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LA RISPOSTA DERMATOLOGICA
ALLE IPERCHERATOSI
Ammonio Lattato 7%
REDUCTION OF SURFACE LIPIOS OURING THE TREATMENT ACTIVITY CARRIEO OUT BY KERATOTAL ACNE ON THE
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9
o....---"•_ 30_ _'--•<_o_oos_wtt.g--'-_...._
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0 ~ 3,0
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~ 30
~ 1,5
8z 1,0
20 ~ 0,5
10
10 12
10 15 20 25 30 Httlmant
gloml
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z 90 + - - - - - - -_,__;___ _ __
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agents (benzoi! peroxide, retinoic acid,
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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
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ADVERSE REACTIONS
In the first days of application transient
For more information effect such as stinging or itching may be
cali to: observed
Mavi Sud Sri
V.le dell'Industria 1
04011 Aprilia (Lt) HOWTOUSE
ltaly Twice a day. Before applications cleanse
Tel. +39.6.92.86.261
Fax +39.6.92.81.523
the skin thoroughly; if stinging occurs,
E-Mail:mavi@colosseum.it reduce application to once a day for the first
URL=http://www.colosseum.it/st81/mavi ten days of treatment
REFERENCES:
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, 137-145
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TIMBRO E FIRMA
SpeclftcarecondlzlonidipagamentoefomireN" Codice Fiscale se è richiesta fattura.
Cosmetic Dermatology
Series Editor: P. Morganti
Volume 2
Every day Problems in Dermatology:
The Cosmetic Connection
This book is comprised of 41 previously unpublished papers dealing with research in various fields
of cosmetic dermatology. The main themes covered are: inter-relationship between drugs and
cosmetic in the skin; the efficacy of, and the raction to, cosmetics; cosmetics in sports and work;
cosmetics in relation to sexuality and pregnancy; and finally, the interconnection existing between
cosmetics and diet. By so comprehensively covering the science of cosmetics, this text is indispen-
sable to those involved in research and development for the cosmetics, toiletries and pharmaceutical
industries. It will also be a great benefit to university and hospital pharmacists and health care pro-
fessionals entrusted with any aspect of skin care.
E DITOR P. MORGANTI
PhD.
SECRETARY GENERAL
INTERNATIONAL SOCIETY of COSMETIC DERMATOLOGY
Via Innocenzo Xl. 41 • 00165 Roma (ltaly) ·Fax +39-6-63.80.839
EDITOR I AL ADVISORY P. AGACHE MD. Prof. of Denmat. Cenire Hosp. Regional de Besançon (F)
BOARO G. BELLOMONTE CChem. Prof. of Chem., Food Depan lsl. Sup. Sanità • Rome (I)
W.F. BERGFELD MD, FACP Cleveland Clinic Ohio (USA)
B. BERRA DSc. Prof. of Biol. Chem. Univ. of Milano (I)
R.CAPUTO MD. Prof. and Chairman. Depan of Dem1a1. Univ. of Milano (I)
O. CARLESIMO MD.• Prof. and Chairman, Depan. of Denmal. Univ. of Rome (I)
D.CERIMELE MD. Prof. and Chainman, Depan. of Derma!. Catholic Univ. of Rome (I)
E. CH IACCHIERIN I CChem, Prof. and Chairman, Dcpan. Techn. of Commerce Univ. of Romc (I)
i.COTTE DSc. Prof. of Cosmel. !PIL Lyon (F)
M.A. DINA MD. Prof. and Chairman, Dcpart. of Phatol. Anat. Catholic Univ. of Rome (I)
G. FABR!ZI MD, Ass. Prof. of Pacdriaiic Dennatologist. Catholic Univ. of Rome ( I)
A. FIDANZA DSc, Prof. and Chairman. Depan. of Physiol. Univ. of Rome (I)
D. GRAFNETTER PhD, lnst. far Clinica! and Exp. Medicine Prague (CS)
J.A.GRAHAM B.Sc, PhD. Dep1. Denmatology Univ. of Pennsylvania (USA)
L. GAGLIARDI Chainman. Depart. of Phanm. Chem. lsl. Sup. Sanità of Rome (I)
B. GUARNIERI MD. Prof. and Chainnan, Depart. of Dcnnal. Un iv. of Messina (I)
A.J.JOUHAR M.B.MRSC Beaconsfield (GB)
F.H. KEMPER MD. Emcritus Prof., Phannacology & Toxicology. Univ. Munster (D)
A.M. KLIGMAN MD. PhD, Prof. of Denma1ol. Univ. of Pennsylvania Philadelphia (USA)
N. LOPRIENO DSc, Prof. of Genetica Univ. of Pisa (I)
S. MADDIN MD, ERCP Clin. Prof. Denmatol. Div. Denmal. Univ. BR. Columbia. Vancouver (C)
G. PUGLISI CChcm. Dcpart. of Pharmacol. and Tox. Univ. of Catania (l)
C.L. MENEGHIN I MD, Prof. and Chainman, Depart. of Denmal. Univ. of Bari (I)
t L. MUSCARDIN MD. Emeritus Prof. of Dennal. Cenlre Hosp. Rcgional IDI Romc (I)
N. ORENTREICH MD, Clin. Prof. of Denmat, New York (USA)
E. PANCONESI MD. Prof. and Chainman. Depan. of Denmat. Univ. of Firenze ( I)
R. PAOLETTI MD, Prof. and Chairman, Depart. of Phannacol. and Tox. Univ. of Milano (l)
\V.E. PARISH MA, PhD. BVSc. Head or Environmental Safety Division. Unilevcr Rescarch Schan brook (GB)
L. PUGLISI DSc. Prof. of Phanmacognosy Univ. of Milano (I)
W. RAAB MD, Prof. and Chairman, Depan. of Denmal. Univ. of Wien (A)
G. RABBIOSI MD. Prof. and Chainman, Dcpan. of Denmat. Univ. of Pavia (I)
A.REBORA MD. Prof. and Chainman. Depart. of Denmal. Univ. of Genova (I)
V. RIZZA PhD. Prof. of Biol. Chem. Univ. of Catania ( I)
G. SALVATORE CChem, De pan. of Tox icol. Jst. Sup. Sanità of Rome (I)
A. SANNA MD, Prof. and Chainman, Depan. of Microbio!. Catholic Univ. of Rome (I)
P. SANTOIANNI MD, Prof. and Chainman, Depan. of Denmat. Univ. of Napoli (I)
H. SCHAEFER Ph.D., Prof. and Scientific Director L'Oreal, Paris (F)
t F. SERRI MD. Emeri1us Prof.. Depart. of Denmal. Catholic Univ. of Rome (I)
A. SERTOLI MD. Assoc. Prof. of Allergie and Occupational Dcrmat. Univ. of Firenze (I)
A. STAMMATI DSC. Depan. ofToxicol. lnst. Sup. Sanità of Rome (I)
I. TADDEI B.Sc., Prof. and Chainnan. Depart. of Pharmacol.Scicnze Univ. of Siena (I)
H. TRONNIER MD, Emcrilus Prof., Dermatology,. Univ. Winen-Herdecke (D)
V. VALKOVIC Ph.D. Prof. of Physic Ruder Boskovic lnSI. of Zagreb (CRO)
GENERAL INFORMATION
The JOURNAL OF APPLIED COSMETOLOGY is an intemational joumal devoted to publisching originai
papers, reviews and other materiai which represent a useful contribution to research on the skin and on cosme-
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It is aimed at cosmetic chemists, dermatologists, microbiologists, pharmacists, experimental biologists, toxico-
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I) Robbins CR, Kellych ( 1970) Aminoacid composition of human hair. Text Res J 40:891 -896
