ALFA 4~ MICOSPUMA. > In pre and post-operative treatment > In post laser treatment >In preventing infection and formation of scar lesions

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2 : A NEW MAVICEUTICAL FOR TOPICAL TREATMENT OF SKIN INFECTION ALFA 4~ MICOSPUMA MILD ANTIMICROBIAL CLEANSER MAXIMUM STRENGTH Caracteristics Autodosable ph balanced Alcohol-free N on-irritating Gentle For more scientific information call to: Mavi Sud Sri - V.le dell'industria, Aprilia (Lt) ltaly Tel Fax mavi@colosseum.it - URL: Indications > In pre and post-operative treatment > In post laser treatment >In preventing infection and formation of scar lesions > To help in healing and revitalizing damaged skin DOES NOT DRY OR IRRITATE THE SKIN

3 ~der A NEW MAVICEUTICAL HCG CITOSTIMOLANTE - CELL ACTIVE DESCRIPTION STERYLIZED WATER SOLUTIDN DF HYALURONIC ACID, VITAMIN C AND BETAGLUCAN. PRESERVATIVES AND FRAGRANCE-FREE. INDICATICNS IT 15 NECESSARY TD PRDMDTE FIRMER, EVEN-TDNED SKIN WITH REDUCED APPEARANCE OF FINE LINES AND WRINKLES. HOW TO USE PDUR 1-2 DRDPS DF HCG INTERESTED. DIRECTLY ON THE AREA Please see a brief summary of prescribing information on next page ~

4 HCG 1 ODO CITDSTIMDLANTE CELL ACTIVE MEANS OF ACTIVITY The balanced combination of the three actives known to further the fibrous component of the dermis, optimizes the normai production of collagen, elastin and fibrine. Betaglucan. as natural cellular protector, is the keystone of the formulation for its efficacy toward the immuno-competent cell ofthe skin. Vitamin C in the form of 1-ascorbic acid arginate is known to stimulate collagen synthesis and help prevent lipid peroxidation of cell membranes due to free radicals. ~dar For more information apply to: Mavi Sud Sri V.le dell'industria Aprilia (Lt) ltaly Tel Fax mavi@colosseum.it URL= Hyaluronic acid. identified as glycosaminoglycan (GAG) favours the hydration of the upper layers of epidermis helping diffusion of Betaglucan and Vitamin C through the skin. UNDESIDERABLE REACTIONS No undesiderable reactions in the use of this product have been reported so far.

5 MAVI IN GINECOLOGIA LA GIUSTA SOLUZIONE MAVI IN GYNECOLOGY THE RIGHT SOLUTION ' f o 8 "' Eli'6E:NCJ - INTIMO I ~ mavi GYNECOLOGY La ricerca scientifica nella dermocosmesi Per Campioni Medici e Documentazione Scientifica scrivere a: MAVI SUD s.r.l. - Direzione Propaganda Medica Viale dell'industria, Aprilia (l T)

6 'Wvi /ntroducing LOTION DIET SUPPLEMENT For the treatment of hair loss if The right reply to on old problem

7 Clinical studies established efficacy with mild to moderate hair loss in the frontal - parietal scalp( 1 ) ncreases mass and numbers of hair in a short period( 1 l IOESSE ~ MEAH PERCEHTAGE VARIATIOH OF TOTAL HAIR MASS PER cm 2 OF PATIEHTS WITH AHDROGEHETIC ALOPECIA TREATED BY GELATIH-CYSTIHE AHD SERENOA REPEHS 611% 60 1ncrease 1n hair mass TOPICAL AHD/OR BY ORAL ROUTE ! u E 20 e.. E 10 "' o n 60 All p values u e hlghly s lgnlflcant (p < 0.005) as b asellne value aa to g ro ups MEAN PERCENTAGE VARIATION -OF HAIR NUMBER PER cm ~ 2 OF PATIENTS WITH ANDROG EHETIC ALOPECIA TREATED BY GELATIH-CYSTIHE AHD SEREHOA REPENS TOPICAL AHD/OR BY ORAL ROUTE n % 1ncrease 1n hair number O weell:s Plocebo(Lotion) All p values are hlghly signiflcan t (p < D.005) as baseline value as to groups *Not~ ~~ -~!P.~ -~ff_~ç_i: _~~-~ -~~s;,ç?,~q~..q AVI sud srl le dell'industria, Aprilia (LT) ltaly l. 06/ Fax 06/ mail=mavi@colosseum.it L= /mavi ) Morganti P., Fabrizi G., James B. And Bruno C. J. Appl. Cosmeto/.16,57,1998

8 A NEW MAVICEVTICAL The originai approach in acne rnanagernent Breaks down the horny layers Reduces significantly the inflammatory lesions ( comedones, papules and pustules) CLINICAL RESULTS ( PRE-TREATMENT WEEK4 KERATOTAL ACNE- THE NEWEST ANTl-ACNE TREATMENT FOR PATIENTS WITH SENSITIVE SKIN, IS: FAT FREE COLOR FREE EMULSIFIER FREE. FRAGRANCE FREE ALCOHOL FREE EXCELLENTTOLLERABILITY 5MOOTHLY ABSORBED NON OCCLUSIVE. 0DORLESS ANO INVISIBLE

9 --;f NEW MAVICEUTICAl Derrnatological/y tested Effective for initial and maintenance therapy ( 1 2 3l Compatible with all the drugs and cosmetics Formulateci to treat mild-to-moderate inflammatory acne, indispensable for patients with sensitive skin CLINICAL RESUL TS > REOUCTION OF SURFACE LIPIOS OURING THE TREATMENT WITH KERATOTAL ACNE 90 ~--" -" 30...:... P_< o_oos _ Wtt.o -=.c"'-'"ootod ;, _ ACTIVITY CARRIEO OUT BY KERATOTAL ACNE ON THE LINOLEIC AGIO ANO SQUALANE CONTENTS OF SURFACE LIPIOS IN SUBJECTS AFFECTEO BY ACNE JUVENILIS n = 30 z ~ so => iil :: ~ giorni lmuntreated Treated I ~ Reduces excess lipids 30 4,0 3,5 ~ 3,0 ~ 2,5 ~ 2,0 ~ 1,5 z 8 1,0 *- 0,5 4 settimane l11 squalene Unoleic Acld I ~ Decreases the Squa/ene content of acne affected skin EFAITG 1,5 z ~ ~ o ~ ~--'---~ o::: ~ so ''-----'---~ I: z #----'---~ ~ ~ ~ I """'""- B111UJlO I WEEK1 WEEK2 ~ Significantly reduces EFA/TG ratio ~ lncreases skin hydration by 97% Please see a brief summary of prescribing information on next page ~

10 BRIEF SUMMARY KERATOTALACNE 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 bi end 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 Aprilia (Lt) ltaly Tel Fax mavi@colosseum.it URL= ADVERSE REACTI ONS 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 forthe first ten days of treatment R EFERENCES: 1,2 - Data on file Mavi Sud 3 - 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,

11 Trimestrale di Dermatologia Cosmetologica Quarterly Review of Cosmetic Dermatology EDITOR-IN-CHIEF P. MORGANTI, Ph.D. Secretary Generai Jntcmational Society of Cosmetic Dermatology Via Innocenzo Xl, Roma (lt aly) Fax SECRETARY EDITOR ADVERTISING M. PASCOLI Via Innocenzo Xl, Roma (ltaly) Fax C.E.C. sas - Anna lebo;..i.:h Viale Legioni Romane, Milano (ltaly) Fax ASSOCIATE EDITORS F.H. KEMPER, M.D. C. JACOBSON. M.D. Professor Emeritus. Past Prcsidcnt - lntemationat Socic1y of Cosmctic Derma1ology Pharmacology and Toxicology 3600 Gaston Ave. Suite 1051 D llas D Mtinster, Domagkstr. 11 TX USA. Fax Fax =kemper@uni-mucnster-de S.D. RANOAZZO, M.D. Professor of Dermatology Univcrsity of Catania Via lacona, Catania (ltaly) Fax M.B. JAMES, M.D. Program Director - ln1ema1ional Socicty of Cosme1ic Dennatology 157 Beacon Strcct #2 Boston, Ma Phone/Fax =AMERx@hey.net SCIENTIFIC SECTIONS AND EDITORIAL BOARD Cell and Tissue Colture G. Biagini (I) L. Di Silvio (UK) N. Stark (USA) Molecular Biology L. Bruckner-Tuderman (D) V. Calabrese (I) T. Krieg (D) J. Uitto (USA) Skin Biology B.Bcrra (I) M. Ponce (NL) Photobiology H. Honigsmann (A) F.P.Noonan (USA) G. Prota(I) Skin lmmunology A. Giannetti (I) Skin Permeation J.P. Marty (F) G. Puglisi (I) Skin Pha rmacology F.H. Kcmper (D) R. Paoletti (!) Skin Toxicology S. Paglialunga (I) M.G. Rozcn (USA) Skin Ageing S. Jablonska (PL) M. Noszczyk (PL) M. Vcrschoore (F) Natural Cosmesis and Balneology G. Agostini (!) B.R. Balda (D) Non-Invasive Methods and Biotechnologies H. Tronnier (D) W. Gchring (D) U. Heinrich (D) Skin and Cosmetic Microbiology J. Kabara (USA) D.Orth (USA) D. Steinberg (USA) Skin Bioengineering L. Andreassi (I) L. Rodrigues (P) P. Elsner (D) Allergy Testing F.K.E. Andersen (NL) B. Santucci (I) A. Sertoli (l) Cosmetic Ma nufacture a nd Contro) L. Nteta (SA) A. Parsons (SA) H.C. Roos (SA) Cosmetics and Fragrances G. Angelini (l) Cosmetics and Environment Retno l.s. Tranggono (Indonesia) P. Suvanprakom (Thailand) Aromatherapy a nd Natural Raw Materials G. Salvatore (I) C osmctics' Safety Evaluation E. Chiaccherini (I) C linica! lnvestigations in Cosmetic Dermatology H. Maibach (USA) Oral Mucosa and Dental Car e Problems E. Benagiano (I) Nail Care Cosmetics R. Baran (F) B. Richcrt (B) A.Tosti(J) Hair Care Cosmetics S. Calvicri (I) W.A.D. Griffiths (UK) C.E. Orfanos (D) Cosmetics and Skin Disorders V. Mordovstev (R) W. Raab (A) T. Ruzicka (D) Plastic a nd Aesthetic Surger y P. Palombo (I) Cosmetic Pediatry G. Fabrizi (I) Y. Kazuya (J) A. Taieb (F) Cosmetic Gynaecology A. Lanzone (I) S. Mancuso (I) M. Massobrio <n

12 GENERAL INFORMATION The JOURNAL OF APPLIED COSMETOLOGY is an international journal devoted to publisching originai papers, reviews and other materiai which represent a useful conuibution 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 contact with the skin and its appendages. The Journal 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 necessarily endorsed by the Editor(s), Advisers, Publishers of Distributors of this Journal. 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 publisher 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 wtitten permission of the Publishers. Sections of J ournal The following sections will be features of the Journal: 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: scientific articles of generai interest to our readers will be considered for publication. These articles should be concerned 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 figures included. Headings ("Materials", "Discussion", etc.) as well as Sumrnaries are to be omitted. If accepted, these submission will appear in print in a very short time. Letter lo the Editor: comments on Journal 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: ali papers should be submitted to: Dr. P. Morganti INTERNATIONAL EDIEMME Via Innocenzo Xl, Rome - ltaly Te!. 0039/6/ Fax. 0039/6/

13 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 Ma11uscripts: submit three copies of both the manuscript and ali illustrative materiai to the above address. Orga11izatio11 of the Mmmscript: investigative studies should be organi zed as follow: title, abstract page, introduction, materiai and methods, results, discussion, acknowledgments, references, legend for figures, tables. Ali pages should 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 international 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 surnames throughout. For multiple authors, use the surnames 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 figures and tables. To allow faster publication only set of proofs will be furnisched 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 summary not exceeding 300 words typed double-spaced. The summary will include statements of the problem, mcthod 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: should be listed consecutively at the bottom of the page on which they fall, designated by the following 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 lndex Medicus. The style of the references must conform to the examples given below: I) Robbins CR, Kellych ( 1970) Aminoacid compositi on of human hair. Text Res J 40: ) Strehler BL ( 1977) Time, cells and aging 2nd edn. Acadcmic Press, New York 3) Ebling FJ, Rook (1972) Ciclic activity of the follicle. In: Textbook of dermatology 11, Blackwell, Oxford, p lllustratio11s: figures should be numbered consecutively using Arabic numerals Tables should be numbered consecutively, using Roman numerals. Ali 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 Experime11ts: 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 European Pharmacopeia. Ifa materials is not listed, then the trademarked name can be used, with the chemical composition given in footnotes.

14 INFORMAZIONI PER L'ABBONAMENTO L'abbonamento annuale comprende quattro numeri. È possibile ottenere abbonamenti a prezzo ridotto da parte dei ricercatori che lavorano presso Istituti che abbiano sottoscritto almeno un abbonamento a prezzo normale. L'Editore potrà fornire a richiesta notizie più dettagliate. Le sottoscrizioni di abbonamento possono essere effettuate mediante assegni postali, bancari, di conto corrente o per contanti indirizzandoli a: INTERNATIONAL EDIEMME - Via Innocenzo XI, 41, ROMA - ITALIA c/c bancario n. 3184/51 Banca di Roma Ag. 1- Aprilia (LT) - ITALIA L'IVA è a carico dell'editore, non detraibile dall'abbonato a norma art. 74 lett. C DPR 633/72 SOTTOSCRIZIONI ANNUALI Italia L Altre Nazioni$ 80 Numero singolo L Numero arretrato L SUBSCRIPTION INFORMATION Subscriptions are entered on a calendar years basis only and include four regular quarterly issues. Half-price subscriptions are available to research scientists whose institutions already subscribe at full rate. Details on application from publisher. Payment must be made in U.S. dollars using bank draft, international postai money order only. Italian residents only may pay by persona! check: INTERNATIONAL EDIEMME - Via Innocenzo XI, 41, ROMA - ITALY c/c bancario n. 3184/51 Banca di Roma Ag. 1- Aprilia (LT) - ITALY ANNUAL SUBSCRIPTION RATE: Italy, Lit Other Countries, $ 80 Additional Air Mail postage rate: $ 15 ISCD Members Free of Charge Additional Air Mail postage rate: Africa and Middle East US$ 12 North, Centrai and South America US$ 14 Far East US$ 15 Oceania US$ 19,50 Statements and opinions expressed in the articles and communications herein are those of the author(s) and not necessarily those of the Editor(s), or publisher. The Editor(s) and publisher, disclaim any responsability or liability for such materiai and do not guarantee, warrant, or endorse any product or service adverised in this publication nor do guarantee any claim made by the manufacturer of such product or service

15 Quarterly Review of Cosmetic Dermatology INFORMAZIONI PER L'ABBONAMENTO L'abbonamento annuale comprende quattro numeri. È possibile ottenere abbonamenti a prezzo ridotto da pane dei ricercatori che lavorano presso Istituti che abbiano sonoscriuo almeno un abbonamento a prezzo nonnale. L'Editore potrà fornire a richiesta notizie più dcnagliate. Le sottoscrizioni di abbonamento possono essere effelluate mediante assegni posrnli. bancari, di conto corrente o per contanti indirizzandoli a: INTERNATIONAL EDIEMME - Via lnnoccnro Xl, Roma e/e bancario n. 3184/51 Banca di Roma Ag. I -Aprilia (LD - halia Abbonamento JOURNAL OF APPLIED COSMETOLOGY Italia L Altre Nazioni $ 80 l s1ruzio11i per/' abbo11a10: O desidero abbo11armi a quesra rivisra per /'a11110 i 11 corso O ri1111ovo a111oma1icame111e il mio abbo11ame1110 per gli a1111i f11111ri ( quesra forma di abbo11ame1110 può essere com1111q11e disdella i 11 og11i mome1110). O desidero ricevere le 11orme ediroriali per eve11111ali collaborazio11i (Scr ivere i11 s1ampa1ello) Nome Indirizzo Città Nazione CAP ~~~~~~~~~~~~~~~~~~~~~~~~~~- -~~~~~~~~~~~~~~~- Quarterly Review of Cosmetic Dermatology SUBSCRIPTION INFORMATI ON Subscrip1ions are cntercd on a calendar ycar basis only and include four rcgular quancrly issucs. Half-pricc sub.scriptions are availablc 10 rcscarch scicntist whose institutions alrcady subsribc at tull rate. Details oo applica1ion from publisher. Paymcn1 must be made in U.S. dollars using bank drafl intemational postai money ordcr only. ltalian residents only may pay by persona! check: e/e bancario n. 3184/51 Banca di Roma Ag. I -Aprilia (Ln-haly Order Form JOURNAL OF APPLIED COSMETOLOGY A a/ subscription raie: ltaly, Lii Other Countries $SO Additional Air Mail postage raie: Africa and Middle East USS 12. North, Centrai and South America US$ 14, Far East USS 15, Oceania USS 19,50 Please Ch eck O I Year s11bscrip1io11 O Renew my s11bscriptio11 a atically in future years (this co111i1111atio11 order is instendedfor subscriber's co111 e11ie11ce 011/y and my be canee/led at any time). O Send me a copy of informationfor Autlwrs. O Please charge this order to my credit card (Al/ arder subjet to credit approml). Delete as necessary: O AMERICAN EXPRESS O DINERS CLUB - Card Number Expiration date.... (Please Print) Name ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Addres~ City Country

16 STAMP spett. Direzione "JOURNAL OF APPLIED COSMETOLOGY" INTERNATIONAL EDIEMME Via Innocenzo Xl, ROMA (ITALY) STAMP Spett. Direzione "JOURNAL OF APPLIED COSMETOLOGY" INTERNATIONAL EDIEMME Via Innocenzo Xl, ROMA (ITALY)

17 Trimestrale di Dermatologia Cosmetologica Quarterly Review of Cosmetic Dermatology Contents Originai Laboratory Studies 125 Role of hyaluronic acid and vitamin C in photoageing A. Di Pietro, G. Fobrizi, U. Gioroli. L. liberi. C. Bruno ond P. Morgonti 135 Study on the in vivo performance of two capacitance system: assessment of the experimental reproducibility and sensitivity Luis Rodrigues, Volando R.Solgueiros, Nuno Golego, Pedro Pinto, Nuno Silvo, Irene Z.Ferro 155 Biochemical studies on a natural antioxidant isolateci from rosemary: dermo-cosmetic implications for its application in human antiaging skin care Calabrese V., Rondozzo S. D., Morgonti P. ond Rizzo V. Generai Articles Cutaneous side-effects of antiblastic chemotherapy: an emerging problem S. Moncuso, S. Greggi. R. Gionnice. P. A. Morgoriti. M. G. Salerno. A. De Dilectis ond G. Scambio Hair and skin photoprotection in China: the cosmetic challenge for the new millenium P. Morgonti lndex to Volume 16, 1998 XIX Announcements ln-cosmetics 1999 Cosmetic science on the brink of a new millenium. The cha//enges and the chances Paris Aprii, 1999 ISCD Conference Program - Day 2 - Aprii 21, Room Diderot 4th Scientific Conference of the Asian Societies of Cosmetic Scientists Cosmetics, Safety and Efficacy: Fact and Dream - Bali lnternational Convention Centre, Nusa Duo, Bali - Indonesia, 7-9 Aprii, 1999 Discover the latest developments in the World of Persona! Care lngredients lntex - Shangai - China, 7-9 Aprii, 1999 The 11 th Annual - Symposium on Aesthetic Surgery San Francisco - California, March 18-20, 1999 CARTA ECOLOGICA ENVIRONMENTALLY PAPER PAPIER ECOLOGIQUE PAPEL ECOLÒGICO ~,,,..,,,.11oi.....i......

