologies adopted or to be adopted to try
to avoid the formation of the unaesthetic keloids.
This first part of the book, consisting of ten chapters, ends with an eleventh chapter which explains
the various complications arising during any cutaneous surgery.
Another seven chapters thoroughl y describe standard procedu res, ranging from an accurate analysis
of how to correctly execute a skin biopsy, to the various suturing and electroepilation techniques, up
to the methods fo r the cryosurgical treatment of c utaneous lesions, of which it outlines the referring
contraindications.
Many chapters dea! with reparative surgery of the various cutaneous areas such as ear, lips, nose or
cutaneous appendages such as nails and hair, of which the different grafting techniques are skilfull y
Book rev1ew
described, from single hair grafting to rotation flap, up to the scalp expansion method.
No technique, whether old or modem , is ever casuall y described. Al! possible treatments, from
simple cosmetic treatments, such as camouflage, to seriou s surgical operations, such as the removal of a malignant melanoma, are always explained and dealt with in a very accurate, simple and
professional way. Nothing is ever accidental, and no interesting subject of dermatologie surgery
has been forgotten.
Laser techniques, cover as much as six chapter which, starting from the basic physical laws, descri be with full details both the operating basis of the various types of lasers and the treatment of telangiectasias or wrinkles, or the difficult elimination of tattoos. Obviously, it also deals with the different techniques for reducing face wrinkles' depth, such as collagen injections or the use of non-collagen-based fi ller materi als, or the techniques used to eli minate fat pads, such as liposuction. Wide
space is left to the various chemical peelings used in cosmetic dermatologie surgery, such as,for
example, trichloroacetic acid, phenol or the more recent g lycolic ac id. Forali treatments, the techniques and referring contraindications are always included. Certainl y, this review does not full y show
the interest this book can raise in readers, whether medicai students or generai practitioners, dermatologists or plastic surgeons. This is not a common plastic surgery book, but a real " bible" for reference before any plastic surgery operation, both cosmetic and reparative. Therefore, this second edition of "Dermatologie Surgery" by Roenigk & Roenigk's should not be missing in the library of ali
those who deal with cosmetic dermatology in a broad sense, as the science conceming the beauty of
the human body, whether non-physicians, such as biologists, chemists, cosmetologists and physiologists, or generai practitioners and dermatology, plastic surgery, pediatry or ginecology specialists.
Pierfrancesco Morganti, Ph.D.
Editor in Chief
Notes
•
CARTA ECOLOGICA· ENVIRONMENTALLY PAPER • PAPIER ECOLOGIOUE • PAPEL ECOLOGICO
f'roçrJ:'ml/M foxnrwcfealt«Jnolo;in,
Chiuso in tipografia: May 2, 1997
Journal of Applied Cosmerology published quarrerly by INTERNATIONAL EDIEMME, Via Innocenzo XI, 41
00165 Roma Iraly. Direttore responsabile: P. Morganri. Direzione, Redazione ed Amministrazione: Via Innocenzo XI, 41
00165 Roma Iraly. Stampa: Grafica Flaminia, Roma. Impaginazione: GRAFO' Comunicazione visiva, Roma. Coperrina:
Dr.ssa M .G. Tucci - Dip. Ricerche INRCA -Ancona Iraly. Sped. abb. Postale Comma 34 arr. 2 legge 549/95 Roma. Aut.
del Trib. di Roma n. 3173/83 del 8-7-83.
THE ANTIAGING LINE
GLYCOLIC ACID ACTIVATED BY
GELATIN - GLYCINE®
TO NORMALIZE THE SKIN TURNOVER
fil .
~
-
; - -~ ·
~- ·~
LA LINEA ANTIAGING
CON ACIDO GLICOLICO H ATTIVATO"
PER NORMALIZZARE
IL TURNOVER CUTANEO
rvV?
LJT.JT.J.
maVI
La Ricerca Scientifica
nella Dermocosmesi
flMVI SUD s.r.l. Aprilia (LT)- ltaly
THE EVOLUTION IN COSMETIC SCIENCE
.Lll1
HYPOALLERGENIC
mav1 COSMETIC PRODUCTS
Ln')
mav
LA RICERCA NON È MAI STATA COSÌ BELLA
,.,
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Cosmetic Dermatology

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th.e new frontier1 in ph.otoprotection

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POLISH SILICONE CREAM Meltonian