2) Strehler BL (1977) Time, cells and aging 2nd edn. Academic Press, New York
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Quarterly Review of Cosmetic Dermatology
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Contents
vivo, dove le cellule sono sparse nella rete della Matrice Extracellulare (ECM). Di conseguenza,
l ' uso del modello 3-D Dermal Equivalent, c he incl ude una matrice collagene-GAG-chitosano quale
maggiore componente dell 'ECM, popolata da fibroblasti, è un modo migliore per stud iare gli effetti
d i nuove molecole sul metabolismo dei fibroblasti e sulla sintesi della ECM. Viene presentata qui
una selezione in vitro di promettenti nuovi principi attivi prodotti dalla biotecnologia. Sono stati
preselezionati quattro biopeptidi dopo aver analizzato gli effetti di 200 biopeptidi sulla stimolazione
della proliferazione cellulare su colture monostrato di fibroblasti. Questi quattro biopeptidi sono sta-
ti poi testati su una coltura monostrato di fibroblasti, sia per la sintesi dei glicosaminoglicani che per
la s intesi proteica. Il più efficace, un biopeptide derivato dal latte, è stato usato per uno studio
sull'efficacia uti lizzando un modello Dermal Equi valent (DE). Questo biopeptide da latte è stato te-
stato sulla DE attraverso applicazioni sistematiche ( 1.25% (v/v) in un medium culturale) per 8 gior-
ni. Il totale proteico, il collagene, i glicosam inoglicani e la sintesi di elasticità sono state poi misura-
te rispettivamente attraverso incorporazione di prolina radioattiva, elettroforesi seguita da saggi di
densitometria e colorimetria. Queste tecniche in vitro hanno portato alla selezione di un principio at-
tivo che la neosintesi dei glicosaminoglicani è capace di indurre, per il trattamento della pelle invec-
chiata e di sidrata.
2
C Augustm. V Fre1. E Pemer, A Huc and O Damour
3
An m vitro select1on of new cosmet1c octive compounds: tram screening test on 3-0 Derma/ Eqwvolent
absence of biopeptides. After 2 to 4 days of cul- of the systemic application period, synthesised
ture, fibroblasts (n=3 wells) were detached by GAGs , tota! proteins and collagenic proteins
incubation with trypsine-EDTA (Sigma, USA) and elastin were assayed on collected and free-
and counted using a Coulter Counter (Coultro- zed culture media and into DEs. Histological
nics, USA). Tota! proteins assay (14): fibrobla- contro!: three DEs were fi xed in Boin 's solu-
sts (23 000 cells/cm 2) were cultured in 75 cm2 tion and embedded in paraffin ; 5 µ m sections
flasks for 7 days with a culture medium contai- were stained with hematoxylin-phloxin-saffron
ning 10% foetal calf serum (FCS) . After this and examined under a ZEISS IM 35 microsco-
proliferation step, the cells were cultured for 7 pe. MTI Test (16): DEs were incubated with 2
additional days with 5% FCS culture medium ml of a solution of I mg MTI/ml of PBS. The
supplemented by 1.25 % (v/v) of each biopepti- reduced MTI dye was extracted with 4 ml of
de except for the contro!. Then, the culture me- HCl 0.04N acidified isopropanol (Merk, USA)
dium was removed, the fibroblast layers rinced and mechanical agitation at room temperature
with PBS (Sigma, USA) and harvested by scra- for 30 minutes. A I 00 µI sample of each extrac-
ping. The cells were lysed with l % Tryton X tion was placed in a 96 wells-plate and the ab-
100 prior to Micro BCA method to determine sorbance was read at 550 nm on a plate reader
the amount of proteins in the suspension. Gly- (DYNATECH MR 4000) with 100 µI isopropa-
co saminoglycans assay ( 15): the fibrobla sts nol as a blank. Tota! and collagenic proteins as-
were cultured in monolayer for 14 days as de- say (17): briefly, on the 7th day of biopeptide
scribed above. In the collected culture medium, culture, 12 DEs were cultured with culture me-
the g lycosaminoglycans were extracted by etha- dium supplemented with 5 µCi/ml of (5-3H)-
nolic precipitation, separated by electrophoresis proline for 24 hours. Half of them were used for
on cellulose acetate gel in a O. I M pH 5 barium determining the tota! incorporateci rad ioactivity
buffer solution and stained with GAGs-specific using a B scintillation counter Packard Tri-carb
alcian blue. The GAGs were identified by com- 2 100 TR. The other half (n=6 DEs) was used in
parison with standards (Hya luronic Acid, Chon- order to measure the radioactivity incorporated
dro"itin-4-Sulphate, Derm atane and Heparane in the non-collageni c proteins, after a specific
Sulphate) and quantified by integrating peak degradation of collagen by collagenase (Advan-
surfaces. The assay was done in triplicate. ce Biofacture, USA). Substracting one from the
others gave the amount of radioactivity incorpo-
Test procedure on Derma/ rated in collagen expressed in radioactivity units
Equivalent (dis integrations per minutes (dpm) per DE).
Human fo reskin fibroblasts (200 000 cells/cm2) Glycosaminoglycans assays: the quantity of gly-
were seeded into freeze dried Derma! Matrix cosaminoglycans synthesised after 5 d ays of
made of 72% bovine collagen types I and III, treatment was assayed as described above in the
8% ovine chondro"itin-4-sulphate and 20% chi- collected culture medium. Elastin assay ( 18):
tosan. DEs were cultured in DMEM supplemen- the soluble fraction of elastin was assayed using
ted with 10% neonata! calf serum, 25 mg/I gen- Fastin elastin kit (B iocolor, Ireland) in the DE
tamycin, 100 000 UI/l penicillin, 1 mg/I ampho- culture medium, sampled on the last day of bio-
tericin B and 40 mmol/l L-glutamine and 50 peptide application (n=6 fractions).
mg/I ascorbic acid (Sigma, USA) for 3 weeks
and the medium was changed twice a week.
Mature DEs were cultured for 8 days in a cultu-
re medium containing 1.25% (v/v) of selected
biopeptide, except for contro! DEs. At the end
4
C. Augustin. V Frei. E. Perrier. A. Huc and O. Damour.
• 4 doys culture
1500 - T
~
E IOCX> - T Fig. I b. Synthesised G/ycosaminog/ycans evaluated by
z" electrophoresis of the culture medium of biopeptide-
J treated monolayered fibrob/ast culture versus contro/
and expressed as activation % versus contro/ (n=3) .