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19 J. Appl. Cosmetol. 16, (October - December 7998) ROLE OF HYALURONIC ACID ANO VITAMIN C IN PHOTOAGEING A Di Pietro', G, Fabrizi', U, Giaroli', L Tiberi', C. Bruno' and P. Morganti' 1. Department of Dermatology "Marchesi" HospitaL Inzago, Milano, ltaly 2. Department of Dermatology, Catholic University of Rome, ltaly 3. R. & D - Mavi Sud S.r.l., Aprilia CLD, ltaly 4. Department of Anatomics and Fisìology, University of Urbino, ltaly Received: November 70, Key words: wrinkling, hyaluronic acid, vitamin C, beta-g/ucan, photoageing, ageing, medicai device, skin implant. Synopsis In order to influence the collagen content of the dermis, for ameliorating the wrinkling appearance of the facial skin, di.fferent active compounds are used, topically applied by cosmetic emulsions or by subdermal injection. Among these, recentely, the more used are: ascorbic acid, beta-glucan, and hyaluronic acid, for their ability to retain large amount of water into intercellular space, to make jelly matrix, to protect celi structure, to hold celi and to defend it against external pollutants and bacteria on infections. These active compounds are used by topica! application or by injection as corrective for wrinkles. The antiwrinkle activity of two new products, HCG 1000 (vitamin C, beta-glucan, hyaluronic acid based) and HY 20 (Bio-technological and purified Hyaluronic Acid) have been controlled "in vitro" and "in vivo" by a double-blind study on 30 volunteer patients in an out-patient office. From the obtained results, it is possible to desume that HCG 1000 may be added to the patient's skin care as a cosmetic routine, before and after the injection of different augmentation materials, to increase the efficacy of the implant. Riassunto Al fine di modificare la presenza di collagene nel derma e per migliorare l'aspetto delle rughe cutanee, si utilizzano generalmente diversi preparati, quali, ad esempio, acido ascorbico, betaglucano e acido jaluronico, sottoforma di cosmetici o di "dispositivi medici". Questi composti attivi, sempre più spesso, vengono utilizzati attraverso applicazioni topiche o attraverso iniezioni intradermiche come correttivi per le rughe. A tal proposito, con uno studio a doppio ceco su 30 volontari, è stata controllata, "in vitro" e "in vivo", l'attività antirughe svolta da due nuovi prodotti, un preparato a base di vitamina C, betaglucano e acido jaluronico, denominato HCG 1000 e un preparato a base di acido jaluronico purificato di origine non animale, denominato HY 20. Dai risultati ottenuti, è possibile desumere che l'hcg 1000 può essere utilizzato, quale coadiuvante dell'attività svolta dall'acido jaluronico o dal collagene, nella terapia topica o iniettiva. 125

20 Rote of hyaluronic OCld and v1tam1n C in photoageing INTRODUCTION The skin is an organ in which aging changes are easily obseived. Tue degree of change varies between individuals and on the different areas, and can be distinguished in intrinsic or chronologic ageing and extrinsic or photoageing. Generally in intrinsic ageing, collagen in the dermis is lost, and reduction of cellular activity and skin thinning occur ( 1-3). Conversely, photoaged skin results thickened and characterized by wrinkles, roughness, tang led degraded elastic fibers and matted hypo-pigmentation and hyper-pigmentation. Both photoaging and intrinsic ageing occur especially in facial skin results in wrinkling, but the ageing changes are also in depending of life-style, such as the amount of sunlight time exposure and quality and quantity of skin-care used. In order to in.fluence the collagen content of the dermis, for ameliorating the wrinkling appearance of the facial skin, different active compounds are used, topically applied by cosmetic emulsions or by subdermal injection. Among these, recentely, the more used are: A) Ascorbic acid, which appears to exert a transcriptional or translation contro! of collagen synthesis and shows promise as a broad-spectrum photoprotectant. ( 4-6) B) The biologica! response modifier Beta-glucan, which seems able to stimulate the skin own defence mechanism, enhancing the macrophage-mediated phagocytosis and the production of cytolytic and cytostatic factors. (7-9) C) Hyaluronic acid for its ability to retain large amount of water into intercellular space, to make jelly matrix, to protect celi structure, to hold celi and to defend it against extemal pollutants and bacterial infection.( I 0-1 3) AIM The aim of this study was to evaluate "in vitro and in vivo" the effect of a balanced and stabilized combination of hyaluronic acid, vitarnin C and beta-glucan (HCG active) in patients showing evidence of photoageing changes in the skin. It was also con- 126 trolled the clinica! effect of a new sterile hyaluronic acid of bio-technological culture origin, used by injection as corrective for wrinkles. MATERIAL ANO METHODS Products HCG (active): aqua, sodium hyaluronate, sodium carboxymethyl betaglucan, ascorbic acid, arginine. HCG (placebo): aqua, arginine. HY : hyaluronic acid mg.20, sodium chloride and disti/led water to 1 ml. CREAM BASE 0 1 : aqua, glycolic acid, propylene glicol, glycerin, lactic acid, stemyl alcohol, cetemyl octanoate, cera alba, cetyl palmitate, cetemyl heptanoate, glycine, gelatin, cetyl alcohol, sodium PCA, stem yl cap1y late, steareth-10, steareth-7, isopropyl myristate, parajfinum liquidum, mùystil alcohol dimethicone, titanium dioxide, pa1fum, imidazolidinyl urea, tocophe1yl acetate, arginine, methylparaben, hydrolyzed collagen, alumina, silica, sodium polyac1ylate, propylparaben, diazolidinyl urea, isopropy/ paraben. CLEANSING LOTIONu>: aqua, isopropyl myristate, ceteareth-6, stemyl alcohol, dimethicone, glycolic acid, octy/ stearate, sorbito!, propylene glycol, glycerin, arginine, tocophe1yl acetale, pa1fum, imida:o/idinyl urea, methylparaben, lactic acid, linoleic acid, linolenic acid, retinyl palmitate, hydroly:ed collagen, disodium EDTA, propylparaben. Patients ALI the 30 volunteer patients (age range years) and of ali both sexes (15 males and 15 females), had attended the dermatology out-patient office, according to Lever et al. (14). The only criterion for entry in the study was the presence of one or more signs of photoageing affecting the face, such as fine wrinkling around eyes, crease lines around mouth and cheeks, telangiectasia, wrinkling of skin (I ) Trade 11ame: QM IDRATANTE JO;MAVI ROMA DAYTIME (2) Trade 11a111e: QM LOZJONE DETERGENTE; MAV/ ROMA GENTLE CLEANSING LOTION

21 A. Di Pietro. G. Fabrizi. U. Giara/i. L. Tiberi. C. Bruno and P. Morganti on the backs of the hands etc, the state of their skin corresponding to degree 3 to 5 on photodigital scale described by Larnier et al. (15). Ali the subjects gave their written inforrned consent in according with the ethics of cosmetic experimentation. Study design "in vitro" It was investigated the activity of the HCG I 000 solution on human keratinocytes obtained by biopsy from the skin of some volunteers of the study group. Keratinocytes The Keratinocytes culture medium was added with the HCG loqo!' active at two different concentrations of 0.01 and 0.02% (w/v) and controlled at 24, 48, 72 and 120 hours according to Zulli et al. (9). The obtained results are reported in figure I. Fibroblasts The fibroblasts culture was added with the HCG 1000 active for 20 hours before being exposed to UV-B radiation at two different doses, 30 and 60 mj/cm2, according to Mori et al. (16) and to our own experience ( 17). Tue obtained results on the influence of human keratinocytes growth, and on the leve! of the cyclic pyrimidine dimers (photo-immuno-protection effect); are reported in figure 2. Study design "in vivo" Tue study was enrolled as a placebo-controlled, double-blind tria! in which the HCG 1000 active and HCG I 000 placebo were randomly applied to the right of left sites of the face and the dorsum of the hands together with the Cream base. Tue patients were given a 15 ml dosable bottle glass container for each site and instructed to apply 3/4 drops of each product to the back of the hand, right or left, and the same amount to the ipsolateral half of the face. Tue Cream base was applied on the tota! surface of both face and hands. Each glass-bottle contained the active HCG l 000 or its vehicle and all the bottles were identica!. Monthly checks were made to ensure that the patients understood the need to avoid contamination of one side by the solution allocated to the other side and the preparations were applied regularly. INFLUENCE OF HCG 1000"' ON THE GROWTH OF HUMAN KERATINOCYTES O Contro! 0.01 % 0.02% TIME (h) Fig

22 Raie of hyaluromc ac1d and vtfamin C in photoageing Treatment Patients were instructed to apply the drops twice a day in the moming and just before retiring in the evening at least 30 min. after cleansing with the specific product supplied. The treatment period lasted 12 weeks during which no other cosmetics or drugs were to be used with the exception of the special Cream base and the Cleansing lotion supplied. Exposure to strong sunlight was avoid. Contro/ assessment and evaluafion Contro! vis it and measurements were performed on lst day (baseline) and after 15 (D1 5), 30 (D30), 45 (D45), 60 (D60), 75 (D75) and 90 (D90) days of treatment with a follow up visit at 120 (D 120) always from the same dermatologist, using a clinica! score method. The individuai signs of photoageing were scored on 0-1 O visual analogue scale with separate scores for each site of the face and each hand. Subjects were also evaluated objectively for the signs and symptoms of skin irritation and subjectively by asking them if there was any itching, sting ing or burning sensation. After the l st, the 2nd and 3 rd month of treatrnent, the subjects evajuated their satisfactory or unsatisfactory scoring the firrnness, softness and the wrinkles appearance, using a scale of O to 4 for each criterion (0: unsatisfactory; 4: satisfactory) according to Berardesca et al. (18) The obtained results are reported on fig. 3-4 and tab. 1-2). Hya/uronic acid (HY 2<P) treatment The injections ofhy 20 were performed only on 1 O peoples of the stuclied group. Because of the nature of the product, composed from hyajuronic acid of no-animai origin, skin testing was not required. Evaluated by " in-vitro" and by preclinical " in-vivo" studies, HY 20~ was found to be biologically compatible, stable in the derma! tissue and persistent in the skin longer than the collagen contro!. Tue viscosity/concentration curve was also another fundamental paramiter controlled (Fig 5)(19). Identified the wrinkles suitable for inclusion, HY 20 was injected to 4 sites per subject into the derma! tissue through a 30-gauge needle by the new Di Pietro methodology (20). AU the subjects treated gave their informed consent prior to entering the study and were free to withdraw at any time. HY 2~ was admi- INFLUENCE OF HCG 1000~ ON LEVEL OF CYCLIC PYRIMIDINE DIMERS AFTER UV IRRADIATION o 0,01 0,02 0,03 HCG 1000' active concentration (% W/V) O UV-8 60 mj/cm 2 UV-8 30 mj/cm 2 Untreated Control Fig2. 128

23 A Di Pietro, G Fabrizi, U. Giara/i, L. T1ben, C. Bruno and P. Morgant1 nistered at the first visit (week 1), and touch-up injections were permitted at the next 2 visit (weeks 2 and 4), when necessary. Clinica/ evaluation Evaluations and assessment were performed at follow up visit at weeks 2,4,6,12, 18 and 24. At each visir the dermatologist and the patient, separately, evaluated the degree of correction for each treatment site, using a 0-10 visual analog scale (0= no correction, 10= tota! correction). The values from each site were added together and the mean value was deterrnined. The obtained results are reported on fig. 6. RESULTS OF 3-MONTH TREATMENT BY HCG 1000 ~ ANO CREAM BASE ON PHOTOAGED SKIN n=30 (DEGREE OF SATISFACTION FROM THE TREATMENT) Eyes fine wrinkles Crease lines (moulh and checks) Hand wrinkling Tcleagiectasia DO Fig 3. Al/ p volues o re significont os to boseline (p<0.05) SUBJECTIVE EVALUATIO AITER 3-MONTH TREATMENT BY HCG 1000" A D CREAM BASE ON PHOTOAGED SKI n=30 (DEGREE OF SA TISFACTION FROM THE TREA TMENT) 4 w ci::: u cn Firmness Softness Hydration Wrinkles O O (Follow-up assessment) Fig 4. Al/ p volues ore significont os to boseline (p <0.05) 129

24 Rote of hvaturomc ac1d and vitamin C in photoagemg Statistica/ analysis A two tailed student's test for paired series was used to analyze the differences between the values obtained before and after the treatments. Product safety The use of both HY 20 and HCG 1000 did not lead to any unwanted skin reaction, demonstrating their complete safety. Table I CLINICAL SIGNS TREATMENT I PERIOD BEFORE AFTER Fine wrinklù1g arow1d eyes vehicle month HCG Crease li11es around mouth a11d cheeks t--- Wri11kli() 011 dorsum o hand vehicle rnonth HCG vehicle month HCG 1000* vehicle 3.3 Telangiectasia 011 the cheeks 3 rnonth HCG 1000" Table I. Mean results ot the 3 month-treatment by HCG 1000' and a cream-base on photoaged skin. Mean results: visual analog score (1-10) tor clinica/ signs. (n=30) Table II CLINICAL SIGNS TREATMENT BEFORE AFTER Fine wri11kli11g around eyes Crease lines arou11d mouth a11d cheeks Wri11kli{) 011 dorsum o hand Tela11giectasia 011 the cheeks vehicle 6.4 HCG 1000" 6.7 vehicle 5.1 HCG 1000" 4.9 vehicle 6.2 HCG 1000* 6.0 vehicle 3.3 HCG 1000" Tab/e Il. Follow-up assessment at week 16 after the HCG 1000' and the Cream base treatment. Mean results: visual analog score ( 1-10) tor clinica/ signs. (n=30) 130

25 A Di Pietro. G. Fabnzt. U Giara/i. L. Ttben. C Bruno and P Morgant1 RESULTS ANO COMMENTS to 75% (p<0.01 (fig.2) skin fibroblasts against DNA damage on leve! of cyclic pyrimidine dimers upon In vitro sfudies UV-B irradiation and promotes the growth of keratinocytes up 200% (fig. l) in depending of the HCG About " in vitro" studies colture experiments showed 1000 concentration used. that HCG 1000 active seems to protect from 50 These results give the chance to think that HCG l~ THE VISCOSITY OF HYALURONIC ACID "HY 20*" SOLUTION RAPIDLY JNCREASES WITH RAJSE IN CONCENTRATJON VISCOSITY/CONCENTRATION CURVE FOR HYALURONIC ACID FCH O-r-~~~~~~~~-.-~~~~~~~~-.-~~~~~~~~~ 0,5 I 1,5 CONCENTRATION % Fig5. IMPROVEMENT OF SCARS ANO WRINKLES TREATED BY"HY 20 9 " n =IO 100 ~ ~ E- z w ::;; w :> o IX Q., ~ 50 o o WEEKS Fig6. 131

26 Rote of hyaluronic acid and vitamtn C in photoageing may have an "in vivo" efficacy to protect skin against oxidative stress induced by UV irradiation with an immuno-protecting effect, and may be able to enhance the renewal rate of the stratum comeum improving skin firmness. In vivo studies As it is showed on tab.i, the studied HCG 1000 solution caused a progressive reduction of the clinical signes of photoaged skin, both on the face and on the dorsum of the hands of the treated subjects. This amelioration of the generai aspects had a positive follow up at week 16 also.(tab.ii) Although ali the clinica! signes started to reduce just after the first 2 weeks of treatment, (fig.3) the differences did not reach statistical significance until after 6 weeks. The differences between treatment with the HCG 1000 or its vehjcle becan1e really pronounced after 12 and 16 weeks of treatment (tab.i-ii). Moreover the clinica! signes from the baseline decrease from about 50% (p<0.005) for the fine wrinkles around eyes and mouth to 21 % (p<0.005) for the wrinkles on the dorsum of the hands or to 29% (p<0.005) for teleangiectasias on the cheeks. The positive mean results obtained with the vehicle treatment from 30% (p<0.05) to 6% (p<0.05), was in our opinion due exclusively to the antiwrinkling activity of the cosmetic base used, rich in glycolic acid, vitamin C, and in other active compound. ffigh satisfaction (p<0.05) has been obtained directly from the patients' response (fig.4) only after using active HCG 1000 in addition to ilie base, whereas not very impressive results have been mentioned after using ilie contro! appljed in addition to the base Crean1. We have considered, then, useful and maybe essential, the contemporary usage of the base cream and the HCG 1000"', mostly in order to facilitate the transcutaneous penetration of the last one. As a matter of fact, if we compare the contro! activity, represented by pure sterile water and arginine used in addition to the base cream, to the HCG 1000"' activity, we'll find that the last one shows more than doubled values, at ilie same time signjficant among them (p<0.005) (tab.1-ii). HY 2<? injections More significant appears the data obtaneid from a sub-group of the same patients treated also by HY 20"' hyaluronic acid injections in order to try to eliminate or to reduce the deeper wrinkles. As it is seen at week 12, patients wiili 85% of scares and wrinkles saw iliem improved at Jeast moderately or better (from 40 to 85%) (fig.6). Moreover ali the patients received an illitial treatment at week O, and 1 touch-up treatment at week 2 or 4 and approximately 50-55% of the treated sites, maintained marked or complete correction (7 5-80%) until week 18 and 24, as compared to baseline (fig.6). In terms of overall level of patient satisfaction wiili HY 20 treatment, 90% of the patients reported higher satisfaction at week 12 and no complications were observed. FINAL CONCLUSION From the obtained results both " in vitro" and "in vivo" it is possible to deduce that the balanced combination of Beta-glucan, VitanUn C and Hyaluronic acid in HCG 1000"' active solution may be added to the patient's skin care as a cosmetic routine, boili before and after the injection of collagen or hyaluronic acid, such as HY 20 or oilier skin augmentation materiai, to increase the efficacy of ilie implant. In addition HCG 1000 may be dispensed togeilier with other selected cosmeceuticals to promote firmer even toner skin with dimillished appearance of fine lines and wrinkles. Author Address: Pierfrancesco Morganti, Ph. D. Via Innocenzo XI, Rome ltaly Tel: Fax: info@mavicosmetics.it 132

27 A 01 Pietro. G Fobnzi. U G1oroli. L T1ben. C Bruno ond P Morgont1 REFERENCES 1. Gilchrest B.A. (1989) Skin aging and photoaging: an overview. J. Am. Acad. Dermatol. 21: Gilchrest B.A. (1984) Skin and aging processes. CRC Press /ne. Boca Raton, USA 3. Webster G.F. and Uitto J.J. (1991) Phannacology of the aging skin. In: Phannacology of the skin. Mukhatar Edr. CRC Press!ne. Boca Raton, USA 4. Darr D., Combs S., Dunston S., Manning T. and Pinnell S.R. (1992) Topical vitamin C protects porcine skin from UV-induced damage. Br. J. Dermatol.127: S. Darr D., Dunston S., Faust H. and Pinnell S.R. (1996) Effectiveness of antioxidants (vitamin C and E) with and without sunscreens as topical photoprotectants. Acta.Derm. Venereo/. (Stoch) 76: Nakamura T. Pinnell S.R. Darr D. et al. (1997) Vitamin C abrogates the deleterious effects of UVB radiation on cutaneous immunity by a mechanism that does not depend on TNF-a. J. lnvesr. Dermarol.109: Babineau T.J., Hackford A., Kenler A. et al. (1994) A phase II multicenter, double-blind randomized, placebo-controlled study of three dosage of an immunomodulator (PGG-glucan) in high-risk. surgical patients. Arca Surg. 129: Zulli F., Suter F. et al. (1996) Carboxymethylated ~-(l-3)-g l ucan. Cosmet.& Toilet.111: (Dic.) 9. Zulli F., Suter F., Biltz H. and Nis.sen H.P. (1998) Improving skin function with CM-glucan, a biologica! response modifier from yeast. /11. J. Cosmet. Sci. 20: Richter A. W. (1974) Non-immunogenecity of purified hyaluronic acid preparations tested by passive cutaneous anaphylaxis. lnt. Arch. Ace. Appll. lmmunol. SO: Agren U.M., Tammi R.H. and Tammi M.I. (1997) Reactive oxygen species contribute to epidermal hyaluronan catabolism in human skin organ culture. Fre. Rad. Biol. Med. 23: (7) Boyle D.E. (1997) lntralesional Hyaluronic acid treatment ofpathological scars. Ann. Plast. Surg. 39:(5) Abatangelo G. (1997) Hyaluronic acid delays or impedes reepithelialization? J. Burn Care Rehabil. 18:(6) Lever L., Kumar P. and Marks R. (1990) Topical retinoic acid for treatment of solar damage. Br. J. Dermatol. 122: Larnier C., Ortonne J.P., Venot A. et al. (1994) Evaluation of cutaneous photodamage using a photographic scale. Br. J. Dermatol. 130: Mori T., Nakane M. et al. (1991) Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4) photoproduct from the same mouse immunized with ultraviolet irradiated DNA. Photochem. Photobiol. 54: Morganti P., Fabrizi G., Bruno C. and Cardillo A. (1998) Fotoprotezione e stress ossidativo. Presented at XXXVII A.O.O.I. National Meeting, Sept. Ischia (Naples) Italy 18. Berardesca E., Distante F., Anthoine P. et al. (1997) Clinica! and instrumental evaluation of the activity of an anti-wrinkle cosmetic product on cutaneous relief and photoaged skin. J.Appl. Cosmetol. 15: Morganti P. (1998) Unpublished data. 20. Di Pietro A. (1998) Proposing a new injection method: microtherapy. Presented at 1 st I.S.C.D. Workshop "Hair loss and skin aging" 5th Asian Dermatologica! Congress. Oct. 17, Beijing, China 133

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29 J. Appl. Cosmetol (October - December 1998) STUDY ON THE IN VIVO PERFORMANCE OF TWO CAPACITANCE SYSTEMS: ASSESSMENT OF THE EXPERIMENTAL REPRODUCIBILITY ANO SENSITIVITY Luis Rodrigues, Volanda R.Salgueiros*, Nuno Galego, Pedro Pinto, Nuno Silva, Irene Z.Ferro* Faculdade de Farmacia da Universidade de Lisboa Laboratorio de Fisiologia Experimental UCTF - Laboratorio de Biologia Cutànea Facultad de Farmacia de Santiago de Campostela. ESPANA Received: June I, 1998 Key words: Hydration, epidermis, capacitance method, in vivo, dynamical analysis Synopsis Tue epidennal water content is one of the most fundamental indicators for cutaneous functional evaluation. The major raie of water in the sk.in physiological and pathophysiological processes is actually well known and recognised and for it, the reinforcement or re-establishment of these properties, through a wide variety of topica! formulations, including cosmetics, is a frequent objective of therapeutical intervention. Among the many different technologies developed by the cutaneous bioengineering to assess the waterrelated variables, the so called electrometric methods are clearly the most frequently used, no matter the criticism and practical limitations brought to light by the continuos experience accumulated on these methods aver the years. Particularly relevant has been the practical impossibility to ensure a previous calibration of the systems which impairs the establishment of direct relationships between the numerica! data provided by those systems, usually expressed by arbitrary units, and the respective physiological equivalents. Moreover this impossibility also affects the operative reproducibility conditions specially when discrete alteration on epidennal hydration occurs. From one of the most frequently used and studied systems based on the detection of the capacitance changes on skin surface, a new system was recently presented. Although based on the same biophysical principles, this new system is referred as an advanced version of the originai device. This perspective motivated the present study which included a detailed analysis on the experimental behaviour of both systems in the in vivo evaluation of regional differences, the effect of room temperature changes, and the epidennal water dynamical changes occurring after specific intervention on nonna! sk.in's physiological state. A clear relationship between the numerica! data resulting from both systems was found but, from a practical operative stand, the new one showed better reproducibility and sensitivity conditions, when compared, within the same experimental methodologies, to the previous version of the same system. 135

30 Studv on the in vivo performance of two copocitonce svstems ossessment of the expenmentol. Riassunto Il contenuto in acqua dell'epidennide è uno degli indici fondamentali per la valutazione della funzionalità cutanea. Il ruolo fondamentale dell'acqua nei processi fisiologici e patofisiologici è attualmente ben nota e riconosciuta, e il rafforzamento o ricostituzione di queste proprietà, attraverso un 'ampia gamma d i preparati topici, inclusi i cosmetici, è un frequente obiettivo degli interventi terapeutici. Tra le varie diverse tecnologie sviluppate dalla bioingegneria cutanea per valutare le variabili relative all 'acqua, i cosiddetti metodi elettrometrici, sono chiaramente quelli più frequentemente usati, malgrado le critiche e limiti pratici portati alla luce dalle continue prove accumulatesi su questi metodi negli anni. In particolare è stata ri levante l'impossibilità pratica di assicurare una previa calibratura dei sistemi che danneggia la possibilità di stabilire relazioni dirette tra i dati numerici fomiti da questi sistemi, generalmente espressi da unità arbitrarie, e i rispettivi equivalenti fisiologici. Inoltre questa impossibilità interessa anche le condizioni di riproducibilità operativa specialmente nei casi in cui si verificano una discreta alterazione o idratazione epidermica. Da uno dei sistemi più frequentemente usati e studiati basati sulla rilevazione dei cambiamenti di capacità sulla superficie della pelle, è stato presentato recentemente un nuovo sistema. Sebbene sia basato sugli stessi principi biofisici, questo nuovo sistema viene definito come una versione avanzata dello strumento originario. Questa prospettiva ha mosso il presente studio che include una analisi dettagliata sul comportamento sperimentale di entrambi i sistemi nella valutazione in vivo delle differenze regionali, gli effetti dei cambiamenti di temperatura ambiente, e i cambiamenti dinamici nell'acqua epidermica intervenuti dopo interventi specifici sul normale stato fisiologico della pelle. E' stato riscontrato un chiaro rapporto tra i dati numerici risultanti da entrambi i sistemi ma, da un punto di vista operativo, il nuovo sistema ha mostrato condizioni di migliore riproducibilità e sensibilità se paragonato, nell'ambito delle stesse metodologie sperimentali, con la precedente versione dello stesso sistema. 136