500 -
results, presented in Fig. l b, were expressed as
percentages of activation versus untreated con-
trai for each glycosaminoglycan detected in the
o- - culture medium: H yaluro nic Acid (HA) and
Unlrooled Whool Milk Soyo Soyc
Contro! I I I 2 Chondro'ltin-4-Sulphate (C4S) . The amounts of
GAGs measured in ali the biopeptide-treated
Fig. I a. Proliferation Rate of fibroblasts cultured in mono- culture media are significantly increased versus
layer according to the different biopeptides added in
the culture medium ( 1.25% v /v) determinated by celi
the untreated contro! (Student's test, p<O.O I ).
count after 2 and 4 days culture (n=3). Milk l biopeptide is the most effective substan-
ce tested regarding the activation percentages of
For ali the tested biopeptides, we observe a si- both HA (39%) and C4S (53%).
gnificant enhancement of the fibroblast prolife- The synthesised total proteins were evaluated
5
An 1n vitro select1on of new cosmet1c octive compounds from screening test on 3-0 Derma/ Equivolent
25
ee: (")
!"I Student s
1
8 test (p<0.01)
] 20
~e
:>
Ql 15
-= ~
6
C. Augustin, V. Frei, E. Perrier. A. Huc and O. Damour.
150
E'
:::>
'i5
Q)
E
Hyoluronic Acid Chondro'ilin·4·Sulphate ~
.2 100
:;
V
UJ
o Contro/ DE Milk l treoted
--.... 15000 culture medium culture medium
2
:::>
.Ee Fig. 3c. Synthesised Elastin evoluoted by colorimetry in
~
Q)
biopeptide-treated DEs culture medium ond contro/ DEs
o.. culture medium (n=6) .
"'oe 10000
·e
]' DISCUSSION
e
·;;;
5000
i:S
7
An in vitro setection of new cosmetic octive compounds: from screening test on 3-D Derma/ Equivolent
In this paper, we present the in vitro techniques on these four selected biopeptides by checking
used for selecting biopeptides, produced by bio- if this proliferation stimulation effect is accom-
technology, which are able to stimulate neo- panied by an increase of the synthesis of extra-
synthesis of ECM components, and especially cellular matrix components such as proteins and
GAGs. As a matter of fact, the decrease in con- glycosaminoglycans. At this stage, two biopep-
centration of those molecules during ageing is tides appears to have potential cosmetic proper-
spec ially linked with dehydratation of aged ties: Soya 2 biopeptide for its effect on proteins
skin. A stimulation of their neosynthesis might synthesis stimulation which is the subject of
be an interesting way of reducing some ageing another study (23), and Milk 1 biopeptide fo r its
effets on ski n structure and properties as the significant activation of GAGs synthesis (39%
polyanionic nature of these macromolecules are increase of HA and 53% C4S). The latter was
responsible for skin turgescence due to their ca- selected for further efficiency study. The aims
pacity to retain water ( 19). of such study are to comfirm and to improve on
The choice of the two in vitro models, mono- the previous results using more sophisticated
layered fibroblast cultu re and Derma! Equiva- analytical methods for measuring dy n amic
lent was motivated by the crucial role of fibro- synthesis of ECM components in a DE, where
blasts in cutaneous ageing mechanism and fibroblasts are in a more physiological environ-
dehydrated skin phenomena. Briefly, cutaneous ment. In this three-dimensional model, the fi-
ageing is characterized by a decrease in the broblasts are non proliferative and surrounded
synthesis of proteins, such as collagens and pro- by their own human neosynthesised extracellu-
teoglycans. Also, a spontaneous and progressive lar matri x, with a very si mi lar organization to
degradation of the elastic fibers take piace (20) that in normai dermis (I 2). After treatment of
and a low cellularity develops (21). Consequen- DEs by Milk I biopeptide, no variation on the
tly, the most visible changes occuring in ageing celi number using the MTT viability test was
skin are in the dermis where the destruction of found versus contro!, and we conclude that the
the relationship between fibroblasts and the in- increase in ECM proteins and GAGs are due to
terstitial matrix occurs (22). Fibroblasts play a a real activation of sy nthesis . The collagen
pivotal role in the morphogenesis and dynamic synthesis was significantly increased (97 %) by
remodelling of the dermis including the synthe- Milk I biopeptide treatment. The effect of Milk
sis of ECM components and specific enzymes I biopeptide on GAGs synthesis was confirmed
involved in ECM degradation. Consequently re- as we obtai ned an increase of 114 % of Hyalu-
search on skin ageing should focus on fibrobla- ronic Acid and 54 % of Chondroi:tin-4-Sulphate
sts, whose role consists in maintaining the ma- synthesis compared with the contro! DE. Mo-
trix structure and fonctionality. reover, the presence of the Milk l biopeptide in
The model used for screening should give rapid the culture medium induced an elastin synthesis
results using relatively easy and inexpensive activation of 64%. Elastin is a macromolecule
analytical techniques. This is the reason why characterized by its high physical and chemical
monolayered fibroblast culture was used for a strength g iving suppleness and plasticity to the
preselection of the most promising biopeptides skin. During derma! ageing, there is a sponta-
among about 200 products of initial interest neous and progressive degradation of the elastic
(Coletica, France) using the criteria of the cellu- fibers. This phenomenon is attributed to a redu-
lar proliferation rate. Four of these biopeptides ced synthesis of elastin molecules and also to a
were able to give very interesting results on fi- concomitant increase in the fiber degradation
broblast renewal, when c ultured in monolayer. susceptibility by proteases (24).
In this paper, we have caffied out investigations Cutaneous ageing is an insidious and progressi-
8
C. Augustin. V Frei E Pemer. A Huc and O. Damour.
ve degenerative process, inevitable in course study and prove the efficiency of new cosmetic
and predictable in outcome. However, cosmetic molecules.
active compounds can slow down the normai
and photoinduced ageing phenomena and can ACKNOWLEDGEMENTS
improve extemal aspects of aged skin. Indeed,
modem cosmetology proposes various active This work was supported by Coletica, DRET
compounds such as alpha-hydroxy acids, cera- n° 95060, CNRS and Hospices Civils de Lyon.
mides or actives extracted from seaweed or Thanks to Jim Torbet for reading th is
plants with different mechanisms of action. To- manuscript.
day, biotechnologies open a way to the disco-
very of a wide range of powerful and innovative
cosmetic active compounds like the Milk I bio-
peptide studied in this paper. New processes ba-
sed on fermentation by different micro-organi-
sms characterized by their highly developed
enzyme systems, give rise to radically different
peptides than those obtained with conventional
chemical method. The biopeptides produced in
this way are highly specific and are structural
analogues of celi mediators like cytokines. They
might be recognised by biological receptors and
can induce biological effects. Moreover, the
biopeptides selected for this study are very
small molecules that penetrate deeper in the
skin than macromolecules such as those are pre-
sent in conventional cosmetic filmogene sub-
stances. Consequently, Milk I biopeptide is a
radically innovative cosmetic moisturizing
agent, because it induces the endogenous restau-
ration of the moisturizing potential of the skin
by stimulating the neosynthesis of glycosamino-
glycans.
CONCLUSION
Modem cosmetology requires the detailed te-
sting of efficacy together with the use of new
and origina] active compounds. Biotechnologies
are very effective in the production of innovati-
ve active compounds. However, the wide range
of products need to be evaluated with fast and
accurate methods allowing the selection of the
most promising molecules. Consequently, the
use of in vitro models are going to be increased
in the future to ensure the safety (25) and to
9
An in vitro selection of new cosmetic oct1ve compounds: from screening test on 3-0 Derma/ Equivolent
REFERENCES
1) Tinois E, Tollier J, Gaucherand M, Dumas H, Tardy M, Thivollet J (1991) In vivo and post
transplantation differentiation of keratinocytes growth on the human type IV collagen film of
bilayered derma) substitute. Exp.Cell Res. 191: 310-319.
2) Cannon L, Neal J, Kubilus J, Klausner M (1993) New epidermal model for derma) irritancy
testing. Symposium "Current trends in in vitro Skin Toxicology and Eye Irritancy testing".
Ottawa, Canada.
3) Rosdy M, Clauss C (1990) Terminal epidermal differentiation of human keratinocytes grown
chemically defined medium on inert filter substrates at the air-liquide interface.
J. lnvest. Dermatol. 409-414.
4) Naughton K, Jacob L, Naughton A (1989) A physiological skin model for in vitro toxicity
studies. In "in vitro toxicology: mechanisms and new technology".
Goldberg A. Ed. New York, Mary Liebert lnc. 183-189.
5) Bell E, lvarson B, Merrill C (1979) Production of a tissue-like structure by contraction of
collagen lattices by human fibroblasts of different proliferative potential in vitro.
Celi Biol. 76: 1274-1278.
6) Laska D, Poulsen R, Horn J, Meador V, Hoover D (1992) An evaluation of TESTSKIN : an
alternative derma! irritation model. In Vitro Toxicol. 5: 177-189.
7) Saintigny G, Bonnard M, Damour O, Collombel C (1993) Reconstruction of an epidermis
on a chitosan cross-Iinked collagen-GAG lattice: effects of fibroblasts.
ActaDerm. Venereo!. 73: 175-180.
8) Shahabeddin L, Berthod F, Damour O, Collombel C (1990) Characterisation of skin
reconstructed on a chitosan-cross-linked collagen glycosaminoglycan matri x.
Skin Pharmacol. 3: 107-114.
9) Augustin C, Damour O (1995) Development of a kit for predicting cutaneous toxicity in vitro
using 3-D derma) equivalent : Phase 1 Reproducibility of derma) equivalent.
J. Cellular Engineering. 1: 58-62.
10) Collombel C, Damour O, Gagnieu C, Marichy C, Poinsignon F (1987) Biomaterials of
collagen, chitosan and glycosaminoglycans; process for preparing them and their application
in human medecine. French patent 8708252, 1987.
European patent 884101948, 1988. US patent PCT/FR/8800303, 1989.
11) Berthod F, Damour O, Collombel C (1993) Collagen synthesis by fibroblasts cultured within
a collagen sponge. Biomaterials. 14: 749-754.
12) Berthod F, Sahuc F, Hayek D, Damour O, Collombel C (1996) Deposition of collagen fibril
bundles by long-term culture of fibroblasts in a collagen sponge. J. Biom. Mat. Res. 32:
87-94.
13) Sahuc F, Nakazawa K, Berthod F, Collombel C, Damour O (1995)
Mesenchymal-epidermal interactions regulate gene expression of type VII collagen and kalinin
in keratinocytes and dermal-epidermal junction formation in a skin equivalent model.
Wound repair and Regenerartion. 93-102.
14) Smith K, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto
E, Groeke N, Olson B, Klenk D (1985) Measurement of protein using bicinchoninic acid.
Anal. Biochem. 150: 76-85.
15) Cappelletti R, Del Rosso M, Chiarugi VP (1979) A new electrophoretic method for the
10
C. Augustm. V Frei. E. Perner, A Huc ond O. Domour.
11
J. Appl. Cosmetol. 15, 13-20 (January-March 1997)
J,G. Camarasa•, P. Anthoine 0 , M.J. Tribo Boixareu•, E. Serra Baldrich* and L. Aubert 0 •
Synopsis
Demonstration of the anti -wrinkle efficacy of a cosmetic product was carried out in 41 women pre-
senting with photoagei ng of facial skin. The subjects applied the product to the whole of the face,
twice a day, for 8 weeks. Evaluation of the state of the skin and cutaneous re lief was caJTied out
before the start of treatment and after 2, 4 and 8 weeks of treatment using a clinical score method,
together with instrume nt measurements: replicas of the area around the eye and Image Analysis.
Evaluation by the subjects was recorded. Skin tolerability was also veri fied.
The clinica! observations showed a significant and progressive improvement in cutaneous rel ief as
well as a significant increase in the firmness and cosmetic qualities of the skin during treatment
These results were confirmed by Image Analysis of the replicas: reduction in the number and depth
of the wrinkles, as well as evaluation of product efficacy by the treated subjects.
Riassunto
La dimostrazione dell 'attività anti-rughe di un prodotto cosmetico è stata svolta su 41 donne che
presentano fotoinvecchiamento della pelle del viso. I soggetti hanno applicato per 8 setti mane il pro-
dotto sull'intera superficie de l volto, due volte al giorno. La valutazione dello stato della pelle e del
rilievo cutaneo è stata svolta sia prima dell ' inizio del trattamento c he dopo 2, 4 e 8 settimane di trat-
tamento utilizzando un metodo di punteggio clinico, insie me a misure strumentali: riproduzioni
dell ' area intorno agli occhi e Im magine Ana litica Computerizzata. Le valutazioni dei soggetti sono
state registrate. É stata anche verifica la tolleranza del la pelle. Le osservazioni cliniche hanno dimo-
strato un significativo e progressivo miglioramento del rilievo cutaneo, insieme ad un aumento si-
gnificativo del tono e delle qualità cosmetiche della pelle durante il trattamento. Questi risultati sono
stati confermati dalla Immagine Analitica Computerizzata delle riproduzioni: riduzione nel numero e
nella profondità delle rughe, insieme alla valutazione dell 'efficacia de l prodotto data dai soggetti trattati.
13
Demonstrat1on of the ant1-wrinkle eff1cacv of a cosmet1c product Correlat1on between .
14
J.G Comoroso. P. Antho1ne M J Tnbo 801xoreu. E Serro Boldnch ond L Aubert
the beginning of the treatment (TO), after 2 The subjects had to judge the efficacy of the
weeks (T 2S), after 4 weeks (T4S) and after 8 product on cutaneous relief (satisfactory or un-
weeks of treatment (T8S) . Evaluation of the satisfactory results obtained), as well as its acti-
state of the skin and cutaneous relief was car- vity on firmness, softness and hydration of the
ried out at each visit A questionnaire concer- skin, according to a scale of O to 4 for each cri-
ning the efficacy and cosmetic aspects of the terion (0: unsatisfactory; 4: satisfactory).
product was given to the subjects after 4 and 8
weeks of treatment. 6 - Tolerability
Skin tolerabi lity was evaluated by the investiga-
5 - Evaluation methods tor after 2, 4 and 8 weeks of treatment.
15
Demonstration of the anti-wnnkle eff1cacy of a cosmet1c product Correlat1on between .