31 L. Rodngues. Y. R. Salguef(OS. N. Go/ego, P. Pinta. N. 51/va. I. Z. Ferro INTRODUCTION Tue quantitative eva1uation of cutaneous hydration is definitely one of the most important objectives for the functional characterisation of human skin. Epidermal water balance is a fondamenta! feature of many different sk:in properties, influencing arnongst others, the epidermal "barrier" function, skin's biomechanical behaviour and, the skin profile (1-3,6-9), and these may explain the extraordinary interest dedicated, specially over the last 10 to 15 years to the study of several forms of impact of extemal intervention (mostly pharmacological and cosmetological) on those variables (11-13). Bioengineering provided fundarnental contributions to the knowledge advancement on these areas, producing severa! technological systems to quantitatively assess cutaneous water content which were progressively applied not only to the clinica I study of human skin but also to the cosmetological R&D. No matter the enormous variety of the existing systems, depending on different biophysical principles, those based on the detection of skin's electrical properties (electrometric methods) were rapidly adopted, probably a direct consequence of being very " user friendly" tools, also providing low analysis costs. These also contributed to look deeper to the many complex and unsolved questions arising from the human skin hydration analysis since this variable does depend from many different factors which affects the individua! and the hydration state of the skin. Endogenous (intrinsic water balance, psychosomatic, anatomica! region, age) and exogenous factors (season, room temperature and humidity) are well known and recognised limiting factors (1-3,6-9, 13). But the differenttechnological systems chosen also introduce new criticai conditionants. In fact, most of these systems use rigid electrodes which ensure value readings after contact with the skin surface. This procedure may preclude the pressure effect or even the effect of other elements (e.g. body hair) which remains between skin's surface and the probe's surface, being also influenced by the intra-individual variation of skin relief (3,8,15). Additionally these systems use distinct electrical frequencies determining the generation of di.fferent electrical fields between the electrodes. Distinct frequencies means different levels of measurement from surface to the deepest cutaneous layers which contain different water compositions, affecting the interpretation of results which cannot be objectively performed without a previous calibration of the systems. Most of these systems evaluate skin's hydration state by the assessment of the impedance or conductance changes, through which resistance, reactance or capacitance can be calculated (1-3,5). However, these physical variables, when obtained from an in vivo model, are difficult to relate with the respective physiological equivalents, which explains why those values are normally expressed by arbitrary units instead of µf (capacitance) or µs (conductance). These considerations are particularly important for the so called cutaneous capacitance a frequencydependent variable unlike "true" electrical capacitance. Cutaneous capacitance is often referred as "capacitance" and, when used, specific operative conditions adopted should be referred. (3). Nevertheless, independently from this aspects, severa! electrometric methods were made comrnercially available. The most expressive examples are found with the Nova DMP900, an impedance method based on the phase angle measurement (2,5) which use frequencies up to lmhz allowing to assess skin water content at the most superficial layers; the Skicon 200 ( 1, 14) which uses high frequencies (3.5 MHz) also allowing to assess skin water content at tl1e most superficial layers; and the Comeometer CM820 (2, 4), a capacitance method using lower operating frequencies (40-75 khz) which means that measurements will probably involve deeper epidermal structures, when compared with the previous systems. Reasonable doubts still remain regarding the measurement deepness performed witl1 this system (estimated between 60 a 100µ) but stili, this is undoubtedly the most frequently referenced system used in ilie field, specially when the quantitative description of epiderrnal hydration is involved. Tue recent evolution to a newly presented system, the Comeometer CM825, was reported as a technological advancement facing the previous model and, the potential advantages implied motivated the 137

32 Studv on the in vivo performance of two copoc1tonce svstems ossessment of the expenmentol present work which, through different experimental methodologies involving epidennal water quantitative description, airned to approach the reproducibility and sensitivity conditions tested for both systems. MATERIAL ANO METHODS The present study was developed using human healthy volunteers (n=7), female, aged from 18 to 26 (mean: 23) years old, exhibiting a normai cutaneous condition, without previous history of any particular cutaneous reactivity. Ali volunteers were chosen following previously defined inclusion criteria, giving informed written consent. Ali measurements were perfonned in a closed room with contro li ed temperature (21±1 C) and relative humidity (60-70%), except for the methodolog ies where the environmental temperature change itself was an experirnental paramete r. In any case volunteers were Jeft in the room for 30 min prior to any measurement in order to ensure full adaptation to room's conditions. During this period the probes from the measurement systems were applied to volw1teers in order to ensw-e sensors adaptation to skin's surface conditions. This was particularly important for the TEWL measurement. The biologica! variables considered to represent the dynamical phenomena involving cutaneous water at skin's surface were the Trans-epidermal water Joss (TEWL) measured through the evaporimetry system Tewameter TM21 0(fM2 15 (CK electronics, Germany) and the epidermal "capacitance" changes assessed through the systems Corneometer CM820 and Corneometer CM825 (CK electronics, Germany). Measurements were performed on the volar aspect of both forearms, using a closed chamber as recommended from reference guidelines published before (9) to avoid exogenous influencing factors. Volunteers, anatomica! sites and measurement sequences were previously randomised through the Latinsquare. Experimental methodologies were designed as follows: Phase I. Evaluafion of fhe measuremenf variabilify in disfincf anatomica/ regions af differenf room femperafures. Measurement areas and sites were previously marked on skin surface in order to establish the different values for epidennal "capacitance" and TEWL corresponding to the different anatomica! regions assessed. At each regi on two contiguous sites (2cm apart) were selected, each corresponding to an area of approxirnately 2 cm 2 One of the sites was used to measure epidermal "capacitance" while the other was used to obtain TEWL readings. In this conditions measurements were perfonned in the following anatomica! regions : right forearm (ventral and dorsal). Measuring sites were marked along a central (longitudinal) line designed between the wrist and elbow joints; right Jeg ( ventral and dorsal). Centrai, at I Ocm from the knee joint; right hand (dorsal and palmar). At a central point considering the hand width and at 4 cm (distai) from the wrist joint; face, at the : front : centrai periorbicular: right and leftjust below the cheek. chin : centra]. Ali anatomical regions were evaluated under different room temperatures. The laboratory selected temperatures were 16, 21 e 24 C, keeping the same referenced relative humidity.. Phase Il. Dynamical evaluafions. These studies were designed to evaluate the behaviour of the two epidennal "capacitance" systems under specifically induced dynarnical changes on the epidermal water balance. The chosen methodologies included: A) POST test - basai TEWL and "capacitance" measurements were obtained, for reference, at previously marked sites on the forearm. 138

33 L Rodngues. Y R Salgue1ros, N Go/ego, P Pinta N 51/va, I Z Ferro The individuals were then submitted to a 6H occlusion patch test (POST - Plastic Occlusive Stress Test) using proper materiai (silicone+ hardener mixture from Hoescht, Germany), The POST - test is a most interesting approach to the characterisation of cutaneous water dynamics (10-12), allowing a detailed decay analysis for each curve drawn from patch removal to full variables stabilisation. Curve profiles vary as a direct function of time, mostly depending from the intrinsic characteristics of each individuai, and also from the sensitivity of the system used to detect these changes (10-12). B) Mathematical modelling analysis using a Glycerol reference solution - basai 1EWL and "capacitance" measurements were obtained, for reference, at previously marked sites on the opposite forearm. Volunteers were then submitted to contact with an acetone + sulphuric ether ( 1: 1) soluti on for 30 min. This procedure is often used to induce full delipidation and dryness of epidermal surface with no sensitising effect (6,11). One hour later different glycerol aqueous diluted solutions (I%, 5%, 10%) were applied at the previously treated sites. Epidermal "capacitance" c hanges were evaluated each 5 minutes for 60 minutes. These data were analysed through the application of a compartmental mathematical model developed by the authors and already presented and discussed (10,11) allowing to identify, from those variables, any dynamical change occurring over time, even if discrete. Ali values regarding epidermal "capacitance" no matter the system used, corresponded to mean values of 5 different measurements performed at the same site, while TEWL values, corresponded to 1 min means after full stabilisation. Descriptive statistics, ANOVA and paired t-student test were applied to data obtained from each methodological phase. A confidence interval of 95% was adopted. Tue Pearson 's correlati on coefficient was also used for the variables correlation analysis. RESULTS ANO DISCUSSION The corneometry system is clearly recognised as the most frequently referenced technological system used to non invasively assess the cutaneous hydration state. Tue good reproducibility experimentally reported added to a fast and easy operation and low cost of analysis are irnportant factors which justifies its popularity in the field. In spite of the irnportant reserves which are know and stili persist regarding the true meaning of the numerica! data obtained from this system (as from the e lectrometric systems in generai) since its difficult to conceptualise the respective physical or physiological correspondences, the fact is that a significant amount of knowledge on human skin physiology and pathophysiology finds its originata great deal of stucties conducted over tl1e years with the Comeometer system. Described as a capacitance method (4) the Comeometer CM820 uses low varying frequencies (40 to 75 khz) around loµa, allowing to detect, in about 20ms (once the system's probe is put in contact with the epidermal surface) skin's dielectric constant changes. Tue probe is applied on skin surface covering an area of 49mm 2 approximately, establishing contact at a constant pressure (3.5N) from which an estirnation of surface water expressed in arbitrary units (AU) is given. Apparently this system is more affected by the water content of deeper layers when compared to the Skicon which seems to be more sensitive to higher hydration levels than the Comeometer. This, in tum, seems to be particularly suitable to assess dry skin (1,2,14). A close relationship between the Comeometer and the Nova DMP is also reported but the Comeometer seems to give consistently highe r values (2,3). Tue new Comeometer CM825 allegedly presents, according with the manufacturer, an irnproved electrical system isolation whic h allows to eliminate parasite c urrents, reducing the influence over the measurement of known interfering factors (at skin's surface) such as salts and ions. lt also operates at low variable frequencies (up to I MHz) and allows a previous calibration which contributes to minirnise the measurement incertitude related with the current dispersion at the different epidermal layers. A recently 139

34 Study on the in vivo performance of two capac1tance systems assessment of the expenmenta/. published paper (1) on the in vitro calibration of the CM825 system, compared to the Sk.icon 200 (conductance), showed that the CM825 detection deepness is lower than the previous CM820. The detected values seem to range µm and in vitro maximal "capacitance" does not exceed 120 arbitrary units (UA). That study also suggested that those values were relatively independent from the chemical composition (15% NaCI sol.) ofthe saturated cellulose fi.lter used for the calibration procedure, from its thickness and ionie or non-ionie characteristics of the used saturated solutions. Experimental data from this new technological assessment tool, in particular from in vivo approaches, is clearly insufficient due to its recent development. For this reason the first experimental phase of the present study was dedicated to reproduce the anatomica! regional differences which are al.ready known and described in terms of TEWL and "capacitance" (1,3) submitting al.i the volunteers to different room temperatures. The obtained results are presented in Table 1 and Figure I. As shown, both "capacitance" systems are able to detect the expected variabilities characterising each anatomica! region. However the CM825 system seems to operate at a lower value scale, independently from the room temperature. Apparent.ly there's also a good relationship between the values obtained from both "capacitance" systems since the observed differences for almost ali the anatomica.i regions are not significant, no matter the room temperature considered (Table II). The exception (leg's anterior aspect and palmar region) may rather be explained by inter-variability factors considering the relatively reduced number of volunteers included in this assessment. Tue frequency distribution histogram (Figure 2) seemed to corroborate these observations suggesting that the moda! distribution at 16 e 24 C was identica! for both systems, although a higher dispersion of results was found for the CM820 system, while at 2 1 C (normai room operating temperature) the CM825 value classes appeared earlier and were more evenl y distributed. Assuming that this non uniform value dispersion could be related with the systems variability which would presumably be affected by the operating room temperature condition, each standard deviation (sd) was normalised according the expression: (sd I mean "capacitance") x 100 and compared with the respective "capacitance" absolute values obtained from the two systems at different room temperatures (Table III, Figure 3). As shown, normalised standard deviation (sci,,) dispersi on along the capacitance scale values was more pronounced for the system CM820 at any of the tested room temperatures (Table ill and Figure 3). On the other hand, values obtained from the system CM825, clearly at a lower "capacitance" interval, presented a broader sd 0 amplitude variation, which may reflect a higher sensitivity for this system expressed through a higher capacity in detecting more intermediate numerica! values within the same capacitance interval, independent.ly from the room temperature considered. This last aspect was further investigated trough the correlation analysis established for both systems within the different hydration levels detected, independently from the anatomica! site regarding the room operating temperature. This study was also important to establish the experimental variation coefficient experimented for both systems (Table IV). This analysis al.lowed to demonstrate the existence of a high correlation for the values obtained from both systems (0,83 at 21 C) even at the extreme room temperature operating conditions (0,87 at 16 C and 0,80 at 24 C), strikingly consistent fora.li the hydration range detected specially at 21and24 C (Figure 4). System 's reproducibility calculated through the variation coefficient (Table IV) also showed to be very similar for both systems. It was found that at 24 C, CM825 variability was lower and thus, less sensitive to extreme values of hydration (0,9 to 8,1%) compared with CM820 (2,4 to 10,2%) which may in fact be attributed to the reported improvement regarding the influence of exogenous influencing factors (as saline components) at skin's surface. Dynarnical evaluations (Phase Il) were also included in the present experimental protocol in order to obtain 140

35 L. Rodrigues, Y. R. Salgueiros, N. Go/ego, P. Pinta, N. 51/va, I. Z. Ferro Table I Temperature : CM820 CM825 Forearm (oc) (AU) (AU) Leg Ventral 16 72,9 (10,5) 46,4 (10,7) 21 67,4 (8,4) 42,8 (8,7) 24 77,7 (14,6) 49,2 (15,5) Dorsa/ 16 66,3 (14,1) 38,5(9,J) 21 58,9 (12,4) 34,l (7,3) 24 70,6 (10,5) 43,6 (14,4) Rand Anterior 16 62,4 (10,2) 4 1,4 ( 10,5) 21 65,4 (13,6) 38,1 (5,7) 24 75,3 (14,4) 45,9 (1 6,2) Posterior 16 73, l (15,2) 46,7 (7,2) 21 74,3 (15,5) 42,8 (9,2) 24 84,0 (14,6) 48,3 (12,2) --- Dorso/ 16 74,2 (30,1) 45,3 (20,4) 21 64,0 (12,0) 33,8 (6,6) 24 68,0 (23,1) 35,6 (9,1 ) Palm 16 74,7 (18,3) 46,5 (10,4) 21 66,7 (17, 1) 39,4 (8,7) 24 82,4 (11,3) 56,4 (12,5) Face Periorbicular (righi) 16 89,7 (10,4) 65,4 (15,2) 21 90,4 (14,0) 66,l (14,9) 24 93,4(11,3) 67, l (12,1) (left) 16 90,6 (10,3) 65,2 (15,3) 21 92,7 (9,7) 64,6 (12,0) 24 96,5 (10,0) 72,0 (8,5) (Front) 16 86,2 (12,3) 65,0 (9,7) 21 87,2 (13,3) 62, I (10,7) 24 92,4 (14,1) 67,3 (18,5) Chin 16 95,5 (12,8) 65,3 (11,6) 21 83,5 (23,6) 56,3 (20,4) 24 96,3 (9,3) 74,7 (20,9) Table /. Cutaneous hydration readings obtained in different anatomica/ regions, at different room temperatures, from two "capacitance" measuring systems (CM820 and CM825) Values shown correspond to Mean ±standard deviation. Mean (SO) standard deviation 141

36 Study on the m vivo performance of two copoclfonce systems ossessment of the expenmentof Front - Chin Periocular Jeft Periorbicular Hand Dorsal 16 C Hand Palm - Leg Poster. LegAnter. Forearm d Forearm v - o CAPACITANCF.(AU) A. CM825 CM820 Front - Chin Periocular Jeft - Periorbicular Hand Dorsal - Hand Palm Leg Poster. LegAnter. Forearm d 21 C Forearm v o CAPACITANCE (AU) B. CM825 CM820 Front Chin Periocular Jeft 24 C Periorbicular - Hand Dorsal Hand Palm Leg Poster. - LegAmer. - Forearm d Forearm v o IO CAPACtTANCE (AU) c. CM825 CM820 Flg. 1. Schematic drawn of the cutaneous hydration volues obtained in different anatomico/ regions at different room temperotures (A. 16 C; B. 21 C; C. 24 C). Colour bors correspond to the two copocitance systems used to quantitotive/y describe epidermal water content. 142

37 L. Rodngues. Y R Salgueiros. N. Go/ego. P. Pinta. N. 51/va. I. Z Ferro Table II CM820 Forearm c c c Ventral 72,8 (10,5) - 67,4 (8,38) 72,8 (l 0,5) - 77,6 (14,6) 67,4 (8,3) - 77,6 (14,6) n.s Dorsal 66,3 (l 4,0) - 58,9 (l 2,4) 66,3 (14,0) - 70,6 (10,4) 58,9 (l 2,4) - 70,6 (I 0,4)* n.s n.s Leg n.s n.s Anterior 62,4 (I 0,2) - 65,4 ( 13,9) 62,4 (10,2) - 75,2 (14,4)* 65,4 (13,9) - 75,2 (14,4)* n.s Posrerior 73,1 (15,2)- 74,3 (15,5) 73,I (15,2) - 84,0 (14,6) 74,3 (15,5) - 84,0 (14,6) Hand n.s n.s n.s Palm 74,2 (30,1)-66,7 (17,1) 74,2 (30, I) - 82,4 (11,2) 66,7(17,1) - 82,4(11,2) n.s n.s n.s Dorsal 74, 7 (l 8,3) - 64,0 (12,0) 74,7 (18,3) - 68,0 (23,1) 64,0 (12,0) - 68,0 (23, I) Periorbicular n.s n.s n.s Righi 89,7 (10,4) - 90,4 (14,0) 89,7 (10,4)- 93,4 (11,3) 90,4 (14,0) - 93,4 (11,3) n.s n.s n.s Left 90,6 (10,3) - 92,7 (9,72) 90,6 (10,3) - 96,5 (10,0) 92,7 (9,72) - 96,5 (l 0,0) n.s n.s n.s Chin 95,5 (12,8) - 83,5 (23,6) 95,5 (12,8) - 96,3 (9,27) 83,5 (23,6) - 96,3 (9,27) n.s n.s n.s Front 86,3 (12,3) - 87,2 (13,3) 86,3 ( 12,3) - 92,4 (14, I) 87,2 (13,3) - 92,4 (14,1) n.s n.s n.s CM825 Forearm c c C Ventral 46,4 ( 10,7) - 42,8 (8,68) 46,4 (10,7) - 49,2 (15,5) 42,8 (8,68) - 49,2 (l 5,5) n.s n.s n.s Dorso/ 38,5 (9,08) - 34,1 (7,31) 38,5 (9,08) - 43,6 (14,4) 34, 1 (7,3 1) - 43,6 (14,4) Leg n.s n.s n.s Anterior 41,4 (10,5)- 38,1 (5,72) 41,4 (I 0,5) - 45,9 (16,2) 38,1 (5,72) - 45,9 (16,2) n.s n.s n.s Posterior 46,7 (7,18) - 42,8 (9,17) 46,7 (7,18) - 48,3 (12,2) 42,8 (9,17) - 48,3 (12,2)* Hand n.s n.s Palm 45,3 (20,4) - 33,8 (6,64) 45,3 (20,4) - 56,4 (12,5) 33,8 (6,64) - 56,4 (12,5)* n.s n.s Dorso/ 46,5 (I 0,4) - 39,4 (8,69) 46,5 (10,4) - 35,6 (9,13) 39,4 (8,69) - 35,6 (9,13) Periorbicular n.s n.s n.s Right 65,4 (15,2)-66,1 (14,9) 65,4 (15,2) - 67, I (12,I) 66, I (14,9) - 67, I (12, I) n.s n.s n.s Left 65,2 (15,3) - 64,6 (12,0) 65,2 (15,3) - 72,0 (8,54) 64,6 (12,0) - 72,0 (8,54) n.s n.s n.s Chin 65,3 (l 1,6) - 56,3 (20,4) 65,3 (l 1,6) - 74, 7 (20,9) 56,3 (20,4) - 74,7 (20,9) n.s n.s n.s Front 65,0 (9,66) - 62,1 (10,7) 65,0 (9,66) - 67,3 (18,5) 62, I (I O, 7) - 67,3 (l 8,5) n.s n.s n.s Table Il. Summorised descriptive stotistics regarding results comparison ofthe epidermal "capacitance values obtained with the two eva/uation systems (CM820 and CM825) in different anatomica/ regions, at different room temperatures (see text and Table I). Mean ± (SD) standard deviation: n.s. non significant: p<

38 Study on the 1n vivo performance of two capaclfance systems assessment of the expenmental. 25 CM n 15 IO 5 O-r-~~-,-~~---.~~~.--~~.---~~--.~~-,-~~---.~~~.--~~.---~~~ IO A. CAPACITANCE (AU) l:r ~ 25 CM825 n B. CAPACITANCE (AU) l:r Fig. 2. Frequency distribution histogram involving the toto/ "copocitonce volues obtoined from the group of vo/unteers, independently from the anatomica/ site considered at different room temperatures (see text). (A. Corneometer CM820; 8. Corneometer CM825). maximum descriptive information on the epidermal hydration state changes occurring following specific extem al inputs. The characteristic "capacitance" curves describing the processes (see ahead) were obtained through the "capacitance" systems CM820 and CM825 and compared under this frame. The POST (Plastic Occlusive Stress Test) metho- dology is particularly useful to look deeper to the dynamical aspects which rule cutaneous water exchange. By an occlusion patch it is possible to over-stimulate water exchange movements to and through the skin, allowing to obtain, after patch removal, different curve profiles which depend not only from the intrinsic characteristics of the individuai 144

39 L Rodrigues, Y R. Salguelfos, N Go/ego, P Pinta, N Si/va, I. Z Ferro Table III l6 C 21 C 24 C CM820 VS CM825 I CM820 VS CM CM820 VS CM825 Forearm Ve/1/ra/ 4,70-5,87 4,50-5, 11 5,14-5,5 1 Dorsal 5,32-5,62 5,55-6,25 5,87-8,16* Leg Anterior 4,40-4,5 1 5, 11-6, 16 5,30-5,67 Posterior I 5,70-9,85 8,02-11,4 5,84-9,02 Hand Pa/111 7,40-9,84* I 7,02-11,6* 6,60-9,02 Dorsal 7,62-12,0* 8,08-8,94 9,52-10,9 Periorbicular Right 8,81-10,0 6,78-8,93 7,30-11,8* Left 5,44-11,8 5,03-10,4* 5, 18-8,75 Face Chin 4,11-8,16* 12,6-1 1,4 5,79-7,77 Front 6, 13-5,50 6,70-9,48 6,08-7,03 Table lii. Normalised standard deviotion stotistics. Volues correspond to doto obtoined from both evo/uotion systems ot different room temperotures (see text ond Figure 3). p< 0.05 itself, specially conceming epidennal functional state, but also from the technological systems used to assess those variables ( l 0-12). Tue POST test is conducted using the TEWL and "capacitance" post-occlusion curve profiles as the sole indicators of the water dynam ical movements occurring at skin's surface. As shown in Figure 5 where the profiles resulting from the present experimental methodology are detailed, the post-occlusive process is primarily characterised by a fast increase of both TEWL and "capacitance" variables, imrnediately after the patch removal, being followed by a slow decrease toward nonna! values. These events primarily depend on the progressive embedding capacity of the epidermis facing the nonevaporated trans-cutaneous water which is being released to the environment, but also from the epiderrnal water retaining capacity which deterrnines the following decay velocities for both variables. Then, through this "stress test" it is possible to establish direct relationships between the different water masses detected on skin's surface over time. Figure 5B. clearly shows that the post-occlusive profùes of epiderrnal "capacitance" obtained from the two systems CM820 and CM825 are equivalent keeping, in spite of the different numerica! values detected, a close relationship during the all experiment (Table V). Similar correlation for the TEWL and "ca p ac i~nce" values could not be demonstrated. Nevertheless, the "capacitance" system's reproducibility was also assessed by the calculated variance coefficients (Table VI) which confirmed to be similar, almost linear, for both systems, under the present experimental conditions (Figure 6). Finally, the sensitivity analysis for both systems was assessed following the application of a mathematical (compartrnental) system analysis previously developed and tested ( l O, 11) which allowed to describe the kinetical aspects of water exchange under specific 145