Table I
CLINICAL SCORES
(mean +/-standard deviation of the mean, % of evolution
with respect to time TO and p determined using Student's t test)
TO: Before starting the treatment; T2S: After 2 weeks of treatment;
T4S: After 4 weeks of treatment; TSS: After 8 weeks of treatment
16
J.G Camarasa. P. Anthome. M.J. Tr1bo 801xareu. E Serra Baldrich and L Aubert
TO T2S
FIGURE I:
Mocrophotogrophs of replicos corried out on two subjects (Fino/ enlorgement: x 10)
Figure lo: subject n° I - Figure Jb: subject n ° 2
TO: Before the start of treotment: T2S: After 2 weeks of treotment; T4S: After 4 weeks of treotment: TBS: After 8 weeks
of treotment;
Table Il
QUANTITATIVE ANALYSIS OF REPLICAS: NUMBER OF WRINKLES PER UNIT
SURFACE AREA AND DEPTH OF WRINKLES IN µM
(mean +/-standard deviation of the mean, % evolution with respect to time TO
and p determi ned by Student's t test).
TO: Before starting the treatment; T2S: After 2 weeks of treatment;
T4S: After 4 weeks of treatment; T8S: After 8 weeks of treatment
17
Demonstrat1on ot fhe ant1-wnnkle eff1cacy of a cosmet1c produci Correlat1on between .
150 100
TO
125 TO 90
• T2S
Il
:;; • JiI
~~ • T4S
-~-
o1 100
T2S
• ~
'O-§
~
V
~
80 • T8S
-=a. -B
~
75
T4S
• :ij 70
•
T8S
50
• 60
4 5 6 7 4 5 6 7
Scores: cutoneaus relief Scores: cvtoneous relie(
Figure 2a: Correlafion between scores (cutaneous re/ief) Figure 2b: Correlation between scores (cutaneous relief)
and depth of wrinkles. and number of wrinkles.
Figure 2: Correlation between scores and the results obteined by lmage Analysls.
TO: Before the start of treatment; T2S: After 2 weeks of treatment: T4S: After 4 weeks of treatment: TBS: After 8 weeks of
treatment;
ber and depth of the wrinkles fo r ali subjects. with respect to improvement in cutaneous relief:
The number of wrinkles was reduced in a signi- in term s of w ri nk le im proveme nt , 92% of
ficant fashion duri ng the treatment (-8, -12 and - subj ects j udged the result obtained to be sati-
20% after 2, 4 and 8 weeks of treatment respec- sfactory from the four th week of treatment
tively) as well as the depth of the wrinkles (- 16, onwards. The subjects also noted a good acti-
-32 and -47%). vity of the product on the firmness, softness and
hydration of the skin.
- 2.3 Correlation between the clinica/
scores and resulfs obtained with lmage 3 - Tolerability
Analysis No undesirable cutaneous reaction was obser-
ved during use of the product: tolerability was
F ig ure 2a represe nts correlati on over time excellent.
between the mean of the clinical scores (cuta-
neous relief) and mean of the v·a lues correspon-
ding to the depth of the wrinkles. DISCUSSI ON
The correlation coefficient r = -0.99 is signifi-
cant at the 0.01 level. This study showed the efficacy of the produci
T here also ex is ts a s ig ni fica nt co rre lati o n studied based on the most characteristic signs of
between the clinica! scores and the number of photoageing of the face: wrinkles and loss of
wrinkles per unit surface area (Figure 2b): fi rmness. The clinical observations showed an
r = -0.98, p = 0.02. improvement in these criteria and the cosmetic
qualities of the skin during treatment. T hey
- 2.4 Subjective evaluation were confirmed by the res ults obtained after
A nalysis of the qu es tionn aires given to the quantitative an alysis of replicas: reduction in
subjects confi rms the efficacy of the product the number and depth of wrinkles. The evalua-
18
J.G. Camarasa. P. Anthoine. M J. Tribo 801xareu. E Serra Baldrich and L Aubert.
19
Demonstration of the anti-wrinkle efficacy of o cosmetic product. Correlation between ..
REFERENCES
1) Gilchrest B.A. (1989): Skin aging and photoagi11g: an overvies,
«J. Am. Acad. Dermatol.» 21: 610-613.
2) Kligman L.H. (1986): Photoaging: manifestations, preventio11 and treatment,
«Dermatol. Clin.» 4: 517-528.
3) Sams W.M. (1986): Su11-induced aging: clinica/ and laboratory observations in man,
«Dermatol. Clin.» 4: 509-516.
4) Baker H., Blair C.P. (1968): Celi replacement in the human strat11m corneum in old age,
«Br. J. of Dermatology» 80: 367-372.
5) Matsuoka L. Y., Uitto J. (1989): A/terations in the elasticfibers in cutaneous aging a11d solar
e/astosis, «Aging and the skin, Raven Press, New York» 141-151.
6) Oikarinen A., KarnovenJ., Vitto J., Hannuksela M. (1985): Connective tissue a/terations
in skin exposed 10 11at11ra/ a11d therapeutic UV-radiation, «Photodermatology» 2: 15-26.
7) Aubert L., Martin R.: Procédé de cultures des bactéries filamenteuses non photosynthétiques
et non fructifiantes, «Brevet FR 94-00425, Série N498.»
8) Raines E. W., Dowen S.K., Ross R. (1989): /11terleukine-l mitogenic activity forfibroblasrs
and smooth muse/e cells is due to PDGF-AA ,«Science» 243: 393-396.
9) Gartner M.H., Benson J.D., Caldwell M.D. (1992): IGF-1and2 expression in the hea/ing
wound, «1. Surg. Res.» 52: 389-394.
10) Mauviel A., Chen Y.Q., Khahari V.M. (1993) : Human recombi11a111
interleukin-1 f3 up-regulates elastin gene expression i11 derma/ fibroblasrs. Evidence for
transcriptional regulation both in vitro and in vivo. «I. Biol. Chem.» 268: 6520-6524.
11) Saufer D.N., Stanulis-Praeger B.M., Gilchrest B.A. (1988): Autocrine growth stimulation of
human keratinocytes by epidermal ce/1-derived thymocyte activating factor: lmplication far
skin aging, «Arch. Dermatol. Res.» 280: 7 1-76.
12) Larnier, C., Ortonne J.P., Venot A., Faivre B., Béani J.C., Thomas P., Brown T.C.,
Sandagorta E. (1994): Evaluarion of cutaneous photodamage using a photographic scale,
«Br. J. ofDermatology» 130: 167-173.
13) Costa C., Rilliet A., Nicolet M., Saurat J. H. (1989): Scoring Atopic Dermatitis: the
simpler, the better? «Acta Dermatovener. Stockholm» 69: 41-45.
14) Makki S., Barbenel J /C., Agache P. (1979): A quantitative methodfor the assessment of the
microtopography of lluman skin. «Acta Dermatovener. Stockholm» 59: 285-29 l.
15) Corcuff P., de Rigai J., Lévèque J.L. (1982): lmage analysis of the cutaneous microre/ief,
«Bioengineering and the skin» 4 (1): 16-3 1.
16) Corcuff P., Chatenay F., Lévèque J.L. (1984): A fully automated system to study skin
surface pattems, «lnt. J. Cosmet. Sci.» 6: 167-176,
20
J. Appl. Cosmetol. 15. 21-32 (January-March 1997)
' MAVI SUD S.r.l. Research and Development Aprilia CLT) - ltaly.
• Dept. of Dermatology. Dermatologists Training School. Il University of Naples. ltaly
2
Dept. of Dermatology, University of Catania - ltaly
' Head Dept. of Dermatology. Umberto I Hospital, Siracusa - ltaly
• Physiology lnstitute. University of Urbino - ltaly
Key words: Acne, phosphatidylcholine, linoleic acid, skin lipids, skin hydration, glycolic acid,
3C System~.
Synopsis
Severa! studies have highlighted as the concentration of linoleic acid in the sebum and in the
epiderm al lipids seems to be strictly connected to the onset of different pathologies, and among
those of acne.
lt has been found also that the wax ester content of the sebum and of the epidermal acylcerami-
des containg linoleic ac id seems to be inversely proportional to the sebum production.
It has been proven that the acylceramides of the comedones and the skin su rface of acne patients
contain much less linoleic acid than the acylceramides from the skin surface of contro! subjects
(6% versus 45% in the normai physiological state).
Other studies seem to suggest that a localized increase of squalene and oleic acid and contempo-
rary reduction in levels of linoleate esterified to ceramide I should be considered as a causative
factor in comedogenesis and acne.
The present study was conducted on 40 vo luntar patients in order to determine whether the sup-
posed overproduction of oleic acid, squalene and sebum, of an acne-affected skin could be in-
fluenced by topica! application of cosmetic emu lsions containing as active ingredients an high
concentration of esterified linoleic acid together with glycolic acid buffered by a special mixture
of aminoacids.
A li the treated patients showed a significant decrease of the free fatty acids/triglycerides ratio
together with decrease of the skin surface I ipids.
From the other hand the obtained clinica! resu lts show that the contemporary use in the same
phospholipidic emulsion of g lycol ic acid partially buffered with aminoacids, of salicilic acid
and chlorexidine digluconate, can effective ly relieve the severity of acne ever since the first
weeks of treatment.
Given the positive results obtained we believe that this cream can be considered as a new co-
smeceutical and a useful aid for the normai acne therapies.
21
Effect of phosphot1dylcholme lmole1c oc1d-nch ond glycoilc OCld m acne vulgons
Riassunto
Diversi studi hanno messo in luce come la concentrazione di acido linoleico nel sebo e nei lipidi
dell'epidermide sembri essere strettamente correlata all'insorgere di diverse patologie, tra cui l'acne.
È stato anche scoperto che il contenuto del sebo e delle acilcerammidi epidermiche contenenti acido
linoleico sembrano essere inversamente proporzionali alla produzione del sebo.
È stato provato che le acilcerammidi dei comedoni e la superficie cutanea dei pazienti affetti da acne
contengono molto meno acido linoleico delle acilcerammidi della superficie cutanea dei soggetti
controllati (6% contro il 45% del normale stato fisiologico).
Il presente studio é stato condotto su 40 volontari con lo scopo di determinare se la supposta sovra-
produzione di acido oleico, squalene e sebo di una pelle affetta da acne potesse essere influenzata da
applicazioni topiche di emulsioni cosmetiche contenenti come principi attivi un 'alta concentrazione
di acido linoleico esterificato insieme ad acido glicolico tamponato da una speciale miscela di ami-
noacidi.
Tutti i pazienti trattati hanno mostrato una riduzione sign ificati va del rapporto acidi grassi liberi/tri-
gliceridi, insieme ad una diminuzione dei lipidi cutanei di superficie.
D'altro canto i risultati clinici ottenuti dimostrano che l'uso contemporaneo nella stessa emulsione
fosfolipidica di acido glicolico parzialmente tamponato con aminoacidi, di acido salicilico e di clo-
rexidina digluconato possono migliorare efficacemente la gravità dell 'acne già dalle prime settima-
ne di trattamento.
Dati i positivi risultati ottenuti riteniamo che questa crema possa essere considerata, quale nuovo co-
smeceutico, di utile ausilio nelle normali terapie dell'acne.
22
PMorgant1, S D.Randozzo, A Giardina, C Bruno, M Vincenti and L T1ben
23
Effect of phosphat1dylcholine linoleic aCJd-nch and glyco/1c acid in acne vulgans
24
P.Morganti. S.O.Randazzo. A. Giardina. C. Bruno. M. Vincenti and L. Tiberi
using solid phase extraction colums, according sions imp roved as follows: A treatment (A
to Cav ina et al. (18). All lipid fractions, redis- cream - active) 33% on average if compared
solved in chloroform, were stored at -20°C un- with the basic values;
ti! required. B treatment (B cream - vehicle) about 17%.
The isolated triglycerides and free fatty acids After three months of treatment improvements
were then quantified separately in order to de- were noticed on ali the lesions counts with the
termine the free fatty acids/triglycerides ratio. A cream (active) and they resulted very high,
equa! to about 80% (Tab. I and Fig. 1).
Skin hydration
The hydration of the horny layer was assessed
by measuring electrical capacitance of the skin
surface by means of the 3C System"' ( 17).
When the probe is applied to the skin (recor-
ding time 0.5 s), the capacitance is displayed
digi tally in arbitrary 3C units. The results are
expressed as mean values of the measurements
performed on fo ur different ri ght or left sites
(cheek, forehead, chin and nose ).
Statistica/ analysis
Student's test was used in evaluation of ali the
data before and after the treatment period. Ali
the analyses were produced using the SAS sta-
tistica! package, version 5. 18 (SAS Institute
Inc., Cary, N.C.). Probabilities less than 0.05
were considered significant.
RESULTS
Clinica/ evaluation
Of the 40 patients enrolled 36 patients (90%)
completed the study. Four patients (3 active
treated, I vehicle-treated) dropped from the
study for the following reasons: two for skin
irritation (active-treated), due probably to gly-
colic acid, and two for persona! reasons. The
mean results of the clinica! parameters evalua-
ted are shown in Table I and Fig. l. FIG 1
Both the treatments, cream A and cream B, The vehicle also, however, has proven to be, as
had significantly reduced ali the acne Jesion it was expected, hig hly effective (46 %)
counts, even if cream A (active) has proven towards ali the checked parameters. This fact
more effective than cream B (vehicle) and ha- has further proven the activity carried out by
ving a q uicker activity. In fac t after the first the high concentration of the linoleic acid in
month of treatment al i the inflammatory le- the liposomal emulsion.
25
Effect of phosphat1dylcholine linoleic acid-flch and glycol1c ac1d in acne vulgaris
Table I
MEAN INFIAMMATORY C OUNTS IN MILD AC NE TREATED BY
PHOSPHATIDYLC HOLINE-C REAM GLYCOLIC AC ID ENRICHED
n=36
Open Comedones
Baseline 34.3
Active (cream A) 22 15 (55%) 12(35%) -65%
Vehicle (cream B) 27 20 17 -50%
Closed Comedones
Baseline 43
Active (cream A) 33 25 20 -53%
Vehicle (cream B) 38 32 26 -39%
Pustoles
Baseline 7
Acti ve (cream A) 5 3 l -86%
Vehicle (cream B) 6 4 3 -57%
Papules
Baseli ne 37
Active (cream A) 20 15 12 -67%
Vehicle (cream B) 29 20 18 -5 1%
Ali p values are significant (p<0.0 1) as to groups and highly {p<0.005) significant as baseline values.