40 Study on the in vivo performance of two capac1tance systems assessment of the expenmental ~ Q 20 "' I5 CM CM825 IO A CAPACITANCE (AU) 80 IOO I ~ Q 20 "' 15 CM CM825 IO B. o CAPACITANCE (AU) 80 IOO I20 24 C c ~ Q 20 "' I5 IO O "a ~ ,.. ~ ~ ~ y, ~._! i! 4l... p.& +-~~~-.~~~~~~~~~~~~~~_;;;:...:~~~_;;;~--'~ o CAPACITANCE (AU) 80 IOO I20 CM CM825 Fig. 3. Graphic presentation showing the relative dispersion tor the normalised standard deviation obtalned at different room temperatures from each "capacitance system (A. 16 C; B. 21 C; C. 24 C) 146

41 L Rodffgues. Y R Salgue1ros. N Go/ego. P Pmto. N Stiva I. Z Ferro C s ~.. "' 60 ::;: u A. o CM820 (AU) C s ~ ~ 60 ::;: u B. o CM820 (AU) C ' ~ ~.. 60 ::;: u c. o CM820 (U) U Fig. 4. Grap hic presentation of the in vivo relationship established for the epidermal capacitance obtained from both systems in different anatomica/ regions, at different room temperatures (see text). 147

42 Study on the 1n vivo performance of two capac1tance systems: assessment of the experimental... A. Temperature: 16 C Table IV em820 ve em82s ve Hydration (AU) (%) (AU) (%) low ± , ±4.03 1, ± , ± , ±2.70 4, ± , ± , ± , ±2.31 2, ±2.13 3, ± , ± , ± , ±0.14 0,17 high ± , ± ,38 B. Temperature: 21 C em820 ve em82s ve Hydration (AU) (%) (AU) (%) low 34,00 ± 2,19 6, ± 1,47 8, ± , ± , ± , ±2.81 7, ± , ±2.91 6, ± , ± , ± , ±2.78 4, ± , ± ,00 high 104.0±2.78 2, ± ,86 C. Temperature: 24 C em820 ve em82s ve Hydration (AU) (%) (AU) (%) /ow ± , ± , ± , ± , ± , ± , ± , ± , ±2.31 3, ± , ± , ± ,76 high ± , ± ,85 Table IV. In vivo hydration va/ues expressed in capacitance AU (mean value ± standard deviation) corresponding to different /evels of epidermal hydration. Data was obtained from ali vo/unteers in different anatomica/ sites at distinct room temperatures (A, B, and C). Calculated variation coefficient (VC) was expressed in percentage. 148

43 ~ L Rodngues. Y R Solgue1ros. N Go/ego, P Pinto. N 51/vo, /, Z Ferro 80,0 70,0,::;-- E 60,0.é so.o ~..J 40,0 ~ E- ""' 30,0 20,0 10,0 ~ ~ ~ "-- I T I[- ~ l I 1 T T T T T" T I I T l.l.l o.o I I o IO 20 I 30 I 40 I so 60 TIME (min) A. -+-TEWL 120,0 5 $ u ""' ;z: 100,0 80,0 ~ -u 60,0 ~ < u 40,0 20,0 o.o o IO TIME (min) 40 so 60 B CM CM825 Fig, 5, Grophic representing the meon evolution ot TEWL (A.J ond epidermol "copocitonce ossessed by the two systems (CM820 ond CM825J (8,) following o POST test (see text). 149

44 Study on the in vivo performance Qf two capaclfance systems: assessment of the experimental... Table V TEWL TEWL TEWL V. V. V. time (min) CM820 CM82S CM820 o 0,466 0,329 0,929 I 0,444 0,377 0,620 s 0,517 0,101 0,620 IO 0,660 0,239 0, ,232 0,268 0, ,305 0,028 0, ,280 0,009 0,606 so 0,322 0,114 0, ,299 0,097 0, ,419 0,088 0,803 Table V. Established correlation (r: Pearson's coefficient) tor TEWL and epidermal "capacitance assessed by the CM820 and CM825 systems following a POST test. Table VI em820 ve em82s ve Hydration (AU) (%) (AU) (%) low 57,8 ± 0,3 0,5 41,9 ±4, ,5 ± 2,7 4,1 55,5 ± 2,8 5,1 73,7 ± 2,5 3,4 62,3 ± 1,9 3,0 83,8 ± 2,1 2,5 74,6 ± 3,8 5,1 92,2 ± 1,8 2,0 80,2 ±0,2 0,3 high 111,2±7,0 6,3 98,3 ± 7,0 7,1 Table V. In vivo hydration values expressed In capocitance AUs (mean volue ± standard deviation) corresponding to different levels of epidermal hydration detected by the CM820 and CM825 systems fol/owing a POST test. Ca/culated variai/on coetticient (VC) was expressed in percentage. conditions. This model regards epidermis as a water reservoir to where water input (gain) is a relatively constant process, while water output (loss) is the lirniting factor which may be altered by topica! substances. Under this perspective, this model is designed to quantitatively screen the total water flux over time, and detect any change, specially if discrete, occurring at the cutaneous water dynamical balance. This model is described through the following expression: Cap = l.(e Ko111 1 _ e Kin*') + F. ( Max.t5) t~0 + t" 150

45 L Rodngues Y R Solgue1ros, N Go/ego. P Pinta N Silvo I Z Ferro $ 80._, < on N ~ u o CM820 (AU) Ffg, 6, Grophic presentotion of the in vivo relotionship estob/lshed for the ep/dermol "copocltonce obtoined from both systems under o POST test experimentol condition (see text), where: I F t Kin;Kout Max t so linear parameter, common to the model 's exponential terms scale factor time I st order kinetical constants Maximal hydration factor time to reach 50% of maximal hydration response curve 's sigmoidicity parameter Tue I A order kinetical variable Kout or elimination constant is a quantifiable parameter, representing an "half-life" time which is used to characterise the relative influence of any factor on skin water balance, particularly when different topica! substances are applied on skin 's surface. In this case, when a topica! formulation is used to reinforce or re-establish epidermal hydration, the expected increase on skin hydration should be measurable fora certain period of time depending on the relative formulation's "hydration power". In other words, the higher the formulation's "hydration power" the bigger the halflife time, meaning lower Kout values. Also, if we accept that the effects of those formulations may persist for longer periods of time, other parameters such as Max and t50 will allow a direct quantification of those etfects even if discrete, allowing an objective comparison of results. Tue mathematical model was applied to the analysis of the capacitance curves obtained with the CM820 and CM825 systems, following the application of different glycerol dilution (1 %, 5% and 10% aqueous solutions) in order to identify the dynarnical changes occurring on cutaneous water balance. Under this circumstances considering the low glycerol concentration chosen, the short-term assay and also, the reduced number of volunteers, one should expect that epidermal "capacitance" changes would be very discrete. In fact, only the curves obtained from the CM825 system were sufficiently defined to provide acceptable information necessary for the kinetical analysis and description. As shown in Table VII the progressive epidermal hydration increase is clearly identified from the variables Kout and Max for the CM825 system, which may confirm the higher sensitivity pointed before for this system, probably due to a better capacity in a detailed detection of lower levels of hydration. 151

46 Study on fhe 1n vivo performance of two capactfance systems assessment of the expenmental Table VII CM820 CM825 - Kout (min 1) Max (min- 1 ) t so (min- 1 ) Kout (m;i Max (m;o J t so (min- 1 ) Glycerol 1% ,686 16,999 0,089 5,294 12,007 Glycerol 5% ,956 15, I 5,375 10,583 Glycerol 10% , ,783 0,078 5,649 14,088 Tabfe V. Kinetic porometers resufting trom mothemoticof modulafion ot epidermaf "capacitance vafues obtained from CM820 and CM825 systems. These parameters were used to characterise the epidermof hydrotion evofution over time (60 min) foffowing the application of different gtycerof difutions on skin 's surface. CONCLUSION Tue overall in vivo resulrs obtained under the present experimental conditions seem to corroborate that the system CM825 corresponds to a generai evolution of its predecessor CM820. Although based on the same biophysical and technological principles, a primary distinction found regards the different nume rical scale range used in each system. Nevertheless, a close relationship, near the linearity, was found during the different phases of the experimental pro cedures. For extremely high values of epidermal hyd.ration, the CM825 seemed to be more reliable, probably for being less sensitive to other influencing factors (such as saline) as reported (l) ; good sensitivity revealed by a better capacity in identifying low hydration Jevels, and good reproducibility con ditions were also apparent. These aspects added to the practical possibility to calibrate the system are truly important positive appreciation factors con firming the interest on this new system. Still, its is necessary to gather more experimental data from this system, which should be experienced in different protocols to establish its full applicability capacities and, hopefully like its predecessor, to continue to contribute to further advances on human skin hyd.ration knowledge. 152

47 L. Rodngues, Y. R. Salgue1ros, N. Go/ego, P Pinta, N. Si/va, I. Z. Ferro REFERENCES 1. Barel A.O., Clarys P. (1997), In vitro calibration of the capacitance method (Comeometer CM825) and the conductance method (Skicon 200) for the evaluation of the hydration state of the skin, Skin Res.Techno/., 3: ; 2. Barel A.O., Clarys P. (1995), Measurements of epidermal capacitance, in "Hand Book of Non Invasive Methods and the Skin" Ed,J,Serup & G.B.E Jemec, CRC Press, Boca Raton, pp.165, 3. Berardesca E. et al., (1997), EEMCO Guidance for the assessment of stratum comeum hydration: electrical methods, Skin Res.Technol., 3: ; 4. Courage W. (1994), Hardware and Measuring Principle: Comeometer. in "Bioengineering of the Skin:Water and the Stratum Comeum", Elsner P., Berardesca E., Maibach H., Eds., CRC Press, ; 5. Gabard B., Treffel P. (1994), Hardware and Measuring Principle: The Nova DPM9003. in "Bioengineering of the Skin:Water and the Stratum Comeum'', Elsner P, Berardesca E., Maibach H., Eds., CRC Press, 177; 6. Imokawa G. (1994), In vitro and In vivo models, in "Bioengineering ofthe skin: Water and the Stratum comeum", Eds. P.Elsner, E.Berardesca and H.I.Maibach, CRC Press, p.23; 7. Leveque J.L. (1989), Measurement oftrans epiderrnal water loss, in "Cutaneous Investigation in Health and disease - Non Invasive Methods and Instrumentation", Ed.New York, M.Decker (p ); 8. Loden M., Hagforsen E., Lindberg M. (1995), Tue presence of body hair influences the measurement of skin hydration with the Comeometer, Acta Derm. Venereo/., 75: 449; 9. Pinnagoda J., Tupker R.A., Agner T, Serup J. (1990), Guidelines for Trans epidermal water loss (TEWL) measurement. A report from the standardization group of the Europ. Soc. Contact Dermatitis, Cont.Derm, 22; 164; 10. Rodrigues L., Melo M., Jaco I., Ara jo A., Abrunhosa S., Pereira, L.M. (1997), Skin Hydration Dynamics Characterised by non-parametric analysis, Skin Res.Technol; 3; P85: 207; 11. Rodrigues L., Pinto P., Galego N., Pereira L.M. (1997), Usefulness of Mathematical Modelling Application to Comparative Testing, Skin Res.Technol; 3; P86: 207; 12. Rodrigues L., Pinto P., Galego N., Amores da Silva, P., Marcelo Pereira L. (1998), TEWL kinetic modelling approach for the parameterization of skin water dynamics, submitted; 13. Rogiers A.V., (1990), Capacitance and TEWL measurements - the need for standardization, Cosm.&Toil., 115; 73-82; 14. Tagami H. (1994), Hardware and Measuring Principle: Skin Conductance. in "Bioengineering of the Skin: Water and the Stratum Comeum", Elsner P., Berardesca E., Maibach H., Eds., CRC Press, ; 15. Yamamura T., and Tezuka T. (1989), The water holding capacity of the stratum comeum measured by I H-NMR., J.lnvest.Dermatol., 93 (1): AUTHOR ADDRESS Prof. Dr Luis A.M. Rodrigues Head of Department Fac. Farmacia da Universidade de Lisboa Av. Forças Armades 1600 Lisboa - Portugal Lrodrigues@ff.ul.pt Te/: ++(351-1) Fax: ++(351-1)

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49 J. Appl. Cosmetol (October - December 1998) BIOCHEMICAL STUDIES ON A NATURAL ANTIOXIDANT ISOLATED FROM ROSEMARY: DERMO-COSMETIC IMPLICATIONS FOR ITS APPLICATION IN HUMAN ANTIAGING SKIN CARE Calabrese V., Randazzo S. D.*, Morganti P.** and Rizza V. lnstitute of Biochemistry Professor (F.R.) of Dermatology. Faculty of Medicine. University of Catania President and Director MAVI SUD. Co. Aprilia (LT) - ltaly Received: July 23, 7998 Key words: Skin aging, oxidative stress, antioxidant defense, antioxidant b iotechnology. Synopsis It is generally accepted that lipid peroxides play an important role in the pathogenesis of free radical-induced cellular injury and that antioxidants such as glutathione, ascorbic acid or alpha-tocopherol are vita! in cellular defense against endogenous or exogenous oxidants. The purpose of this work was to investigate the effectiveness of a natural extract derived from rosemary to control free radical-induced lipid peroxidation and tissue damage of skin. In the present study we provide evidence that an alcoholic extract of rosemary leaves is endowed with a strong antioxidant activity and is capable of inhibiting free radical-mediated reactions, as evaluated by both in vitro and in vivo biochemical systems. The present study is a preclinical perspective on the interface between the biochemical properties of a natural extract isolateci from rosemary leaves, a better understanding of the endogenous antioxidant potential of skin and the real validity of a natural antioxidant biotechnology in the antiaging management of skin. Riassunto È generalmente riconosciuto che i perossidi svolgono un ruolo importante nella patogenesi del danno da radicali liberi e che gli antiossidanti come il glutatione ridotto, I 'ascorbato o la vitamina E sono essenziali nelle difese cellulari contro ossidanti endogeni o esogeni. Lo scopo del presente lavoro è stato quello di esaminare l'efficacia di un composto naturale, derivato da Rosmarinus officinalis L, nella protezione della cute dal danno da radicali liberi. Nel presente studio dimostriamo che un estratto alcolico derivante da foglie di rosmarino possiede una notevole attività antiossidante, essendo capace di limitare significativamente reazioni da radica!i liberi, come osservato da test effettuati sia in vitro che in vivo. Lo studio offre una prospettiva preclinica all'interfaccia tra proprietà di un estratto naturale isolato dalle foglie di rosmarino, potenziale antiossidante della pelle ed efficacia di una biotecnologia antiossidante naturale nel trattamento antiaging della cute. 155

50 Biochemical stud1es on a natural antioxidant isolated from rosemary... INTRODUCTION In assessing dietary modulation of disease processes focus shifted over the years from the recognition in the late 1800s that diseases could be prevented by replacement of missing components, to concems in the I 960s regarding toxic food constituents. Recently the pendulum has swung back to recognizing that much of the impact of diet on disease can be attributed to the presence or absence of dietary protective agents. Among the dietary "chemoprotective" agents, compounds endowed with antioxidant properties, such as carotenoids, ascorbate, and tochopherol have been intensively investigated (1-5). It has been now extensively demonstrated that oxygen derived active species cause damage to DNA, structural proteins, carbohydrates, enzymes and, especially, the lipid components ofmembranes (6), and there is growing evidence that free radical-induced perturbation of oxidant/antioxidant balance in cell is a primary pathogenic event in a number of human diseases such as Alzheimer's disease, ataxia telangiectasia, atherosclerosis, Parkinson 's disease, cataracts, aging and carcinogenesis (7-10). Previous studies have also indicated that spontaneous ultraweak light emission from biologica! systems, due to the decomposition of long-lived intermediates produced by oxidative processes at the cellular leve!, and attributed to the relaxation of electronically excited states generated from biochemical pathways involving the interaction of free radicals and enzymatic reactions, is a reflection of in vivo oxidative stress. Consequently, the development of an in vivo oxidative stress condition may lead to enhanced chemiluminescence, as found in experimental pathological situations such as chronic ethanol intoxications, ischemie and post-ischemie brain damage or barbital treatment ( 11). Skin, highly differentiated and certainly complex organizationaj structure, is particularly vulnerable to free radical damage, because of its contact with oxygen and with other environmental stimuli. It also have been demonstrated that UV light irra- diation leads to a considerable decrease in the concentration of antioxidants naturally present in the skin. As a result, a large increase in lipid peroxidation occurs. Among the reactive oxygen species, interest in the biologica! effects of singlet oxygen has been stimulated by results suggesting that singlet oxygen is involved in the pathogenesis of various photodermatologic disorders (12-14 ). In addition to endogenous photosensitizers including porphyrins, flavins, and nicotinamide adenine dinucleotides, the skin has access to an increasing number of exogenous photosensitizers in cosmetics, medications, drugs, plants, and industriai emission. Reactive oxygen species have been implicated in the pathogenesis of the severe connective tissue dan1age present in severa! photode1matologic disorders, including drug induced phototoxicity, porphyrias, and photoaging (15). However, the free radical-related tissue changes are detennined not only by an enhanced generation of reactive metabolites, but also by the activity of the different antioicidant systems, including the endogenous cellular enzymes as well as the exogenous scavengers. In recent years we have been engaged in studies in which, using stress agents, we have modulated cellular redox state of different cellular systems, including the skin, in order to assess the possible antiaging effects of natural compounds (16-23). The presence of antioxidants in rosemary (rosmarimus officinalis L.) is well known, and severa! compounds endowed with antioxidative properties have been isolated and identified as phenolic diterpenes in th e lipophilic fracti on of rosemary extracts (24). In the present study we previde evidence that a new compound isolated from the hydrophilic fraction of a fifty per cent acqueous methanol extract of the leaves of rosemary, which we indicate as fraction or compound F is endowed with a strong antioxidant activity and is capable of inhibiting free radical mediated reactions and stress-induced damage of the skin. 156

51 Calabrese V.. Randazzo S. D.. Morganti P. and Rizza V. MATERIAL ANO METHODS Reagents Linoleic acid (18:2), Fiske-Subbarow reagent, ammonium molybdate, thiobarbituric acid, hydrogen peroxide, cytochrome c (horse-heart type VI), nitro-blue tetrazolium, hypoxanthine, superoxide dismutase (bovine) were obtained from Sigma (St. Lol!is, USA). Xanthine oxidase was from Boehringer. Ali other chemicals were analytical grade and were obtained from Merck (Germany). lsolation of fractions from rosmarinus officinalis leaves Rosmarinus officinalis L. was obtained from the locai growers in the region of Siracusa. The leaves were harvested and homogenized with 50% methanol at room temperature, and the residue was reextracted with 50% methanol. The extract was washed with n-hexane and after adding water to make the concentration of 10% methanol the combined extract was chromatographed over MCI-gel CHP 20P using a stepwise gradient elution of water and methanol. Tue 30% MeOH fraction was applied to a Sephadex LH-20 column and eluted with 50% MeOH to obtain the fraction F which was used in the course of this study. Thin Layer Chromatography (TLC) Milligram quantities of compounds present in fraction F were prepared by TLC on Silica Gel (Merck 60 F254). Resolution was accomplished with chloroform-meoh-h20 (7:3:0.5). After TLC, the band, visualized under UV light (254 nm) or by sprayng I 0% H 2 S0 4 reagent followed by heating, was scraped and eluted with 50% methanol. Eva/uation of antioxidant activity A) Tue antioxidative activity of fraction F was evaluated by a ferric thiocyanate method, according to (24). Briefly, different amounts of samples dissolved in 120 µi of ethanol were added to a reaction mixture in a screwcap via!. Each reaction mix ture consisted of 2.88 ml of 2.51 % linoleic acid in ethanol and 9 ml of 40 mm phosphate buffer (ph 7.0). The via] was placed in an oven at 40 C in the dark. At intervals during incubation, a O.I ml aliquot ofthe mixture was diluted with 9.7 ml of 75% ethanol, which was followed by adding O.I ml of30% ammonium thiocyanate. Precisely 3 min after the addition of 0.1 ml of 20 mm ferrous chloride in 3.5% HCI to the reaction mixture the absorbance at 500 nrn was measured. B) Antioxidant activity of fraction F purified by TLC was also evaluated for their quenching activity on the superoxide radical generated in vitro by the hypoxanthine-xanthine oxidase system. The reaction was carried out essentially as described by Halliwell et Al. (25). The reaction rnixture in a tota! volume of 3 ml contained O. I ml of 30 mm EDTA, 10 µl of 30 mm hypoxanthine, 100 µj of 3 mm cytochrome c or 3 mm Nitro-blu tetrazolium (NBT). The solution was brought to volume with 50 mm potassium phosphate buffer ph7.4. The reaction was started by adding 0.2 ml of xanthine oxidase freshly diluted in phosphate buffer ph 7.4 to l unitimi. Rates of cytochrome c or NBT reduction were measured at 560 nm or 550 nm respectively at 25 C. Tue antioxidant activity of the fraction isolated from the hydrophilic fraction of rosemary was measured on the basis of the percent inhibition in the reduction of cytochrome c or NBT. The concentration of cytochrome c in solution was calculated by measuring the absorbance at 550 nm before and after addition of excess sodium dithionite, using a molar reduced minus oxidized extinction coefficient of 1.85 x 104 at 550 nm. Xanthine oxidase activity was monitored in the absence of cytochrome e or NBT by measuring the assorbance at 290 nm due to ureate production. This assay was included in order to ascertain that the compounds tested did not interfere with xanthine oxidase activity. 157

52 81ochemical stud1es on a natural ant1ox1dant 1solated from rosemary. Sampling of skin surface lipids from healthy volunteers Tue study was limited to 30 adult male volunteers. The age of the subjects ranged from years old. The mean age in the group was 33 ± 11. The subjects were requested to avoid the use of hair lotions or other oil containing ointments during the duration of the experimental peri od. The experirnental procedure was designed to evaluate the effectiveness ofthe hydrophilic fraction isolated from rosemary in preventing lipid peroxidation of skin surface lipids, as compared to vitamin E. Tue study was carried out in the Experirnental Dermatology Clinic at the University Hospital. Tue 30 volunteers were divided randomly into two groups (A, and B) of 15 individuals. A sample of skin surface lipids was obtained from the forehead of each individua!. as control. Then group A was asked to apply fora week vitamin E dissolved in ethanol 20%, whereas the group B was asked to use fraction F dissolved in 20% ethanol, for the same period. Tue experimental groups were asked to wash the forehead thoroughly with neutral soap before treatments, which were repeated each day at the same hour (10:00 p.m.). Tue moming following, respectively, the first, third and the last treatment, samples of skin lipids, obtained by the swabbing technique as described below, were taken. The surface lipids were obtained by swabbing an area of the forehead (3 cm x 3 cm) with a cotton swab. The procedure was standardized such that the area was swabbed three times horizzontally and three times vertically for each individuai. Extraction of lipids The cotton swabs were extracted for their lipid content with 3 ml of chloroform:methanol mixture (1:2.5). The extraction procedure was a lowed to continue at room temperature for 2 hours in the presence of heneicosanoic acid (10 µg) as internal standard. Tue cotton was removed and re-extracted with fresh chloroform:methanol solution (l ml). Tue extraction solutions were combined and 1%NaCIin0.01 M HCI was added to the mixture and centrifuged. Tue chloroform layer from both e tractions was pooled and washed with 3 ml methanol:water (1: 1). Tue solution was centr fuged and after phase separation, the chloroform layer was recovered and evaporated to dryness under a stream of nitrogen gas in the dark. The dried lipids were dissolved in 3 ml chloroform-methanol solution (2:1) and the solution was stored at -20 C in the dark unti! analysis of lipid content and lipid peroxidation studies. Analysis of lipid conteni The content of amount of lipid extracted was assessed by the determination of phosphate, as described elsewhere ( 16) Peroxidafive stress of skin surface lipids and analysis of chemiluminescence Tue sensitivity of skin surface lipids extracted from individuals in the three experimental groups was evaluated by measuring the susceptib lity of the lipids to oxidative stress in the presence of t-butyl hydroperoxide (3 mm). Oxidative damage to lipids generally leads to the formation of long-lived intermediates, attributed to the relaxation of electronically excited states generated from free radicals reactions, and hence enhances chemiluminescence, as a reflection of in vivo oxidative stress, which has been investigated extensively as a marker for lipid peroxidation processes either in vitro or in vivo. Measurement of chemiluminescence was accomplished with a Tumer TD 20(20 luminometer, according to (26) and results were expressed as per cent of peroxidation. Statistica/ Analysis Results are the means ± S.E.M. of four to six independent experiments. Data were analyzed by oneway ANOVA, followed by inspection of ali differences by Duncan 's new multiple-range test. Dif- 158

53 Calabrese V, Rondozzo S O, Morgont1 P ond Rizzo V ferences were considered significant at P<0.05. RESULTS Figures 1-2 show the antioxidant activity obtained with the acqueous extract of rosmarinus officinalis L. Tue results are reported as percent inhibition of cytochrome e (Fig. l) or NBT reduction (Fig. 2) in the presence of µ g amounts of the fraction examined. Alpha-tocopherol was used for comparison of the antioxidant activity. As can be seen, significant inhibition of the rate of reduction of cytochrome e at 550 nm was observed in the presence of fraction F. Similar results were obtained when testing for reduction of NBT. In both cases a dose-dependent inhibition was found relative to contro) values. However, this result was somewhat lower than the antioxidant effect found 350 INHIBITION CYTOCHROME C REDUCTION IJV o o O- Comp,F -0- Vit.E Concentration (µg) Fig INHIBITION NBT REDUCTION IJV o o Comp,F -Q- Vii.E Concentration (µg) Fig

54 Biochem1ca/ stud1es on a natural antioxidant 1solated tram rosemary. with corrispondent amounts of vitamin E. Antioxidant activity of this fraction was investigated using the model of peroxidation of linoleic acid (18:2), as revealed by the thiocyanate method. As shown in Figure 3, the effects of different concentrations of compound F on the time course of peroxidation of 18:2 was monitored. Peroxidation of linoleic acid led to a time-dependent oxidative cleavage ofthis fatty acid, which was oxidized progressively during six days of incubation at 40 C in the dark. Addition of increasing amounts I O to I 00 µ g of rosemary extract delayed considerably and in a dose-dependent manner the oxidative brakdown of linoleic acid. The susceptibility of lipids to peroxidative stress was evaluated using lipid samples collected in the study performed with human volunteers. As can be seen from Figure 4, lipids collected from ANTIOXIDANT ACTIVITY OF FRACTION F o lncubation time (hr) -0- Contro! -6:- IO µg -<:)- 50 µg -tr- 100 µg 180 Fig. 3. 0,5 LUMINESCENCE PRODUCTION FROM SKJN SURFACE LlPIDS SUBJECTED TO OXIDATIVE STRESS AND ANTIOXIDANT TREATMENT 0.4 Ci: o E ;3...:l 0, ,1 o Time (min) -0- Contro! -6:- Vii E -tr- froci. f 80 Fig.4 160

55 Calabrese V., Randazzo S. D.. Morganti P and Rizza V. the fore head of healthy volunteers, were subjected to in vitro oxidative stress, performed by incubation of lipid extract with 1 mm hydrogen peroxide and the chemiluminescence measured during the time course. Altematively, Iipid extract was studied with the thiocyanate method during six days of incubation as described above, and the results showed in Figure 5. As can be seen from both figures, comparable protective effects were obsejved either in the presence of rosemary extract or vitamin E towards lipid exposed to oxidative damage. In Figure 6, we report the interesting finding of a protection afforded by topic application of the hydrophilic extract from rosemary on skin surface lipids, against oxidative challenge of the skin. In fact, lipids extracted at different times, after topic treatment with the rosemary extract exhibited a signi- 120 ANTIOXIDATIVE ACTIVITY OF DIFFERENT CONCENTRATIONS OF FRACTION F OR VIT. E ADDED TO SKIN SURFACE LIPIDS e e: o u '"" o ~ o Contro! Vii.E 100 µg * Significantly difterent from contro! (P<O.OS) Vii.E so µg Frac!. F 100 µg Fract. F so µg Fig. 5. % PEROXIDATION OF SKIN LIPIDS COLLECTED AT DIFFERENT DAYS OFTREATMENT % * 15 IO 5 o 0 Control lst E) Vii.E l'ract. F 3st 7st * Significanlly difterent from control values (P<O.OS) Days Fig

56 81ochem1cal stud1es on a natural ant1ox1dant 1solated from rosemary ficant higher resistence towards lipoperoxidative chain reactions. In the sarne figure are reported the results of application of vitamin E, which showed to be of comparable extent respect to the rosemary extract. This demonstrates that treatment with an hydrophilic fraction derived from rosemary extract significantly inhibited peroxidative damages to skin lipids. DISCUSSI ON It is increasingly evident that free radicals play a key role in determining the generai appearance of the skin. Skin, highly differentiated and certainly complex organizational structure, is particularly vulnerable to free radical damage, because of its contact with oxygen and with other environmental stirnuli. Tue prirnary target are the unsaturated lipids in the stratum corneum, the outermost layer of the skin. Most of these lipids, consist of ceramides with unsaturated fatty acyl side chains and cholesterol which also has unsaturated double bonds (27). Tue stratum comeum also contains squalene, a hydrocarbon which is readily peroxidized upon irradiation with U.V. light in the presence of oxygen. lt also have been demonstrated that UV light irradiation leads to a considerable decrease in the concentration of antioxidant naturally present in the skin ( 15). As a result, a large increase in lipid peroxidation occurs. This sensitive marker of free radical attack well correlates with irreversible damage of cellular lipid, protein and nucleic acid constituents. Moreover, lipid peroxides resulting from lipoperoxidative processes, are themselves irritating to skin, as demonstrated in human studies. The prevention of oxidative reactions in the skin lipids, through exogenous supplementation of antioxidants could have cosmetic benefits, in that may represent an efficient tool to mitigate the consequences of skin photoaging. In the present study we employed peroxidative systems to mime pathogenic conditions supposed to be involved in skin aging processes, in order to evaluate the effectiveness of a natural antioxidant, isolated from rosemary leaves in inhibiting both in vitro and in in vivo free radical-mediated reactions. In this study we provide evidence that the compound F isolated from a hydrophilic extract of rosemary leaves is endowed with strong antioxidant activity, as revealed by the in vitro tests of inhibition of reduction of cytochrome e or nitro-blu tetrazolium, as well as by studing the susceptibility of linoleic acid (18:2) to peroxidative breakdown. Tue present compound revealed a strong antioxidant activity, of level comparable to vitamin E. This activity was observed also in vivo either by exposing skin surface lipids to oxidative stress, performed in absence and in presence of added antioxidants, or after topical applicati on of this compound to the skin of healthy human volunteers, whose surface lipids taken and analysed for their resistence to oxidative stress. In this last case, a Jower susceptibility to oxidative stress was found, and this was even higher than the effect of vitamin E, as revealed by chemiluminescence analysis. Our data clearly demonstrate that the fraction F is endowed with antioxidant activity and therefore it may afford an efficient pharmacological tool to control lipoperoxidative changes of the skin, pointing out the irnportance of a natural antioxidant biotechnology in the anti-aging treatment of skin. 162

57 Calabrese V. Randazzo S O. Morgant1 P and Rizza V REFERENCES 1. Krinsky N.I. (1989) Antioxidant functions of carotenoids. Free Rad. Biol. Med. 7: Krinsky NJ. (1991) Effects of carotenoids in cellular and anima! systems. Am. f. Clin. Nun: 53(1):238S- 246S; 3. Bendich A. (1988) The safety ofb-carotene. Nutr. Cancer 11: Pryor W.A. (1991) The antioxidant nutrients and disease prevention-what do we know and what do we need to find out? Am. f. Clin. Nutr. 53(1):391S-393S. 5. Ziegler R.G. (1989) A review of epidemiologie evedence that carotenoids reduce the risk of cancer. J. Nutr. 119: Orrenius S. (1993) Mechanisms of oxidative celi damage, In: Free RadicaJs: From Basic Science to Medicine. Poli G., Albano E., and Dianzani M.U., eds. Birkhauser Verlag, Basel; Davison A.J., Tibbits G., Shi Z., Moon J. (1988) Active oxygen in neuromuscular disorders. Molec. Celi. Biochem. 84: Joenje H., Nieuwint A.W., Taylor A.M., Harnden D.G. (1987) Oxygen toxicity and chromosomaj breakage in ataxia telangiectasia. Carcinogenesis 8: Luc G., Fruchart J.C. (1991) Oxidation of lipoproteins and atherosclerosis. Am. f. Clin. Nutr. 53(1):206S- 209S. 10. Cutler R.G. (1991) Antioxidants and aging. Am. J. Clin. Nutr. 53(1):373S-379S. 11. Flecha B., Llesuy S., Boveris A. (1991) Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle. Free Rad. Biol. Med., 10, Miyachi Y. (1987) Reactive oxygen species in photodermatology. In: Hayaishi O., Imamura S., Miyachi Y (eds.). The biological role of reactive oxygen species in skin. Elsevie1; New York, Black HS (1987) Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem. Photobiol. 46: Gomer CG, Luna M., Ferracio A, Rucker N (1991) Increased transcrption and translation ofheme oxygenase in chinese hamster fibroblasts following photodynamic stress or photofrin II incubation. Photochem Photobiol 53: Vile G.F., Tyrrell R.M (1993) Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin. J. Biol. Chem. 268: Calabrese V., Dinotta F.S., Randazzo S.D., Rizza V. (1995) Evaluation of a lipid soluble fraction isolated from lemon oil extract capable of inhibiting oxidative damage to the skin. J. Appl. Cosmetol. 13: Mangiameli S., Calabrese V., CantarelJa G., Milazzo G., Nicolosi D., Rizza V. (1995) Compatibility of reduced glutathione (GSH) with different solutions currently used in anesthesia and reanimation. Minerva Anestesia/. 61: Calabrese V., Calderone A., Ragusa N., Rizza V. (1996) Effects of metadoxine on cellular status of glutathione and of enzymatic defence system following acute ethanol intoxication in rats. Drugs Exptl. Clin. Res. 22: Calabrese V., Renis M., Calderone A., Russo A., Rizza V. (1996) Stress proteins and SH-groups in oxidant-induced celi damage after acute ethanol administration in rat. Free Radical Biology and Medicine 20: Renis M., Calabrese V., Russo A., Calderone A., Barcellona M.L., Rizza V. (1996) Nuclear DNA strand breaks during ethanol-induced oxidative stress in rat brain. FEBS Letters 390:

58 Biochem1cal stud1es on a natural antiox1dant 1solated from rosemary. 21. CaJabrese V., Ragusa N., Antico A., Mangiameli S., Rizza V. (1997) Cysteine-induced enhancement of lipid peroxidation in substantia nigra: comparative effect with exogenous administration of reduced glutathione. Drugs Exp. Clin. Res. 23: Calabrese V., Randazzo G., Rizza V. (1998) Regulation of heat shock protein synthesis in human skin fibroblasts in response to oxidative stress. Relationship with the antioxidant system. Derm Clin 1: Calabrese V., Renis M., Calderone A., Russo A., Reale S., Barcellona M.L., Rizza V. (1998) Stress proteins and SH-groups in oxidant-induced celi injury after chronic ethanol administration in rat. Free Radical Biology and Medicine 24: Okamura N., Haraguchi H., Hashimoto K., Yagi A. (1994) Flavonoids in Rosmarinus officinalis leaves, Phytochemistry 37: Halliwell B., Gutteridge J.M.C. (1989) Free Radicals in Biology and Medicine. Oxford: C/arendon Press. 26. Calabrese V., Spadaro F., Dinotta F., Ravagna A., Randazzo F., Randazzo G., Ragusa N., Rizza V. (1998) Long-term ethanol adrninistration enhances urinary ultraweak lwninescence and age-dependent modulation of redox in centrai and peripheral organs of the rat. f. Tissue Reactions 20: Pryor W.A. (1987) The involvement of free radicals in chemical carcinogenesis. In: Cerutti, P.A.; Nygaard, O.F.; Simic, M.G., eds. Anticarcinogenesis and radiation protection. New York: Plenum Press; l

59 J. Appl. Cosmetol (October - December 1998) CUTANEOUS SIDE-EFFECTS OF ANTIBLASTIC CHEMOTHERAPY: AN EMERGING PROBLEM S. Mancuso, S. Greggi, R. Giannice, P. A. Margariti, M. G. Salerno, A. De Dilectis and G. Scambia Dept. Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, ltaly Received: November 7, Presented at 5'h Asian Dermatologica/ Congress, october , Beijing, China Key words: antiblastic drugs, chemotherapy, alopecia, skin toxicity, acne, erythema, hyperpigmentation, phlebitis, photosensibility, onycholysis. Synopsis Locai cutaneous complications following chemotherapeutic agents administration are generally due to accidental extravasation of drugs, while systemic toxicity may be caused by: i) a direct toxic effect, ii) hypersensitivity reactions and iii) dissemination of inflammatory response mediators by necrotic cells. Alopecia is the most common cutaneous systemic effect of antiblastic chemotherapy. Other lesions are maculo-papular eruptions, cutaneous plaques, bullae, hyperpigmentation, folliculitis and nail dystrophy (e.g. red and brown transverse lines, Mees' strips, Beau-Reil lines and onycholisis). Dermatological side effects rarely represent the dose-limiting toxicity of antiblastic drugs. Nevertheless, some of these agents, even when used at standard dose, may cause severe cutaneous side effects requiring treatment modification. With the introduction into clinical practice of new drugs and innovative treatment st:rategies and the implementation of effective hematological support, the toxic profile of chemotherapy has changed with non-hematological side-effects being the most common dose- limiting toxicity. In particular, dermatologica! toxicity involves the patient body image, with a peculiar influence on the patient's quality of life. Therefore, cutaneous side-effects increasingly involve the oncology clinical practice and require a multidisciplinary team for an adequate treatment. Controlled clinica! trials are worthwhile to explore effective means of prevention and therapy. Riassunto La tossicità cutanea da terapia antiblastica può manifestarsi a livello locale e sistemico. Le complicanze cutanee locali sono dovute a stravaso accidentale del farmaco antiblastico mentre la tossicità sistemica può essere causata da: I) un effetto tossico diretto, 2) una reazione di ipersensibilità e 3) una liberazione dei mediatori della risposta infiammatoria da parte delle cellule necrotiche. L'alopecia è l'effetto sistemico cutaneo più comune causato dalla terapia antiblastica. Altre lesioni comprendono eruzioni maculo-papulari, placche e bolle cutanee, aree di iperpigrnentazione, follicolite e distrofia ungueale (linee marroni e rosse trasversali, linee di Mees, linee di Beau-Reil e onicolisi). La tossicità dermatologica raramente rappresenta un fattore dose-limitante della chemioterapia. Tuttavia alcuni agenti antiblastici, anche se a dosi standard, possono causare effetti collaterali, a carico 165

60 Cutoneous s1de-effects of ont1blost1c chemotheropy: on emerging problem della cute e degli annessi, tali da richiedere una modificazione del trattamento. Con l'introduzione, nella pratica clinica, di nuovi farmaci e strategie di trattamento e con l'utilizzo del supporto ematologico il profilo tossico della chemioterapia è cambiato e gli effetti collaterali non ematologici sono diventati la causa più comune di riduzione della dose. In particolare la tossicità dennatologica, coinvolgendo l'immagine della paziente, esercita una particolare influenza sulla sua qualità di vita. Perciò gli effetti cutanei da chemioterapia antiblastica diventeranno di interesse crescente per il medico oncologo richiedendo un approccio multidisciplinare per un trattamento adeguato. Studi clinici controllati dovranno verificare l'effettivo significato di eventuali terapie preventive. 166

61 S Mancuso. S Greggi. R G1annice. P A Margant1. M G Salerno. A De 01!ect1s and G Scambia INTRODUCTION Tue effects of antiblastic drugs on slcin and its appendages can be either locai or systemic. Locai side-effects can be due to accidental extravasation of the drugs with different degrees of damage which can be either reversible or, occasionally, irreversible. Systernic side-effects include itching, hypo or hyperpigmentation, cutaneous eruptions, ulcerations, necrosis, alopecia, folliculitis, nail changes and radiotherapy recali reactions (1). Tue evaluation criteria of cutaneous toxicity proposed by the National Cancer Institute of Canada are detailed in table l [2]. Dermatologica! side-effects do not generally represent the dose- limiting toxicity of antiblastic drugs. Nevertheless, some of these agents, even when used at standard dose, may cause severe dermatolog ica) effects which require a dose reduction or, occasionally, discontinuation of treatment (3-5]. The introduction of human growth factors into the clinica! practice, and the increasing adoption of auto Jogous peripheral stem celi support, made it feasible to administer high-dose chemotherapeutic regirnens (6,7]. With this approach, however, non-haematological toxicity has now often become the dose-lirniting factor. Moreover, unexpected and new toxicities are observed, not seen following conventional therapies. In addition, concurrent radio-<:hemotherapeutic regimens are increasingly employed in locally advanced tumors with promising therapeutic results, but at the cost of a higher toxicity, also at the cutaneous Jevel (8). Based on the above mentioned considerations, nonhaematological toxicity and, particularly cutaneous complications, have become more frequent and increasing attention must be payed in evaluating the dermatological toxic pro file of the chemothe- Table I Toxicity GO Gl G2 G3 G4 Alopecia No loss Mild hair l~nounced or tota! Total body hair loss - head hair loss >- - Ski11 cha11ges None Localized Generalized Subcut, fi brosis or Generalized pigmentation pigmentation c~a nges localised shallow ulcerations or I changes or atrophy ulceration necrosis I Desquamati on None Dry desquamation I Moist desquamation Confluent moist - desquamation -- I Dryski11 None Mild Moderate Severe - Rash / Jtch None Scattered macular, Scattered macular or Generalized Exfoliative or papular eruption or papular eruption or symptomatic macular, ulcerating dermatitis asymptomatic erythema erythema with pruritus papular or vesicular or other associated eruption symptoms I Locai toxicity I None Pain Pain and swelling Ulceration Plastic surgery with infl ammation or indicated phlebitis Nail cha11ges None Mild Moderate Severe - -- Table I. Dermatologie Toxicity by Expanded Common Toxicity Criterio (From Brundage 1993) 167

62 Cutaneous s1de-effects of ant1blast1c chemotherapy on emerg1ng problem rapeutic regimens currently employed. In this article we review the state-of-the-art on dermatologica! side-effects caused by antiblastic chemotherapy. Locai Cufaneous Toxicify The cutaneous and subcutaneous damage is generally due to the accidental extravasation of antiblastic drugs which may cause at the site of injection eiythema, pain, urticaria! eruptions, phlebitis, ulcerations and necrosis. Drug extravasation may occur because of diffusion through the blood vessel wall or by direct injury with the needle to the vessel. The degree of morbidity depends on the type of drug used, its dose, its concent:ration in the adrninistration vehicle, and on the span of time between the occurrence of extravasation and the adoption of specific therapeutic measures. The onset of symptoms can be either early or delayed, from days to weeks. Early symptoms include loca] discomfort, pain, buming and eiythema, whereas late symptoms can be chronic pain, edema, ulceration and necrosis. Extravenous diffusi on of most cytotoxic drugs causes Joss of cutaneous compactness and, especially in regions with scanty subcutaneous fat, such as the volar surface of the hands, may cause severe damage to nerves, tendons and muscles. Conceming local side-effects, antiblastic drugs have been classified as irritating and vesiculating [9]. Initating drugs produce locai phjebitis with or without cutaneous reaction. The effects are transient and necrosis never occurs. Yesiculating agents produce a severe inflammation and necrosis is common. Rudolf and Larson classified antiblastic drugs on the basis of their property to bind DNA In fact, drugs which do not bind DNA produce mild loca! sideeffects that heal spontaneously, whereas drugs which bind DNA cause long lasting side-effects including progressive ulceration and necrosis and often require cosmetic surgical treatment. This latter group of drugs include anthracyclins, mechloretharnine, actinomycin D, rnithramycin, vinblastine, mitomycin and streptozocin [10]. Patients at risk for locai cutaneous complications from antiblastic chemotherapy are those with previous vascular diseases, under-nourishment, previous chemotherapy adrninistration, polychemotherapy adrninistration, axillruy dissection and previous irradiation at the site of infusion. Preventative measures include selection of a site of injection further from nerves and tendons (e.g. avoid injecting into the distai and palmar part of the forearm, volar hand surface and the antecubital fossa). In generai, it is advisable to avoid the sites of recent injection of other antiblastic drugs, irradiated areas or the veins in arms subrnitted to axillruy node dissection (especially ifwith lymphoedema). When ali sites of injection, at low risk of extravasation, are not available, the use of a central venous catheter is advisable. The occurence of drug extravasation is not very common for skilled nurses ( < I%), however it increases with repeated administrations[i]. Once antiblastic drug extravasation has occurred, drug administration must be promptly stopped. Prirnruy care should include blood aspiration in order to reduce the drug concentration at the site of injection before removing the needle. In the case of acute phjebitis without clear evidence of extravasation, the prompt administration of a saline solution is advisable to cleanse the vessel. Other therapeutic measures include freezing ofthe injured area for minutes, four times a day for ali drugs except alkaloids of vinca (Periwinkle). For the latter drug, heating and local infiltration with l ml hyaluronidase is the treatment of choice. In addition, the injured area must be promptly infijtrated with other specific antiblastic drug antidotes: for doxorubicin sodium bicarbonate followed by corticosteroids or locai application of dimethyl-sulfoxide and a-tocopherol for one week; for chloroethamine sodium thiosulphate followed by corticosteroids [1,9]. Only when the necrotic area is well established and delineated, a plastic surgery procedure may be considered. Sysfemic Cufaneous Toxicify Chemotherapy-related systernic cutaneous toxicity may be caused by: i) a direct toxic effect, ii) hypersensitivity reactions, and iii) dissemination of inflam- 168