Skin surface lipids These val ues seem to prove what stated by
Ali the patients showed at baseline an higher G hyczy et al (7): phos pholipidic e muls ions,
content of surface lipids (casual level) ( 175 ± when rich in linole ic acid, are able to reach the
50 µ g/cm 2) . cells of the sebaceous g land inducing them to
At week 2 the va lues a lready dec reased o f reduce the sebum secretion.
a bo ut 7 0 % for both th e ve hi c le and ac ti ve The high percentage of linoleic acid, entering
cream. As seen in Fig. 2 and Tab. II both the into competion wit h the o leic acid , due to a
ve hi c le (B cream ) and th e ac ti ve cream (A mec hani s m yet un k now n , w o ul d ac t as a
cream) are able to reduce the skin lipids casual " brake", red ucing its excessive presence also
level since the first month of treatment (- 70%) at level of the surface lipidic film .
proving a remarkable topic effectiveness.
26
P.Morganti. S.D.Randazzo. A. Giardina. C. Bruno. M. Vincenti and L. Tiberi
Table Il
RIDUCTION OF SURFACE SKIN LIPIDS BY
A PHOS PHATIDYLCH OLINE-C REAM GLYCOLIC AC ID ENRIC HE D
(µg/c m 2) n=36 t=22°C RH=50 %
Weeks
o 2 4 6 8 10 12
Active 195±50 138±33 110±24 94±24 80±1 8 78±21 59±16
baseline
Vehicle 187±48 134±35 121 ±3 1 107±27 95±25 85±22 7 1±18
baseline
Ali p values are not signi ficant as to groups and highly significant (p<0.005) as to baseline values.
All p values are not significant as to groups and highly significant {p<0.005) as to baseline values
Fig2
27
Effect of phosphatidylcholine linole1c acid-r1ch and glycolic acid in acne vulgaris
Table lii
EFFECT OF TOPICAL APPLICATION OF PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON FREE FATTY ACIDS/TRIGLYCERIDES RAT IO
n=36 t=22°C RH=50%
Ali p values are highl y significant both as to groups (p<0.005) and to baseline values.
0,9
0,8
o 0,7
:;::::;
...
11:1
0,6
...J
(!)
I- 0,5
LL
LL 0,4
0,3
0,2
0,1
o
o 2 4 6 8 10 12
WEEKS
IDVEHICLE (CREAM B) •ACTIVE (CREAM A) I
All p values are highly significant (p<0.005) both as to groups and to baseline values
Fig3
28
PMorganti, S.D.Randazzo. A Giardina. C. Bruno. M Vincenti and L.T1ben
Table IV
MEAN VALUES OF SKIN HYDRATION IN ACNE PATIENTS TREATED BY
A GLYCOLIC ACID ENRICHED PHOSPHATIDYLCHOLINE CREAM
n=36 t=22°C RH=50 %
Weeks
o 2 4 6 8 10 12
Active 78±11 ns±11 123±14 L28±21 126±19 132±20 134±24
Ve hicle 74±9 I LO± 128±16 133±17 138±18 141±23 140±22
Ali p values are not significant as to groups and highly significant (p<0.005) as to baseline values.
140
Ciì - -
:t::
e: 120
~ - -
:::>
~ ~
. ra
-:e
...
...
100
80 /
~
V
"':::>w
....I
60
~ 40
o
('?
20
o
o 2 4 WEEKS 6 8 10 12
1-- ACTIVE (CREAM A) - - VEHICLE (CREAM B) I
All p values are not significant as to groups and highly significant (p<0.005) as to baseline values
Fig4
29
Effect of phosphatidylchoilne linoleic acid-rich and glycolic ac1d m acne vulgaris
The contemporary presence of the g lycol ic al (7) the use of soybean derived phospatidyl-
acid would facilitate, moreover, the transcuta- choline seems to be able to nonnalize excess of
neous pene tration of phospholipids, improv ing sebum.
in this way the ir activity. Ali the treated patients showed a significant de-
crease of the free fatty acids/triglycerides ratio to-
Free fatty Acids/Triglycerides gethe r with decrease of the skin surface li pids
ratio (Tab IIl and Fig. 3).
This particular phospholipidic emulsion has pro- From the other hand the obtained clinica] results
ven to be highly active also towards another para- show that the contemporary use in the same pho-
meter of the acne development, namely the pre- spholi pid ic emulsion of glycolic acid partially
sence of free fatty acids, that are the main cause buffered with aminoacids, of salicilic acid and ch-
of the abnonnal inrrafollicular keratinization. Free lorexidine digluconate, can effectively relieve the
fatty acids have an inflammatory effect and beco- severity of acne ever since the first weeks of treat-
me potential irritants, wh ich when released into ment. It has been noted an high reduction of a li
the dennis cause cysts to fonn. the inflammatory acne lesions such as small or
As see n in Fig . 3 a nd Tab. III the free fatty large solid bumps, pus pimples, " blackheads" and
acids/triglycerides mean ratio decreases of 4 1% whiteheads (Fig. I and Tab. I). The use of the gly-
just after two weeks of treatment, and further re- colic acid, even if in rather high concentration
duces up to about 7 1% after three months. (I 0%) did not cause clear irritating forms or stin-
During this time period it seems that this activity ging activity.
is perfonned only by the active cream (A cream) Only two patie nts had to stop immediately the
for the probable antiseptic activity carried out by treatme nt, consideri ng it not suitabl e for the ir
the glycolic acid, and particularly by the salicilic skin type.
acid and the clorexidine digluconate. In fact, as it The high bearableness of the used compound is
has been proven e lsewhere, there is a significant due, in our opinion, both to the strong presence of
reduction of propionibacterium acnes colonies, phosphol ipids and to the special buffer we already
with a subsequent drop in the production both of used to neutralize the aggressiveness of the glyco-
enzyme esterase and of free fatty acids ( 19). lic acid (12).
Un like a li th e acne treatments utili zed, thi s
Skin hydration phospholipidic preparation not only reduces in
Ali the patients showed a significant increase in drasti c way the presence of lipids and acneic le-
moisture content and maintained these values un- sions, but rehydrates the skin since the first pe-
ti! the end of the study. The results are shown in riod of application, mantaining it e lastic (Fig . 2,
Table IV and Fig. 4. 4 and Tab. II, IV).