63 S Moncuso, S Greggi, R G1onn1ce, P A Margont1, M G. Salerno, A De Oilect1s and G Scambia matory response mediators by necrotic cells [l]. Alopecia is the most common cutaneous systemic effect of antiblastic chemotherapy. However, other dermatologica! lesions are reported, such as maculopapular eruptions, cutaneous plaques, bullae, hyperpigmentation, folliculitis and nail dystrophy [l], Alopecia is the typical dermatologica! complication directly related to the cytotoxic effects of chemotherapy. In fact, it is due to the drug-induced damage of the rapidly growing cells of the root of the hair follicles. Tue cells responsible for hair growth have a very high metabolism and growth rate. The hair follicle produces 0.35 mm of hair every 24 hours and during this time interval most ofthe cells replicate. The antiblastic drugs which exert their cytotoxic effect during DNA synthesis and the M (mitotic) phase of the cell cycle do not distinguish between normai and tumor cells and, therefore, hair loss occurs due to severe damage of actively replicating cells [8]. The chemotherapeutic agents which induce alopecia are cyclophosphamide, camptothecin-11, taxol, taxotere, doxorubicin, daunorubicin, epirubicin, vincristine, actinomycin-d, etoposide and topotecan. Conversely, the following drugs are less frequently associated with hair loss: 5-fluorouracil, bleomycin, ifosfamide, methotrexate, melphalan, mitomycin C, mitoxantrone and teniposide [9]. However the dose, schedule and mode of administration make it quite difficult to predict the extent and the time of occurrence (e.g. complete alopecia fouows intravenous but not oral administration of cyclophosphamide ). Moreover, the impact ofthe interaction of drugs on the hair loss must be taken into account when the expected toxicity of a combination regimen is considered (Table 2). Alopecia caused by chemotherapy is mostly reversible. Often hair re-growth starts during the treatment and sometimes new hair reappear within 2-3 months. This early re-growth can possibly be explained by a proportion of hair follicles being in a quiescent phase at the time of drug administration. Indeed, the antiblastic d.rugs eliminate actively growing hair, which represents about 85%, and because the anagen phase lasts 3 months, hair re-growth can require 3-5 months. Tue psychological irnpact of hair loss is relevant to the patient and, therefore, detailed counseling may prove effective to reduce patient' s stress. Sometimes, changes in hair colour and shape occur, and usually hair re-growth is curly. It should be recommended the patient use of a soft brush, a delicate shampoo and a soft pillow cover in order to reduce hair trauma. In addition, in cases of complete baldness, the use of protection against U. V. rays is advisable [9]. Another attempt to prevent hair loss is based on the fact that the scalp is supplied by superficial blood Table II Regi men l % CMF 10 Carboplatin/5-Fluorouracil 25 Docetaxel/Ifosfamide 34 CAV 40 CMFVP 40 MOPP 42 CAE 45 Topotecan MOPP/ABVD 68 VP-1 6/Cisplatin 90 FAC 90 CMF/adriamycin 96 Taxotere I CMF: Cyclophosphamide, 5-Fluorouracil, Megestrol. CAE: Cyc/ophosphamide, Doxombicin, Etoposide. MOPP: Mechlorethamine, Vincristine, Procarbazine. CAV: Cyclophosphamide, Doxorubicin, Vincristine. CMFVP: Cyclophosphamide, Methotrexate, 5 j/11oro11racil, Prednisone, Vincristine. FAC: 5-Fluorouraci/, Doxorubicin, Cisplatin. ABVD: Doxorubicin, B/eomycin, Vinb/astine, Cisplatin. Tab/e //, lncidence of Alopecia by Different Single Agents And Polychemotheropeutlc regimens (Modified from Fischer DS, Tlsh Knobf M ond Durivoge HJ. The concer chemotheropy hondbook, IV Edition.) 169

64 Cutaneous s1de-effects of ant1blastic chemotherapy: on emergmg problem vessels which can be occluded for a short time without damage to the tissues. This may be possible by reducing with pressure the blood flow to hair follicles unti! drug plasma concentration is reduced. However, the effectiveness of this measure is stili debated. Some authors tried also to reduce hair toxicity by causing vasoconstriction of the scalp using a freezing cap. This device may prove useful, especially in the case of rapid administration of antiblastic drngs with a short half!ife (doxorubicin, vincristine, vinblastine, etc.). However, the use of scalp hypothermia is presently forbidden by F.D.A. (Food and Drug Administration) in the United States because concem exists in the presence of hematological malignancies, tumors at high risk of brain metastasis and sol id tumors which can metastasize to the cranial theca [ 11, 12]. A preventative measure to reduce hair loss could be the locai use of minoxidij, a drug effective in male baldness or alopecia areata. Duvic et al. used locai application 2% minoxidil in a randomized tria! and reported a significantly longer interval unti! maximal hair loss and a shorter interval from baseline to maximal re-growth in those patients treated with minoxidil (13]. After alopecia, the cutaneous lesions most frequently observed are erythematous to dusky eruptions, often beginning on the hands and feet. These lesions can be due to a hypersensitivity reaction and, in most instances, are represented by urticarioid, itching and edematous lesions which are confluent and involve lhe entire trunk and face (up to a generalized edema) in the most severe cases. The lesions have a rapid evolution and generally disappear in three to five weeks, leaving hyperpigmentated or desquamated areas (4,6, 14,15]. The dermatologica! systemic side-effects induced by some of the drugs more frequently associated with cutaneous toxicity, except for alopecia, are reported in table 3. Systemic cutaneous side effects, produced by a single chemotherapeutic agent, are detailed in table 4. In spite of the fact that cutaneous reactions are not infrequent, they rarely require a reduction in dosage or discontinuation of treatrnent. Predictors for those patients more prone to reactions are, at present, unclear, although a higher incidence in the oldest patients has been reported. Moreover, increased peak plasma concentrations seem not to be co1related with severity of reaction to docetaxel. This suggests that these reactions are either idiosyncratic or, more probably, related to tissue concentration via locai blood flow (3). Tue hypersensitivity reaction to taxol and teniposide originates from either teniposide I taxol-induced histamine release or Cremophor-EL-mediated release. It has been proven tlmt Cremophor-EL is responsible for the majority of cases [15, 16). Similar reactions have also been reported with the use of other agents formulated in this and other excipients (e.g. Tween- 80 for etoposide) [6]. These reactions have been greatly reduced by premedication regimens containing steroids ( e.g. methylprednisolone 40 mg i.v.) and anti-histamines (e.g. clorphenamine 20 mg i.v.) [5,17,18]. A particular form of erythematous reaction is represented by the aerai erythema, an unpredictable reaction characterized by an aerai, livid-red, painful, tender eruption preceeded by edema and tingling (also known as pajmar-plantar erythrodysestesia). Erythematous lesions induced by docetaxel are similar to acral-erythrodysestesia but are generally not limited to the aerai areas [3]. Treatrnent for erythrodysestesia and other cutaneous complications of taxanes was for presented symptoms or not required. Mediun1 to low-potency topica! corticosteroids are used in patients with mildly itching lesions, while cool compresses are used in edematous plaque-type lesions. Aerai erythema has been treated witli steroids with vruiable success. There is evidence fora reduced incidence of erythrodysestesia fouowing steroids-contain ing premedication (3). The use of pyridoxine is currently under evaluation as a possible tllerapy for erytllrodysestesia induced by docetaxel [ 19]. Disorders of cutaneous pigmentati on are more frequent among the black race, often associated witli changes in the nail colour, and caused by different type of antiblastic drugs. Sometimes the hyperpigmentation is transient and disappears after treatrnent. [ 1,9]. Polycyclic pigmentati on of the scalp after daunorubicin chemotherapy has been described (20]. Skin biopsies of such pigmentated lesions revealed an increase of melanin, thought to be of major impor- 170

65 S Mancuso, S Greggi, R G1annice, P A Marganf1, M G Salerno, A De Dilect1s and G. Scambia tance. Since black people have a higher number of melanocytes in the skin and nail matrix, they are predisposed to an increased incidence of this sideeffect. Tue use of busujfan has been frequently associated with generalized darkening of the skin which resembles that of Addison disease [ 1]. Photosensitivity reactions to methotrexate, 5-fluorouracil and cyclophosphamide produce a persistent skin suntan in sun-exposed areas. In addition, 5- fluorouracil produces hyperpigmentation along the course of the injected veins [ 1,9,2 1]. Sometimes doxorubicin and actinomycin-d cause hyperpigmentation of the tongue and ora I mucosa as well as of the skin between the fingers. A bizarre hyperpigmentation pattern on the trunk (linear strips) has been reported in approximately % ofthe patients treated with bleomycin. Often sclerodermia-like alterations of the skin occur with hyperpigmentation. Also, elevated doses of carmustine may produce cutaneous erythema which ends in persistent hyperpigmentation resembling a suntan. A prolonged exposure to hydroxyurea can cause hyperpigmentation of skin areas which are under pressure (e,g, the posterior). The hyperpigmentated areas subsequently become dihydrated and atrophic [I]. Tue drugs which most frequently cause nail changes are cyclophosphan1ide, anthracyclins, 5-fluorouracil and bleomycin. Tue use of polychemotherapy increases the incidence of this kind of toxicity (Table 5), - I Drug Authors Toxicity Table III +3. Teniposide I Kellie, 199 l Urticaria, maculopapular rash, itch, edema 30 - Etoposide Kellie, 1991 Urticaria, maculopapular rash, itch, edema 28 - Carboplatin Chang, 1995 Itching, urticaria Topotecan Kudekka, 1996 Maculo-papular erythema I 20 Taxol* Olson, 1998 Hypersensitivity cutaneous reactions Taxotere Hoesel, 1994 Erythema 70 Burning 5 ltching 35 Desquamation 35 Maculas-Papulas 30 ~ - Taxotere* Pronk, 1998 Maculo-papular erythema * Corticosteroid premedication Table lii. Skin Toxicity lncidence by Some Chemotherapeutic Drugs 171

66 Cutaneous s1de-effects of ant1blast1c chemotherapy on emergmg problem Drug Toxic effect Table IVa --- Acti11omicy11-D Amsacrine Azacytidine Bleomycin 811s11/pha11 Carbopla1i11 Carm11sti11e Cyc/ophosphamide Cisplatin Cytarabine Camptothecin Dacarbazine Acne, erythema, hyperpigmentation, alopecia, extravasation necrosis Phlebitis, injection site pain, erythema, urticarioid eruptions Alopecia, infrequent itching erythematous eruption Pain in the site of injection, phlebitis Hyperpigmentation, dry skin, alopecia, urticarioid eruptions, erythema lnfrequent erythema, urt icarioid eruptions and alopecia Alopecia, hyperpigmentation Alopecia, nail and skin hyperpigmentation, dermatitis Alopecia, phlebitis Erythema, infrequent a lopecia, maculopapular lesions Alopecia, erythema Rare alopecia, maculas-papulas, -- - Da1111ol'llbicin Doxor11bicin Edatrexate Epil'llbicin Etoposide Floxuridine F/11darabine Maculas, papulas, urticarioid eruption, alopecia, phlebitis, extravasation necrosis, nail pigmentation Alopecia, nail hyperpigmenauion, recai! reactions, vesciculating, urticarioid eruptions Rare maculas, papulas, alopecia Alopecia, nail and skin hyperpigmentation, recali,reaction, phlebitis, extravasation necrosis. flushes Alopecia,maculas, papulas, itching, recali reaction, phlebitis, hyperpigmentation Rare alopecia, erythema, maculas, papulas, hyperpigmentation, itching ---,_ Rare alopecia, maculas, papulas 5-F/11oro11racil Hexamethylmelami11e Hydro).y11rea /darubicin lfosfamide Nail dystrophy, photosensibilization, dry skin, hyperpigmentation, maculas, papulas, venous pigmentation - Erythema, alopecia (infrequent) Maculas, papulas, erythema, hyperpigmentation, itch, alopecia, recali reaction Alopecia, extravasation necrosis, urticarioid reactions, recali reaction Alopecia, maculas, papulas, nail dystrophya, hyperpigmentation Table /Va. Systemic Cutoneous Side-Effects by Chemotheropeutic Drugs 172

67 S Mancuso, S Greggi, R G1annice, P A. MargarJ/1, M G. Salerno, A De D1/ect1s and G Scambia Table IVb Drug Lomustine Mechlorethamine Melphalan Menogaril Toxic effect Infrequent alopecia Vein hyperpigmentacion, extravasation necrosis, erythema, hyperpigmentation, itching, buming ltching, maculas, papulas, rare alopecia Alopecia, phlebitis, itching, urticarioid eruption, bullas Mercaptopurine Extravasation necrosis, maculas, papulas, hyperpigmentation -- Methotrexate Erythema, maculas, papulas, itching, urticarioid eruption, rare alopecia, photosensibilization, hyper-hypopigmentation, acne, bullas, folliculitis ~ Mitoguazone Extravasation necrosis, phlebitis -- Mitomycin-C Alopecia, photosensibil ization, erythema, maculas, papulas, itching, extravasation necrosis, phlebitis Mitoxantrone Navelbine PALA ~ Pentostatin Alopecia, itching, dry skin - - Alopecia, phlebitis -- - Maculas, papulas, recali reaction Maculas, papulas, dry skin Plicamycin Prednimustine Procarbazine Extravasation necrosis, phlebitis, hyperpigmentation Alopecia, maculas, papulas Maculas, papulas, urticarioid eruption, photosensibilization Streptozocin Phlebitis -- - Suramin Maculas, papulas, itching, urticaroid reaction Taxol Alopecia, hypersensitivity reaction. -- Taxotere Alopecia, maculas, papulas, hypersensitivity reaction Teniposide Alopecia, maculas, papulas, hypersensit ivity reaction Tiothepa Rare a lopecia, maculas, papulas, erythema, hyperpigmentation - Thioguanine Topotecan -- Trimetrexate Drug Macula, papulas Alopecia, maculas, papulas. itch, urticarioid reaction Maculas, papulas, itching, hyperpigmentation, alopecia, recali reaction Tox ic effect - Uracil Vinblastine Alopecia, itching, hyperpigmentation Rare alopecia, extravasation necrosis, maculas, papulas, photosensitivity Vindesine Zidovuldine I Alopecia, extravasation necrosis ltching, maculsa, papulas Table /Vb, Systemic Cutoneous Side Effects b y Chemotheropeutic Drugs 173

68 Cutaneous side-effects of antiblostic chemotherapy on emerging problem The disorders are represented by changes in nail pigmentation, increased fragility, transverse ridgings, subungueal erythema, subungueal hemorrhaging and onycholysis. Tue pathogenesis of nail dystrophy remains unknown. The changes in the nail colour can be represented by white transversal strips (so called Mees' strips) followed by white nail furrows (so called Beau 's lines)[22,23]. Altematively changes are represented by brown transverse bands or red trasverse lines [20,25]. Ben-Dayan et al. described Mees' stripes and Beau 's lines after polychemotherapy containing etoposide, doxorubicin, cyclophosphamide, vincristine, bleomycin followed by DVIP protocol (dexa- Drug Bleomycin Cyclophosphamide Doxorubic in Table V Nail side-effect Loss Hyperpigmentation Hyperpigmentation 5- Fluorouracil Dystrophya and loss Taxotere Daunorubic in Mitoxantrone Doxorubicin R ubidomycin Hydroxyurea Onycholysis Subungueal erythema/haemorrage Onycholisis Hyperpigmentated transversal saips Mees' saips (white transversal saips) Beau-Reil lines (white furrows) Onycholysis Red transverse lines Red transverse lines Fragility Modifi ed from Fischer DS, Tish Knobf M and Duri vage HJ. (The cancer chemotherapy handbook, IV Edition.) Tab/e Il. Noi/ Chonges by Some Chemotheropeutic Agents cort, etoposide, ifosfamide, cisplatin and mesna) with no change in nail colour and nail resurning their normal appearance during chemotherapy-free intervals [22]. Tue occurence of the Mees' strips has al so been reported following daunorubicin administration, and a dose-dependent correlation has been demonstrated [20]. Red transverse lines are described after doxorubicin and rubidomycin [24,25]. Dark strips have been reported with histological evidence of increased pigmentation of the ventral ungueal plate: the pigment accumulates at the nail base and progressively proceeds with the growth ofthe nail [1,20]. A possible mechanism involved in the pathogenesis of altered nail pigmentation is the increased activity of melanocytes in the nail matrix, perhaps due to increased levels of the Melanocyte-Stirnulating Hormone (MSH). Lastly, MSH could conjugate with daunorubicin and then binds to MSH receptors stimulating an increased production of melanin [20]. Subungueal erythema (and sometirnes even subungueal hemorrhaging) have also been described, resulting in subsequent nail loss, especially during taxotere administration [3,16]. Onycholysis is the separation of the nail from the ungueal bed which frequently occurs in cases of psoriasis, infections, thyroid dysfunctions, trauma and antiblastic chemotherapy such as with bleomycin, taxotere and others. Recovery usually starts 3 to 4 months after chemotherapy terrnination and many months are needed to retum to normai. Few cases of onycholysis have been reported following mitoxantrone in combination with methotrexate and mitomycin-c, but a possible underreporting of onycholysis has been suggested by some authors [22,26]. Folliculitis is rather frequent after actinomycin-d administration and occasionally it has been described after high dose methotrexate [l]. Antiblastic drugs can cause severe damage to irradiated tissues. This cumulative toxicity, defined as recali reaction, is especially evident in the skin. Dactinomycin is the drug most frequently involved in such post-irradiation skin reaction [1,9]. Tue reaction occurs usually within 1 week after radiotherapy but sometirnes it may occur even Iong after. Other agents which may cause this 174

69 s. Mancuso. S. Greggi. R. Giannice. P. A. Margariti. M. G. Salerno. A. De Dilectis and G. Scambia kind of toxicity include doxorubicin, 5-fluorouracil and hydroxyurea [1]. From the clinica! standpoint, this reaction is characterized by erythema, edema and subsequent desquamation and, in more severe cases, even vesicles, necrosis and painful ulcers are described. CONCLUSIONS Dermatologica! toxic effects induced by cytotoxic drugs are increasingly observed in the every-day oncologica! practice. Although cutaneous toxicity is not a freq uent cause of treatment modifications, it has a relevant impact on the patient quality of life. This implies that effective prevention measures and the optimal management of these complications can substantially improve the tolerability and compliance of chemotherapeutic treatment. A multidisciplinary team is required for an adequate approach to cutaneous side-effects, and efforts have to be made in order to achieve a better knowledge of the clinica! picture and of the therapeutic measures. Controlled clinica! trials are stili needed to demonstrate the efficacy of the possible means of prevention and therapy. 175

70 Cutaneous side-effects of ant1blastic chemotherapy: on emerging problem REFERENCES 1. Bonadonna G., Robustelli Della Cuna G. (1996) Medicina Oncologica, V Ed. 2. Brundage J.L., Zee B. (1993) Assessing the rialibility of two toxicity scales: implications for interpretating toxicity data. J Nati Cancer Inst; 85: Zimmerman G., Keeling J.H., Burris H.A., Cook G., Irvin R., Kuhun J., McCollough M. L., Von Hoff D.D. (1995) Acute cutaneous reactions to docetaxel, a new chem~therapeutic agent. Arch Dermatol; 131: Huinink W.W.B., Prove A.M., Piccart M., Stewart W., Tursz T., Wanders J., Franklin H., Clave! M., Verweij J., Alakl M., Bayssas M., Kaye S.B. (1994) A phese II tria! with docetaxel (taxotere) in second line treatment with chemotherapy for advanced breast cancer. Ann Onco!; 5: Chang S.M., Frybergger S., Crouse V., Tilford D., Prados M.D. (1995) Carboplatin Hypersensitivity in children. Cancer; 75: Beyer J., Grabbe J., Lenz K., Weisbach V., Strohscheer I., Huhn D., Siegert W. (1992) Cutaneous toxicity of high dose carboplatin, etoposide and ifosfamide followed by autologous stem cell reinfusion. Bone Marr Tras; 10: Benedetti-Panici P., Pierelli L., Scambia G., Foddai M.L., Salerno M.G., Menichella G., Vittori M., Maneschi F., Caracussi U., Serafini R., Leone G., Mancuso S. (1997) High dose carboplatin, etoposide and melphalan (CEM) with peripheral blood progenitor celi support as late intensification for high risk cancer: non-haematological, haematological toxicities and role of growth factors administration. Br. f. Cancer; 75: Adelstein D.J., Saxton J.P., Lavertu P., Tuason L., Wood B.G., Vanamaker J.R., Eliachar I., Strome M., Van Kirk M.A. (1997) A phase III randomized trial comparing concurent chemotherapy and radiotherapy with radiotherapy alone in resectable stage III-IV squamous celi head neck cancer: preliminary results. Head and Neck; 25: Fischer D.S., TishKnobfM., Durivange H.J.. Tue cancer chemotherapy handbook,fourth Ed. Mosby Year Book. 10. Rudolf R., Larson D.L. (1987) Etiology and treatment of chemotherapeutic agent extravasation injuries. A review. J Clin Onco!; 5: Keller J.F., Blausey L.A. (1988) Nursing issues and management in chemotherapy-induced alopecia. Onco! Nurs Forum; 15: Siepp C.A. (1987) Scalp hypotermia: indications for precaution. Onco! Nurs Forum;l0: Duvic M., Lemak N.A., Valero V., Hymes S.R., Farmer K.L., Hortobagi G.N., Trancik R.J., Bandstra B.A., Compton L.D. (1996) A randomized tria! of minoxidil in chemotherapy-induced alopecia. J Am Acad Dermatol; 35: Pronk L.C., Schrijvers D., Schellens J.H.M., de Bruijn E.A., Planting ASTh, Locci-Tonelli D., Groult V., Verweij J., van Oosterom A.T. (1998) Phase I study on docetaxel and ifosfamide in patients with adavnced solid tumors. British J Cancer; 77: Kellie S.J, Crist W.M., Pui C.H., Crone M.E., Fairclough D.L., Rodman J.H., Rivera G.K. (1991) Hypersensitivity reactions to epipodophyllotoxins in children with acute lymphoblastic leukemia. Cancer; 67: Weiss R., Bruno S. (1990) Hypersensitivity reactions from taxol. J Clin Onco!; 8: Pronk L.C., Stoter G., Verweij J. (1995) Docetaxel (taxotere): single agent activity, development of combination treatment and reducing side-effects. Cancer Treat Rev; 21:

71 S. Mancuso. S. Greggi. R. Giannice. P. A. Margariti. M. G. Salerno. A. De Dilectis and G. Scambia 18. Olson J.K., Sood A.K., Sorosky J.I., Anderson B., Biller R.E. (1998) Taxol Hypersensitivity: rapid retreatment is safe and cost effective. Gynecol Onco!; 68: Vukelja SJ., Lombardo F.A., James W.D. (1989) Pyridoxine for the pa!mar-plantar erythrodysesthesia syndrome. Ann Intern Med; 111: Anderson L.L., Thomas O.E., Berger T.G., Vukelja S.J. (1996) Cutaneous pigmentation after daunorubicin chemotherapy. J Am Acad Dermatol; 37: Dunagin W.G. (1982) Clinica! toxicity of chemotherapeutic agents: dermatologie toxicity. Semin Onco!; 9: Ben-Dyan D., Mittelman M., Floru S., Djaldetti M. (1994) Transverse nail ridgings (Beau's Iines) induced by chemotherapy. Acta Hematol; 91: Lemez P. (1994) Transverse nail ridgings (Beau 's lines)induced by chemotherapy. A dose- dependent phenomenon. Acta Haematol; 92: Morris D., Aisner J., Wiernik P.H. (1977) Horizontal pigmented banding of the nails in association with adriamycin chemotherapy. Cancer Treat Rep; 61: Hanada T., Ohata Y., Miyazaki M., Yoda Y., Tanoue K., Abe T. (1979) Horizonatal pigmented bandings of the nails with daunorubicin therapy. Nippon Naika Gakkai Zasshi; 68: I Makris A., Mortimer P., Powles TJ. (1996) Chemotherapy induced onycholysis. Eur J Can; 32: Van Hoesel M., Verweij J., Catimel G., Clave! M., Kerbrat P., van Oosterom A.T., Kerger J., Tursz T., van Glabbeke M., van Pottelsberghe C., Le Bail N., Mouridsen H. (1994) Phase II study with Docetaxel (taxotere) in advanced soft tissue sarcomas of the adult. Ann Onco!; 5: Kudelka A.P., Tresukosol D., Edwards C.L., Freedman R.S., Lavenback C., Chantarawiroj C., Gonzales de Leon C., Kim E., Madden T., Wallin B., Hord M., Verschragen C., Raber M., Kavanagh J.J. (1996) Phase II study of intravenous topotecan as a 5-day infusion for refractory epithelial ovarian carcinoma. J Clin Onco/; 14: Creemers G.J., Bolis G., Gore M., Scarfone G., Lacave A.J., Guastalla J.P., Despax R., Favalli G., Kreinberg R., Van Belle S., Verweij J., W.W.T. Huinink (1996) Topotecan, an active drug in the second-line treatment of epithelial ovarian cancer: results of a large european phase II study. J Clin Onco!; 14: Cunningham D, Gilchrist NL, Forrest GL, Soukop M. (1985) Onycholysis associated with cytotoxic drug. Br Med J; 290: AUTHOR ADDRESS S.Mancuso Department of Obstetrics and Gynecology Catholic University of Sacred Heart Largo Gemelli no. 8, Rame Tel Fax:

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73 J. Appl. Cosmetol. 16, I (October- December 7998) HAIR ANO SKIN PHOTOPROTECTION IN CHINA: THE COSMETIC CHALLENGE FOR THE NEW MILLENIUM P. Morganti* Secretary Generai lnternational Society of Cosmetic Dermatology, Rome ltaly President/Director R&D Mavi Sud Sri Aprilia (LT) ltaly Received: November 7 O, Key words: Cosmetic Dermatology, Hair, Skin, Photoprotection, Sunscreens, Hair Protection Factor, Carotenoids, Hyaluronic acid, Vitamin C, 3C System, Phospholipids, PUFA. Skin hydration. In the wonderful frame of Beijing, majestic capitai of the Chinese Popular Republic, the l 4th of October tlùs year 1998, The Fifth Asian Dermatological Congress opened its works with the subject: Orientai Medicine Towards the World (Fig. 1). Fig 1. Beijing - Archgate of Kindness and Happiness and the Three Archstone Bridge, The Summer Po/ace. In this magie and channing piace rich in history, the diligent organizers didn't forget to give a big room to the new medicai field: Cosmetic Dermatology, interested above ali in preventing skin pathologies and in mantaining healthy the skin structure and its appendages, hair and nails. The first workshop on Cosmetic Dermatology, coorganized by Tue Intemational Society of Cosmetic Dermatology - ISCD - started with a long introduction of Professor Constantin Orfanos, President of The ISCD.After thanking Professor Hong-Duo Chen, President of the Congress, and ali the Chinese coordinators for their hospitality, and after briefly introducing the tasks carried out over the years by Tue ISCD, I have been asked to talk. The subject designed for me was : UV and Hair Weathering, what happens to the hair continuously stressed by UV rays and air pollutants. Of course, the hair structure suffers obvious deteriorations according to the oxidants, to the quantity and the quality of the UV exposing time (Fig. 2). Tue keratin tiles disappear from the surface, the cortical fi.bers appear and the hair splits (split tips) and "breaks" (Fig. 3). Furthermore, clorinated water pools, frequent brushings, hot phon temperature, coloring and waving increase this degradation process. (Fig. 4). What should we doto improve hair condition? New 179

74 Hair and skin photoprotection in China: the cosmetic cha/lenge tor the new millenium Fig2. Fig3. Fig4. sugar-like non-ionie tensides, such as the polyglucosides, may be used because less aggressive and with a high derma! and eco-compatibility. The right UV screen, able to reduce sun-damages, has also to be added in the shampoo compositions. According to a specific double-blind study, results on volunteers' non-treated hair have been showed by the Mavi's research team from Rome, demonstrating how the right concentration of the new screen used (the dimetyl pabamidopropil laurdimonium tosilate) filming around the hair acts, both as UV neutralizing substance and as balm-soothing-discharged substance. It seems to be walk on air, but both the screen and the polyglycoside raw materiai shampoo used, have been on the market only lately. Using this new filter it has been possible determining through a new method, the so-ca!jed hair protective factor (HPF) (Fig. 5). It has been obtained, then, a HPF 15, equa! to a 95-98% protection, comparable to a "maximum protection", just as happens for the skin UV screens. Following the lecture above, Professor Jacobson, a dermatologist from Texas, spoke about the fruit-acids: a-~ hydroxy-acids. After few years of massive and non-correct use of glycolic acid, now we come to conclusions. This a -hydroxyacid is very active as epidermolityc in cutaneous peelings that must be done only by skilled doctors, as dermatologists and plastic surgeons, but it has been of such utility its introduction not over a 10% in the antiageing cosmetics. What has been stressed by Dr. Jacobson is the importance ph has in the cosmetic preparations based on glicolic acid. As a matter of fact, if we want to obtain a better transdermal penetration, ph leve! must be around In concentration of 20% it can be used as weekly long-term "lunch-time peel". Regarding the ~ hydroxy-acids, the rediscovered salicylic acid, if used in low percentrage, is able to stimulate the cellular tumover without causing manifest erythema, as also Professor Albert Klingman from Pennsylvania University, underlined in a lecture held in a parallel session. Mr. Guarneri, Professor and Head Departement of the Dermatologica! Institute in Messina, together with Mr. Caruso, a dermatologist from Palermo, insi- 180

75 P Morgant1 sted on the necessity in using sun screens with at least a 15% protective factor, in order to protect the skin from sun damages. He also stressed the task mothers have in protecting their children from sun since childhood, that because we store up sun damage during our lifetime, expecially in the first twenty years. He mentioned, then, how purified and yeast cell wall derived ~ 1,3 glucan and polyphenols extracted from green tea leaves, are able to neutralize free radicals at cutaneous level and to have a cyto-photoprotecti ve effect protecting skin cells against the depletion of antioxidant molecules upon UV-A irradiation, promoting the growth of keratinocytes and activating macropages and neutrophils cells. As a matter of fact, it is necessary to remember that in the derrnis, free radicals produced by UV-A radiation increase the peroxidation of phospholipids into DNA, thus accelerating skin ageing. Moreover,these new active principles have been recently included also in the innovative up-dated sun cosmetics, in order to avoid the problems above and to protect the skin longer. Besides we don 't ha ve to forget the use of carotenoids, taken as nutritional supplement, first of all ~ -carotene and lycopene, that, if regularly taken at the right dosage, avoid the usual photo-allergic and/or photo-sensitiveness problem during the first sun exposures, and protects people "at risk": white complexion or affected by peculiar pathologies. Dr. Paolo Palombo, a well-known plastic surgeon from Ortopedie Hospital "Alesini" in Rome, pointed out the necessary use of shower-bath able to destroy the body bacterial charge, before the surgical operations, in the orthopaedic field also. It would be a good habit if the surgeons would advice their patients to adopt this tipology of medicai or cosmeceutical protections, at least one week before anykind of operation. As a matter of fact, a complete and continous cleansing of the whole body and hair should avoid for long terms the frequent golden staphilococcus appearance, that often hides as unpleasant host in the nose mucous membrane, in order to reproduce and reappear later on all over the body during the staying in bed. These prophylactic cleansings seem to be able, then, to reduce the problems above, providing that their use go on also in the hospital until the operation day. This kind of shower-bath is now available on the market as self-dosing foam useful mostly for the frequent cleansing of small skin areas and hands. The use of this emollient and no-defathing foam for the hands is useful also for the surgeon, because it is able to erase the colonies of bacteria and pathogeneous fungi, affecting the lipidic surface film. In this way it is possible to avoide the xerosis forms, frequent expecially among surgeons and dentists. These lectures have been followed by two interesting works made 0, :z 0,45 o -00 0,4 i=.. ""'z t:;i;l o~... t:;i;l 00 i:.:: o-.....s: > s _ s SE, ""'::i:: 0,3 2 3 Treated virgin hair Untreated virgin hair EXPOSURE TIME (time) z w o...j 9 60 E=: < u u w VJ... b 6 40 ~ :i: 3 20 o o ~ Fig5. 181

76 Hair and skin photoprotect1on m China: the cosmetic challenge tor the new millenium by Dr. Lacharriere and Dr. Rainer Schmidt, from L'Oreal laboratories in Paris, on the characterization of different skin typologies of different races living in different geographic areas (towns or countrysides). They also studied how to reproduce "in-vivo" the epidermis in order to obseive in!ab the activity done by the different active principles of cosmetic or pharmaceutical use. Dr. Lacharriere's results demonstred how dry or oily skin differently reacts according to the transcutaneous penetration of the different active principles of cosmetic use, and how the consume of the different cosmetic cathegories changes whether living in town or in the countryside. It couldn't miss the clinic inteivention on the medical use of herbs peculiar of the Chinese medicine. Professor Liu Ji-He, a well-known hystopathologist from Nanking University, presented an interesting study done on 334 acne vulgaris affected patients, who have been treated with a cosmetic mask made out from a decoction composed by three different herbs: the coptis, the suctellaria and the zhuberb. Tue obtained results showed a global improvement of 97% on comedons, papules and pustules, without negative side effects. Tue section concluded with the intervention of Professor John Kabara, a well-known American researcher, who demonstrated how it is possible to produce preservative-free cosmetics provided that specific conditions are respected in choosing the ph of the products and the typology of the raw materials used, such as lipids, emulsifiers, antioxidants, flavours, or the possible and specific active principles. These new way of thinking the compositions, will give the chance to set up new cathegories of safer cosmetic products side effects-free. The second workshop has been organized by Professor Yasuo Asada. Tue introductive lecture of Professor Constantine Orfanos, the well-known dermatologist from Berlin University, pointed out how the achieved new knowledge about the structure of the skin, expecially about the homy layer and its lipids, opened a new way to the modem cosmetology. Tue new beauty products have been enriched with biological photoprotectives (such as carotenoids) or with new anti-free-radicals compounds, such as the green tea polyphenols and the grapes pro-antocianines, or with ceramides, fundarnental skin barrier components. Ali these active principles are usually added in the modem updated cosmetic formulations. Another important factor is the environment surrounding us. Many studies demonstrated how UVA and UVB cause skin damage and fast aging, that is why the cosmetics are enriched with sun filters, both organic and inorganic, indispensible to reduce the damages above mentioned. Tue Modem dermocosmetics must be considered, then, not only beauty products able to keep the skin in a good state, but proper means to prevent skin damages and pathologies. In the near future, the new molecular biology technics and the latest knowledge acquired, will scientifically go on stimulating also the cosmetic dermatology new field. Professor Masafumi Iijirna, from Tokio University, pointed out how the majority of the Japanese middle-aged women desire to rub out from their face the cutaneous hyperpigmentations, such as melasma and solar lentigo, improved by the living in the open air. The 38% of these patients use cosmetics bought in big stores and only the 11 % ask the dermatologist a proper cure. Therefore, it is necessary that the companies deeply study these cathegories of products, in order to better evaluate the efficacy and the safety in their usage, since the 70% of the female people desire elirninating this "skin defect" aestethically very bad considered. With regards to that, Professor Jenny Hun Leei, from Seul Yosei University, said how extracellular calcium has a strong effect on the keratinocytes' prolipheration and differentiation, influencing remarkably the skin barrier, whose efficacy can be evaluated also through the contro! of these methaloids. Professor Hirohito Akamatsu, from Moriguchi Kansai University (Japan), underlined how irnportant is the cosmetics role, both for skin irnprovement and for acne vulgaris aggravation, that, as everybody knows, affects a high percentrage of young people during their puberty. That's why the cosmetics have to be scrupolously evaluated to eliminate ali the acnigenous compounds in their composition. If that will be done, the cosmetics could be considered safe in their usage and also 182

77 P Morganti useful as "prevetive" products able to reduce the percentrage of the severa! tipologies of acne. Dr. Bruce Semple, a chemical researcher from the Cincinnati Procter & Gamble, concluded this session underlining the relevance of using correct methods apt in demonstrating the real "non-aggressivity" of the cleansing cosmetic products. Too often the methods used, classify the products as "irritative" when, in normal use, they are not. It has been proposed, then, a new methodology that, well considering both the cutaneous biotype and the water hardness, is able to show quickly the aggressivity or the "non-aggressivity" of the studied cleansers. To the cosmetologica! dermatology subject has been given a big room, also in the posters session presented by The ISCD. About sun and photoprotection Professor Pinne!, from The American Duke University, talk.ed also.the use of inorganic filters is very important because they are not reactive. Among them, it has been demonstrated of such utility the use of the zinc oxide in protecting from the UV-B and UV-A, at the same time. In order to avoid phenomema of ionization and agglomeration of the zinc oxide particles, these last ones are covered with a polymer that isolates and makes them completely not-reactive to the biologica) structures. These new inorganic fùters, combined with the right organic fùters, allow to formulate sun products with a high protective factor. Dr. Antonino Di Pietro, from Marchesi Hospital (Milan - Italy), together with Dr. Giaroli showed the resujts obtained from hyaluronic acid used both by injection and/or topically applied. Tue new HJ20 collagen injectioned, resulted very efficacious as filjing in eliminating JittJe wrink:jes and "crow's feet" round lips and eyes, and in improving lips 'turgidity. Very good results have been obtained also using a HCG 1000 based on ascorbic acid, hyaluronic acid and betaglucan as coadjuvant in ali the corrective operations. Used before the sessions, HCG improve the hyaluronic acid activity reducing its painfulness, while massaging it directly on the skin already treated, it seems to prolong its activity improving its stability. Again on wrinkles subject, Caridad Ponce-Luna, from Manila, underlined the positive effects obtained in reducing the number and the depth of the wrinkles through the skin resurfacing method, after a treating term of six months. The usage of phospholipids in the modem cosmetology stili gives good results even in treating pathologies as acne.this is what results from the work presented by Professor Fabrizi, from the Dermatologica! Departement of Catholic University in Rome, and Professor Randazzo, from the University of Catania. The phospholipidic emulsion rich in linoleic acid, proves itself to be again very active in reducing the sebum from the skin surface of acne affected people, being a therapeutic means, both preventive and curative, in the juvenilis acne therapy (Fig. 6). Being extremely innocuous, the usage of this new phospholipidic lamellar structure, if used since puberty, seems to be also useful as prophylactic means in reducing acne incidence during the hormonaj production increase. The continuous usage could avoid the next pharmacological treatment, both topica! and systemic. These obtained results perfectly suit the other ones recently obtained by my research team, in ltaly, and by Mr. Ghyczy's team, in Germany. On polynsaturated fatty acids subject, Mrs. Valenzano Ferraris, in collaboration with me, underlined the importance in using ffi -3 and ffi -6 oils in the modem daily linolenic diet. As a matter of fact, both the a-linolenic and the y-linolenic produce the forrning of many prostaglandines, indispensable for their antiinflarnmatory activity. Tue importance of linolenic acid component of ceramide-1 as regulator of the Fig 6. Before and after anti-acne treatment 183

78 Hair and skin photoprotection in China: the cosmetic chal/enge tor the new millenium perspiratio insensibilis, is well-known. Both the w- 6 and the w-3 can be included in the innovative cosmeceuticals or nutriceuticals useful in reducing or avoiding wants far the necessary PUPA. Stili on the innovative nutriceuticals, together with Professor Giuseppe Fabrizi, from The Catholic University in Rome, I insisted on the interesting activity done by the carotenoids combined with the polyphenols, in reducing the oxidative stress. Using a simple and quick non-invasive new method, the ROS-meter System, it has been possible to demonstrate clinically, the depth activity anti-ros (reactive oxygen species) obtained on a group of volunteers exposed to sun radiation, who took the in-studing dietetic, compared to a control group. It has been showed how the carotenoids and the polyphenols, if taken in the right proportion and quantity, are able to modify the immunologica! answer to the sun]jght, opposing to the immuno-depressing activity typical of the UV. Beside that, according to the latest studies, made in Germany by Professor Tronnier with his equipè of researchers, it has been demonstrated that carotenoids are able also to stimulate directly the melanine production, speeding up its forming. Carotenoids decrease the rate of hydroperoxides formation and they are imp]jcated in the regulation of tyrosinase activity and, therefore, in the melanosomes' melanization.on free-radicals subject, Professor Calabrese, from The Biochemistry Departement ofthe Catania University, pointed out the interesting anti-free-radicals activity done by the rosemary officinalis extract. This hydrosoluble extract, that seems to act as a powerful anti-oxidant, can represent a newborn subject of cosmetic use, both locai and generai. Licia Tiberi and Alessio Fionda, from the Italian Mavi Sud Company researching team (Aprilia - Rame), underlined the possibility of using specific molecules far speeding up the transcutaneous absorption of the cosmetic active principles, inserted in a new dermocosmetic system : the Transdermal Cosmetic De]jvery System (TCDS). Working in the right way, in a near future it will be possible to produce patches of cosmetic use, efficacious, for example, in reducing the wrinkles' depth. Professor Luis Rodrigues, from Pharmacy Departement of Lisbon University, insisted on the importance of the Trans Epidermal Water Loss for the study of the cosmetic activity, mostly in verifying "invivo" the correct course of the skin barrier function. Of, course, to make the measurement correct and efficacious, it is necessary to know well the TEWL kinetic with regards to the area to be controlled, the time and the environmental temperature, well knowing also its mutability connected to lower or higher hydration of the normai or pathological skin. Skin hydration has also been the subject of two posters presented by Dr. Feng-Xin Zong, a dermatologist from the Second Shangai Medicai University. It has been studied the hydrating efficacy of an emulsion and its safety in using. The hydration improves in 40% even after 24 hours, while, at the same time, the skin decreases in dryness and improves in elasticity. This cream, called Thermal-S by the Vichy laboratories from L'Oreal, demonstrated to be safe in the usage and of high cosmetic tollerance. Using the 3 C System, a new medicai device far measuring skin hydration and superficial skin ]jpids, Dr. Cucinelli and his colleagues, from the Gynecology Departement of the Catholic University in Rome directed by Professor Salvatore Mancuso, controlled the skin of 51 menopaused women treated with extradiol through patch (50mg. daily of 17 Beta-extradiol far 24 weeks). Sebum and skin hydration of these patients have been controlled before and after the treatment. The treatrnent with extradiol causes a ]jght dehydratation of face and body, and an improvement in the con- Fig 7. 3C System" Dermotech 184

79 P. Morganti Fig 8. Xian - The Terracotta So/diers centration of the superficial skin lipids on the entire area of the body, both covered and uncovered. However, the usage of the estrogen improves, in its complex, the quality of the skin of all the patients treated, and the 3 C System can be used as a fast and precise means for controlling, since its easy manageability and its high accuracy in measuring (Fig. 7).Another interesting poster, presented by Professor Mancuso and his colleagues, showed in a very unexceptionable way, ali the side effects caused at a cutaneous level by the systemic chemiotheraphy, from total alopecia to different high inflammato1y reactions, from locai hyperpigmentations to axillary folliculitis and nails dystrophy. Since these therapies cause even remarkably changements on the generai patient's image, it should be desirable the inte1vention of a team of researchers able to evaluate first the aestethical damages and, second, to intervene with corrective cosmetic means also. lt seems to us of some utility to conclude the report of the cosmetological works presented at Tue Fifth Asian Dermatological Congress, with this clinic work thai shows how the cosmetic dermatology, even if in its interdisciplinariety, constitutes an irnportant branch of the dermatology not to be ignored or underestimaled, since it is useful even in the presence of important pathologies such as the tumoural ones. As it has been illust:rated, t:his Fifth Asian Dermatological Congress gave a big room to the cosmetologic dennalology also, and this represents a big award for the Chinese Dermalological Society, expeciall y for 1he President of the Congress Professor Hong-Duo Chen and for the entire scientific commission. A special thank goes 10 hong-duo Chen, chairman of the Congress and to the entire organiz.ative structure Fig 9. Beijing - The Great Wall Fig 10. /SCD Dermatologist's Group partecipating The Congress 185

80 Hair and skin photoprotection 1n China: the cosmetic challenge tor the new mil/enium of the Congress that allowed the attendees, not only to visit the wonders enclosed in the magie city of Beijing, but to breathe the magnificence of the imponent Great Wall and Xian. For many of us that had the chance to visit the city of Xian, the view of the terracotta soldiers (Fig. 8) represented a big fountain of emotional unforgettable feelings. It has to be remember that the terracotta soldiers, with their horses and chariots were built to guard the tomb of Qin Shi Huang, the first emperor of unified China. lt was Qin who began building the Great Wall, who razionalized weights and measures, who created a- single Chinese script, adopted a single currency and even gave his name to the nation of China (Fig. 9). An unforgettable Congress for an unforgettable experience (Fig. l 0). 186

81 J Appl Cosmetol 16, (October - December 1998) lndex to Volume 16, 1998 Contents: Originai Laboratory Studies Experimental study on the melanogenic effect of some cosmetics. Huang Tian, Zhong Xun. 1 Comparison of physiology parameters of the skin of face in normaj people under two different conditions. Jiang Yihong, Wang Xuemin, Lin Yinfen, Le Jiayu, 7 A comparative double-blind vehicle controlled within subject study of the efficacy and tolerability of a topica! treatment of fat deposits and cellulitis on the thighs. Stig Bruset, Erling Thom. 11 Effects of UVR on immune response of skin and evaluation of sunscreen Zhou Hua, Zhu Huigang, Zhang Zhennong 27 Sunscreen formulation: a study of siliconeemulsifiant concentration V. Gallardo, N. Aybar, A. Hemandez, M.A. Ruiz 37 Effect of gelatin-cystine and serenoa repens extract on free radicals leve! and hair growth P. Morganti, G. Fabrizi, B. James, C, Bruno 57 DPPG liposomes as preferential vehicles for "human-identical" ceramides Pedro Amores da Silva, Lufs Monteiro Rodrigues and Andre Moes 65 A peat of pajeozoic origin as a multifunctional ingredient for skin care P. Morganti, G. Fabrizi, B. James 73 Role of hyajuronic acid and vit?min C in photoageing A. Di Pietro, G. Fabrizi, U. Giaroli, L. Tiberi, C. Bruno and P. Morganti 125 Study on the in vivo performance of two capacitance system: assessment of the experimental reproducibility and sensitivity Luis Rodrigues, Yolanda R.Salgueiros, Nuno Galego, Pedro Pinto, Nuno Silva, Irene Z.Ferro 135 BiochemicaJ studies on a natural antioxidant isolated from rosemary: dermo-cosmetic implications for its application in human antiaging skin care Calabrese V., Randazzo S, D., Morganti P. and Rizza V. 155 Generai Articles Essential oils and the antioxidant compounds from Rosmarinus officinalis L.: their rational use in cosmetics. M.Prevedello, E.Veggetti, S. Rapelli. 17 A new diffusion system through the mucous membranes, skin and hair P, Morganti, A. Lanzone, L. Tiberi 45 Prospects for cutaneous wound healing in ageing skin. A working hypotesis: chitosan and ceramides M.G, Tucci, M. Mattioli Belmonte, R. Muzzarelli, G. Ricotti, A. Giacchetti, G. Biagini

82 lndex to Volume Traumatic nail abnormalities M. La Placa, A. Tosti 81 Cutaneous Side-Effects of Antiblastic Chemotherapy: an Emerging Problem S. Mancuso, S. Greggi, R. Giannice, P. A. Margariti, M. G. Salerno, A. De Dilectis and G. Scambia 165 Hai r and Skin Photoprotection in China: The Cosmetic Challenge for the New Millenium P. Morganti

83 Author lndex Author I ndex Amores da Silva P., DPPG liposomes as preferential vehicles for "human-identical" ceramides, 65 Aybar N., see Gallardo V., 37 Biagini G., see Tucci M. G., 51 Bruno C., see Morganti P., 57; see Di Pietro A., 125 Bruset S., A comparative double-blind vehicle controued within subject study of the efficacy and tolerability of a topica! treatment of fat deposits and cellulitis on the thighs, 11 Calabrese V., Biochemical studies on a natural antioxidant isolated from rosemary: dermo-cosmetic implications for its application in human antiaging skin care, 155 De Dilectis A., see Mancuso S., 165 Di Pietro A., Role ofhyaluronic acid and vitamin c in photoageing, 125 Fabrizi G., see Morganti P., 57; see Morganti P., 73; see Di Pietro A., 125 Ferro Irene Z., see Rodrigues L. M., 135 Galego Nuno, see Rodrigues L. M., 135 Gallardo V., Sunscreen formulation: a study of silicone-emulsifiant concentration, 37 Giacchetti A., see Tucci M. G., 51 Giannice R., see Mancuso S., 165 Giaroli U., see Di Pietro A., 125 Greggi S., see Mancuso &., 165 Hernandez A., see Gallardo V., 37 Hua Zhou, Effects ofuvr on immune response of skin and evaluation of sunscreen, 27 Huigang Zhu, see Hua Zhou, 27 James B., see Morganti P., 57; see Morganti P., 73 Jiayu Le, see Yihong Jiang, 7 Lanzone A., see Morganti P., 45 La Placa M., Traumatic nail abnormalities, 81 Mancuso S., Cutaneous Side-Effects of Antiblastic Chemotherapy: an Emerging Problem, 165 Margariti P. A., see Mancuso S., 165 Mattioli Belmonte M., see Tucci M. G., 51 Moes Andre, see Amores da Si Iva P., 65 Morganti P., Effect of gelatin-cystine and serenoa repens extract on free radicals leve! and hair growth, 57; A peat of paleozoic origin as a multifunctional ingredient for skin care, 73; see Di Pietro A., 125; see Calabrese V., 155; A new diffusion system through the mucous membranes, skin and hair, 45; Hair and Skin Photoprotection in China: The Cosmetic Challenge for the New Millenium, 179 Muzzarelli R., see Tucci M. G., 51 Pinto Pedro, see Rodrigues L. M., 135 Prevedello M., Essential oils and the antioxidant compounds from Rosmarinus officinalis L.: their rational use in cosmetics, 17 Randazzo S. D., see Calabrese V., 155 Rapelli S., see Prevedello M., 17 Ricotti G., see Tucci M. G., 51 Rizza V., see Calabrese V., 155 Rodrigues L. M., see Amores da Silva P., 65; Study on the in vivo performance of two capacitance system: assessment of the experimental reproducibility and sensitivity, 135 Ruiz M.A., see Gallardo V., 37 Salerno M. G., see Mancuso S., 165 Salgueiros Y. R., see Rodrigues L. M., 135 Scambia G., see Mancuso S., 165 Silva Nuno, see Rodrigues L. M., 135 Thom E., see Bruset S., 11 Tian Huang, Experimental study on the melanogenic effect of some cosmetics, 1 Tiberi L., see Morganti P., 45; see Di Pietro A., 125 Tosti A., see La Placa M., 81 Tucci M.G., Prospects for cutaneous wound healing in ageing skin. A working hypotesis: chitosan and ceramides, 51 Veggetti E., see Prevedello M., 17 Xuemin Wang, see Yihong Jiang, 7 Xun Zhong, see Tian Huang, 1 Yihong Jiang, Comparison of physiology pararneters of the skin of face in normai people under two different conditions, 7 Yinfen Lin, see Yihong Jiang, 7 Zhennong Zhang, see Hua Zhou,

84 Sub1ect lndex Subject lndex Acne, treatment,93; efficacy of phosphatidylcholine to treat, 108; 162; treatment by herbs, 182; pathogenesis, 122; role of cosmetics in, 122 Ageing, 125 AHA, 92 Alfa-Bisabolol, 110 Aloe Vera, 12 Alopecia, 97; 165 Alpha-linoleniç acid, antiinflammatory activity of, 183 Androgenetic alopecia, 57; treatment of, 59 Angiogenesis, regression of, 54 Antiblastic drugs, 165 Antioxidant, activity, 19; natural, 162 Antioxidants, 17; flavonoids as, 101; natural, 118 Arachidonic acid, release, 20 Arbutin, as whitening agent, 119 Arginine, and skin hydration, 98 Aromatherapy, 17 Aroma-chology, 20 Ascorbic acid, stable solution of, 96; for collagen synthesis, 126; coadiuvant in skin injection, 183 Basocellular carcinoma, 112 Bath-foam, as prophylactic mean, 181 Beta-carotene, to neutralize free radicals, 181 Beta-extradiol, in menopaused women, 184 Beta-glucan, defence mechanism of, 96; 110; 125; as skin-defences stimulant, 126; as skinprotectant, 181; 182 BHA, 19; 92 BHT, 19 Bio-Mud, organic Peat, 76 Biotechnology, 155 Body Mass lndex, 13 Borneol, 19 Borneol acetale, 19 Cadinene, 21 Caffeic acid, 19 Calcium, and epidermal barrier, 121; in skin barrier, 182 Capacitance, methodology, 135 Carcinoma, basocellular, 112 Carmine, 3 Carnosic acid, 19 Carnosol, 19; inhibition of nitric oxide by, 20; as photoprotectant, 118 Carotenoids, as nutriceuticals, 104; as photoprotectans, 118; 179; as antioxidants, 182 Cellulite, 11 Ceramides, in skin ageing 51; and chitosan, 55; 65; in skin barrier, 118 Ceramide III, 69 Ceramide III b, 69 Ceramide VI, 69 Chemotherapy, skin toxicity of, 97 Chitosan, carboxy butyl, 54; and celi proliferation, 54 Cinnamic aldehyde, 3 Citrus bergamium, 18 Cleansing, the skin, 91 Collagen, and photodamage, 33 Contact Dermatitis, in China, 120; induced by cosmetics, 120 Cosmeceutical, 57; a new class of, 59; a new, 91; to treat acne, 108 Cosmetic, toxicology, 17; natural, 22; acne treatment by, 93; quality, 99; and skin dryness, 99; whitening, 119; safety, 182; cleansing, 183; preservative-free, 182 Cosmetic dermatitis, 3; induced by cosmetics, 5 Cosmetic Dermatology, 179; the future of, 182 Cosmetic toxicology, 17 Cream, activity, 75 Crease lines, treatment by collagen, 130 Criptococcus neoformans, 20 Damage, UV light, 90 Defense, 155 Dermatitis, induced by cosmetics, 4 Diet supplement, and hair growth, 57 Diffusion System, 45; Dimethicone, to coat ZnO, 100 Dipalmitoylphosphoglycerol, 67 DNA, damage, 20 Dynamical analysis, 135 Dystrophy, nail, 97 Elageno~ A, 47 Enviromental pollution, 29 Epidermis, 135 Epirosmanol,

85 Subject lndex Erythema, 165 Estradiol, postmenopaused treatment by, 102 Eucalyptus globulus, 18 Exfoliation, by poly hidroxy acids, 92 Fat, 11; measurement by infrared technique, 13 Ferulic acid, 19 Fibroblast, 32; and UV, 32; inhibited proliferation of, 111; protection, 127 Flavonoids, as antioxidants, 101 Foam, cleansing, 91; self-dosing, 181; Foodstuff, 18 Free radical, 73; and skin, 101; carotenoids as anti, 104 Gamma-linolenic acid, antiinflammatory activity of, 183 Gelatin-cystine, and hair, 47; and free radicals, 62 Gelatin-glycine, and skin hydration, 47 Great Wall, in China, 185 Green tea, as photo protectant, 94; polyphenols from, 94; 182 Hair, splits 47; absorbtion; 48; increase of, 61; weathering, 90; and UV light, 90; damage, 90; tensile strenght, 90; 179; degradation, 179; sun damage of, 180 Hairgrowth, 57 Hair Protection Factor (HPF), 179 Hand, washing, 91 HCG 1000, as skin protectant, 131 Herb, chinese, 93; acne treatment by, 93; decoction to improve acne by, 182 Hesperidin, 19 Horny layer, 46 HPF, hair protective factor, 180 Hurdle technology, to preserve cosmetics, 95 HyaJuronic acid, activity, 96; 125; to make jelly skin matrix, 126; injection, 128; in ageing skin, 129; 179 Hydration, skin, 98; reduction of, 102; 135 Hydroxy acids, 92 Hyperpigmenta tion, 5; 165 HY 20, safety of, 131; viscosity, 131; scar improvement by, 132 Injection, 128 In vivo, 135 Iontophoresis, to change epidermal calcium gradient, 121 Irradiation, 32 Isorosmanol, 19 Keratin, 46 Keratinocytes, 32; growth induced by HCG 1000, 127 Keratosis, and skin dressing, 54 Koilonychia, 81; 86 Kojic acid, as whitening agent, 119 Lanolin, 3 Lavandula officinalis, 18 Limocitrin, 3 Limonene, 21 Linoleic acid, in phosphatidylcholine, 108; for skin barrier, 109; oxidative cleavage of, 160 Lipid, peroxides, 73; skin, in menopausa! women, 184 Lipids, the prevention of oxidative reactions of, 162 Lipid peroxides, role of, 155 Lipolysis, 12 Liposomes, 65; incorporation activity of, 68; as vehicle for ceramides, 70 Lycopene, to prevent the photosensitized damage, 104; as anti-free radicals conpounds, 181 Mask, chinese herbs in, 93 MDS, 45 Medicai device, 125 Melanin, 1; granules containing, 5 Melanogenesis, 1 Melanogenic effect, of cosmetic ingredients, 3 Melanonychia, 83 Merthiolate, 3 Mevalonic acid, 18 Microbial flora, skin and, 91 Microtherapy, as new injection method, 116 Moisture index, 7 Mucous Membranes, 45 Mufa, monounsaturated fatty acids, 109 Mycobacterium intracellulare, 20 Nail, princer, 81; meclian dystrophy, 82; Heller's canaliform dystrophy, 83; ingrowing, 84; brittleness, 86 NIR, near infrared technique, 13 Non-aggressivity, a new method to contro! the cosmetic cleanser, 183 NutraceuticaJ, 59; safety parameters of, 60; or nutriceutical?, 104 Nutriceutical, 57; 59; as an active diet supplement, 104; Pufa as,

86 Subject lndex Oil, essential, 17 Oncology, cutaneons side effect, 97 Onychogriphosis, 85 Onycholysis, 81; 87; 165 Onychodistrophy, post traumatic, 83 Onychomycosis, 87 Onychophagy, 82 Onychotillomania, 83 Ozone layer, 29 Oxidative stress, 20; induction, 101; as cell damage, 104; 155; 160 Parabens, 3 Paronychia, chronic, 86 Patch-Test, role of cutaneous ligands in, 115 Peat, of lake origin, 73; 30,000 years old, 74 Persona! cleanser, irritation potential, 123 PCA, and skin hydration, 98 ph, 7 Phenolic diterpens, 20 Phlebitis, 165 Phosphatidylcholine, lamellar emulsion, 108; safety of, 108 Phospholipids, 179; in acne therapy, 183 Photoageing, skin, 96; treatment of, 125 Photocarcinogenesis, 32 Photodamage, 32; by UV-B, 32 Photoonycholysis, 87 Photoprotectants, vitamin C and E as, 118 Photoprotection, by sunscreens, 94; 179; by carotenoids, 182; UV, 183 Photosensibility, 165 Photosensitivity, avoided by caroteinoides, 181 Polyglicosides, as shampoo raw materiai, 180 Polyphenols, as immunostimulants, 110 Pufa, 20; from diet, 109; 179 Qin Shi Huang, the China unifier, 186 Reactive oxigen species, 32 Rejuvenation, by skin resurfacing, 103 Resurfacing, skin, 103 Retinoids, to treat photoaged skin, 111 Rosemary, 17; stimulant, 18; oil as skin protectant, 155; antioxidant activity of, 159; extract as antifree radicals, 184 Rosemary extract, detoxifying activity of, 20 Rosmanol, 19 Rosmarinus officinalis, 17; antibacterial activity of, 21; as skin protectant, 155; antioxidant activity of, 159 Rosmariquinone, 19 ROS, determination, 60 ROS-meter, 60; system to contro! reactive oxygen species, 184 Seborrhea, 57 Sebum, increased by estradiol treatment, 102 Sebum index, 7 Self-preservation, microbial, 95 Senile lentigo, in elderly, 119 Serenoa repens, 57; activity of, 59; and hair loss, 59 Serine, and skin hydration, 98 Sesquiterpens, 18 Silicone, surfactants, 37 Silicone emulsion, stability of a, 39 Skin, physiological parameters, 8; stimulant, 18; aging, 29; irradiation, 32; 129; 155; hydration, 45; penetration, 46; dressing, 54; repair, 54; hydration, 75; 98; 179; 184; free radicals, 76; depigmentation, 76; surface lipids, 76; firmness, 76; aged, 77; cleansing, 91; microbial flora, 91; treatment by AHA, 92; ageing, 94; resurfacing procedure, 103; profile analysis, 105; banier function, 55; 107; barrier homeostasis, 109; implant, 125; injection therapy, 116; cleansing, i1titation, 123; wrinkling appearance, 125; evaluation of hydration, 135; scavengers, 156; 179; lipids in menopausa! women, 184; toxicity, 165 Solar lentigo, in Japanese women, 119 SPF, determination, 30 Staphilococcus aureus, how to reduce its growth, 181 Stratum granulosum, high leve! of calciurn in, 121 Stress protein, as molecular chaperone, 101 Sun, exposure, 31 Sunburn, 31; in childhood, 31 Sunlight, 31 Sunscreen, evaluation, 27; topica! and ora!, 94; coated ZnO as, 100; zincoxide as, 183 Sunscreens, 32; 38; wash off test of, 33; 179 TCDS, transde1mal cosmetic deliveiy system, 110; 184 Telangiectasia, treatment of, 130 Terpenoids,18 Terracotta Soldiers, in Xian (China),

87 Sub1ect lndex TEWL, measurement, 106; kinetical description of, 106; curve profile, 107; kinetic of, 184 Thigh, circumferense, 11 Tinea Capitis, treatment, 114 Tioveil, 37 Titanium dioxide, as sunscreen, 100 Tolerability, cream, 15 Toxicity, skin, 165 Transition metal, role of, 115 Tricophyton Mentagrophytes, culture, 114 Urocanic acid, immune response of, 33 Ursolic acid, 19 UV, 27; hair damage by, 90; damage repair, 94; oxidative darnage by, 104; damage, 179 UVR, 27; 31; and keratinocytes, 32; and fibroblast, 32 UV-A, rays, 94; damage, 94 UV-B, protection, 94 Vaselin, white, 3 Vitamin C, 73; cream, activity, 75; activity, 96; as whitening agent, 119; 125; for collagen synthesis, 126; 179 Zincoxide, as sunscreen, 100 Water, epidermal content of, 105 Weathering, hair, 90; 179 Would healing, chitosan in, 51 Wrinkling, 125; treatment, Cineole, 19 3C System, to contro! the skin heajth status, 102;

88 Announcement IN - COSMETICS lnternotionol So<ietv of Cosmeti< Dermatology IN-COSMETICS 1999 Paris, Aprii, 20/ ' Cosmetic Science on the brink of a new Millanium The challenges and the chances The 1999 IN-COSMETICS from Aprii stands on the brink of the new millenium. What is the future of cosmetic science? How will the cosmetic product develops? What is the client of the J d millenium? What will the marketing of Cosmetic and Toiletries products look like? Send or fax to: Verlag rur chemische Industrie, H. Ziolkowsky GmbH, Postfach , Ausburg, Germany. Tel: Fax lnternational Society of Cosmetic Dermatology, Dr. Pierfrancesco Morganti, Via Innocenzo Xl, Rome- ltaly. Tel: Fax iscds'colosseum.it

89 Announcement ISCD Conference Program Day 2 - Aprii 21, Room Diderot Session on Scientific Cosmetics Organized by the lnfernafional Society of Cosmefic Dermafology (ISCD) In cooperation with Verlag tor chemische Industrie Working Agents in Cosmetics Chairman: C.E. Orfanos, Germany C.E. Orfanos, Berlin (D) lntroduction: "Working" cosmetics. What is the future? H. Maibach, San Francisco (USA) "Developments in cosmetic formulations" G. Pierard, Liège (B) "Preacne and acneiform conditions: Mikrocomedo imaging" Coffee Break P. Morganti, Rome (I) "Vehicles and bases for sensitive skin" J.H. Saurat. Geneva (S) "Topica! retinoids in cosmetics" Lunch Break Chairman: B. Giannotti, ltaly B. Giannotti. Florence (I) lntroduction: Dermatological aspects W. Bergfeld, Cleveland (USA) "CIR-Safety regulations and alpha-hydroxy acids" H. Tronnier, Dortmund (D) "New developments in carotenoids" L. Kanerva, Helsinki (F) "Allergie dermatoses due to skin and hair cosmetics. An overall evoluti on" F.H. Kemper, Munster (D) "The progress of in vitro safety evaluation for cosmetic ingredients"

90 Announcement Discover the latest developments in fhe World of PERSONAL CARE INGREDIENTS ~-~~IW~A~~~---~~~~~~~ 1'\- ~ 1- J1'77 ~~~~~OJfiiJffm~~.:f-\1' 'Y 7- lntex - Shangai - China, 7-9 Aprii, /B) ~ ~ \ Persona I Care lngredients l1<f'1a An infernafional exhibifion and conference in fhe Asia Pacific Region feafuring raw maferials and ingredienfs for persona/ care producfs Request for more informations Should you wish lo discuss any points on Persona/ Care lngredients Asia, please contaci one of the fo/lowing addresses: HeadOffice Persona/ Care lngredients Asia The Studio, Lower Green Road, Rusthall, Tunbridge Wells, TN4 8TT UK Te/ Fax mail@stepex.co.uk Internet: Sales Office Ingrid Smith van der Linden Te! Fax For the Asia Pacific Region Persona/ Care lngredients Asia Clara lnternational Beauty Group, 91 Jalan Su/fan, Kua/a Lumpur, Malaysia Te/ Fax In China Persona/ Care lngredients Asia lntex Shangai, 88 Lou Shan Guan Road, Shangai , P.R. China Te/ Fax

91 Announcement The Division of Plastic Surgery University of California School of Medicine San Francisco, California and Davies Medicai Center, San Francisco present The 1 lth Annua/ SYMPOSIUM ON AESTHETIC SURGERY FEATURING UVE SURGERY Endorsed by the American Society of Aesthetic Plastic Surgery (ASAPS) San Francisco - California, March 18-20, 1999 Symposium Chairman: John Q. Owsley, MD Program Chairman: Bernard S. A/pert, MD Te/evised Surgery Coordinator: Issa Eshima, MD For information Lynda McNeal Office of Continuing Medicai Education University of California, San Francisco, California Telephone: + 1 (415) Fax:+ 1 (415)

92 Chiuso in tipografia: 27 gennaio 1999 Journal of Applied Cosmetology published quarterly by INTERNATIONAL EDIEMME, Via Innocenzo XI, Roma, Italy. Direttore responsabile: P. Morganti. Direzione, Redazione ed Amministrazione: Via Innocenzo XI, Roma, Italy. Stampa: Grafica Flaminia, Roma. Impaginazione: GRAFO' Comunicazione visiva, Roma. Copertina: Dr.ssa M.G. Tucci - Dip. Ricerche INRCA - Ancona Italy. Sped. abb. Postale Comma 34 art. 2 Legge 549/95 Roma. Aut. del Trib. di Roma n. 3173/83 del

93 ADVANCES IN CHILDREN'S SUN PROTECTION For a safe skin protection in children aged O to 16 L'EVOLUZIONE NELLA PROTEZIONE SOLARE PEDIATRICA per una protezione sicura della pelle da O a 16 anni NO CHEMICAL SUNSCREENS NON CONTIENE FILTRI CHIMICI PRESERVATIVE FREE NON CONTIENE CONSERVANTI ALCOHOL AND FRAGRANCE FREE NON CONTIENE ALCOOL E PROFUMO sun atock NO CHEMJCAI. sunscreen WATER RESISTANT ~ SPF 15. ' ' r 4 ~I. ~~... Mavi sud - Viale dell'industria, Aprilia (LT} - ltaly Tel Fax mavi@colosseum.it

94 PHOTOSTABLE TANNING LINE NO CHEMICAL SUNSCREENS ADDED WATER PROOF LINEA SOLARE FOTOSTABILE TOTALMENTE PRIVA DI FILTRI CHIMICI RESISTENTE ALL'ACQUA Sun sensitive skin. Pelli intolleranti al sole. Drug photosensitization. Fotosensibilizzazioni da medicamento. Photoageing prevention. Prevenzione Fotoinvecchiamento. Chloasma. Cloasma. External exposure conditions. Condizioni esterne di esposizione. Scar and stretch mark protection. Protezione delle cicatrici e delle smagliature. Protection from photodermatosis. Protezione dalle Fotodermatosi. Protection from photoallergies. Protezione dalle Fotoallergie. Prevention of sun exacerbated dermatosis. Prevenzione delle dermatosi aggravate dal sole. btl7. m VI se NrE SUN SERVlt<IG LA SCIENZA 4.l SERVIZIO DEL SOLE Mavi sud s.r.l - Viale dell'industria, l l l Aprilia ILTJ - ltaly Tel Fax E moil: movi@calosseum.it