Given the positi ve resu lts obtained we be lieve
DISCUSSION that this cream can be considered as a new cosme-
ceutical and a usefu l aid for the nonna! acne the-
In acne, as known, defects in the keratinization of rapi es.
the "epithelial" lining of the pilosebaceous follicle
block eagress of sebum, leading to "plugging". Author address:
Propionibacterium acnes, exerts lipolytic effect on P. Morganti, PhD
stagnant sebum, releases free fatty acids and cau- Via Innocenzo Xl , 41
ses inflammation when they escape from the pilo- 00165 Roma l taly
sebaceous follicle. E-mail=mavi@colosseum.it
In this study, as also suggested from Ghyczy et URL=http://www.colosseum.it/st 81 lmavi
30
P.Morganti, S.O.Randazzo, A. Giardina, C. Bruno, M. Vincenti and L. Tiberi
REFERENCES
1) Wertz P.W., Mietake M.C., Long S.A., Strauss J.S. and Downing D.T. (1985)
The cornposition of the ceramides frorn human Stratum Corneum and frorn cornedones,
J. lnvest. Dermato/., 84:410-412.
2) Wertz P.W., Madison K.C. and Downing D.T. (1989) Covalently bound lipids of human
straturn corneum, J. lnvest. Dermato/., 92: 109-11 l
3) Colarow L.J. (1992) Quantitation of cerarnides with essential fatty acids rnoieties in the
human skin surface and blood plasma lipids. Planar Chrom. 3: 126-32
4) Stewart M.E., Grahak M.O. and Cambier L.S. (1986) Dilutional effect of increased
sebaceous gland activity on the proportion of linoleic acid in sebaceous gland esters and
in epiderrnal acylceramides. J. Invest. Dermatol., 87:733-736.
5) Perisho K., Wertz P.W., Madison K.C., Stewart M.E. and Downing D.T. (1988),
Fatty acids of acylceramides frorn cornedones and frorn the skin surfaces of acne patients and
contro! subjects. J. lnvest. Dermatol., 90:350-5
6) Morello A.M., Downing D.T. and Strauss J.S. (1976), Octadecadienoic acids in the skin sur
face lipids of acne patients and normai subjects, J. lnvest. Dermatol., 66: 319-323
7) Ghyczy M., Nissen H.P. and Biltz H. (1996), The treatrnent of acne vulgaris by phosphatidyl-
choline frorn soybeans, with an high content of linoleic acid. J. Appl. Cosmetol., 14: 137-145
8) Brooks G. and ldson B. (1991), Skin lipids, Int. J. Dermatology, 13: 103-113
9) Morganti P. (1996) Alpha hydroxy acids in cosrnetic dernrntology, J. Appl. Cosmetol, 14:35-41
10) Morganti P. (1996), USA Pat. Appl. 60/009045 (Provisional) corresponding to RM 95A000807
11) Burke B.M. and Cunliffe W.J. (1984) The assessrnent of acne vulgaris: the Leeds tecnique.
Br. J. Dermatology, 3:83-92
12) Morganti P., Randazzo S.D., Fabrizi G. and Bruno C., (1996), Decreasing the stinging
activity and irnproving activity of AHAs, J. Appl. Cosmetol., 14:79-91.
13) Van Scott E.V. and Yur J., (1974) Contro! of keratinization with alpha hydroxy acids and
related cornpounds - Topica) treatrnent of ichtyotic disorders, Arch Dermatol., 110:586.
14) Murad H., Shamban A.T., Moy L.S. et al. (1992), Study shows that acne irnproves wi th
glycolic acid regirnen, Cosm. Dermatol., 5:32.
15) Wallhausser K.H. (1984), Praxis der Sterilization - Disinfection - Konservierung. 3, Auflage
Thierne, Stuttgart, p. 27.
16) Morganti P., Randazzo S.D., Bruno C. and Cardillo A. (1988), Ethyl lactate and benzoyl
peroxyde in acne vulgaris, J. Appl. Cosmetol., 6: 15-30.
17) Cardillo A. and Morganti P. (1994), Fast and non-invasive method for assessing skin hydration,
J. Appl. Cosmetol, 12: 11-16
18) Cavina et al.(1965), Ann. Ist. Super. Sanità, 1, 566
19) Morganti P., Agostini A., Bruno C. and G. Fabrizi, (1997), Role of topica! glycolic acid and
phosphatidylcholine linoleic acid-rich in the pathogenesis of acne. linoleic acid versus squalene.,
J. Appl. Cosmeto/. 15:33-41
31
J. Appl. Cosmetol 15. 33-47 (January-March 7997)
Synopsis
Acne is a disease that commonly occurs during adolescence with the production of testosterone and
the activation of the sebaceous glands. Factors such as stress, the environment, drugs, greasy cosme-
tics and mechanical irritants may contribute to or aggravate this disease.
Many facts indicate that free fatty acids released in sebaceous follicles through the action of bacte-
rial lipases on sebaceous triglycerides play an important role in the overall pathogenesis of acne.
Moreover, it has been shown that there is a significant decrease in the levels of linoleic acid in se-
bum of patiens with acne and a contemporary increase of squalene and oleic acid.
Supported by our recent data that demonstrate an anti-acne activity carried out by an emulsion based
on phosphatidylcholine and glycolic acid buffered through a special mixture of aminoacids we con-
trolled TEWL, corinebacterium acnes colonies and the presence, at cutaneous level, of linoleic acid
and squalene on 20 patients with an average age of 18±2 with a mild to moderate acne.
The data obtained proved a remarkable decrease of squalene and a contemporary increase of linoleic
acid in the stratum comeum lipids, together with a considerable improvement of the skin 's look of
the patients.
Therefore it seems possible to assert that phospholipidic creams particularly rich in linoleic acid can
be used as new cosmeceutical means adjuvant in the acne juvenilis therapy.
Riassunto
L'acne è una malattia che si verifica di solito durante il periodo dell'adolescenza con la produzione
di testosterone e l'attivazione delle ghiandole sebacee. Fattori come lo stress, l'ambiente, i farmaci, i
cosmetici grassi e gli irritanti meccanici possono contribuire o aggravare la malattia.
Molti fattori indicano che gli acidi grassi liberi rilasciati nei follicoli sebacei attraverso l'azione di
lipasi batteriche sui trigliceridi sebacei giocano un ruolo importante nella patologia complessiva
33
Raie of topico/ glyco/ic ocid ond phosphotid1/coline /Jnoleic ocid-rich 1n the pothogenesis of acne
dell 'acne. Inoltre è stato provato che si verifica una diminuzione significativa nei livelli di acido li-
no leico nel sebo dei pazienti affetti da acne ed un contemporaneo aumento di squalene ed acido
oleico.
Sostenuti dai nostri recenti ri sultati che dimostrano un 'attività anti-acne svolta da un 'emulsione ba-
sata su fosfatidilcolina e acido g licolico tamponato da una speciale miscela di aminoacidi, abbiamo
controllato la TEWL, le colonie di corinebacterium acnes e la presenza, a livello cutaneo, di linolei-
co e squalene su 20 pazienti con una età media di 18±2 affetti da acne debole o media.
I risultati ottenuti hanno dimostrato un a considerevole diminuzione di squalene ed un contempora-
neo aumento di acido linoleico nei lipidi dello strato corneo, insie me ad un miglioramento conside-
revole dell 'aspetto cutaneo dei pazienti.
Per questi motivi sembra possibile affermare che le creme fosfolipidiche particolarmente ricche di
acido linole ico possono essere utilizzate come nuovi mezzi cosme ti ci ad iu vanti nella te rapia
dell'acne juvenilis.
34
P Morganti. A. Agostirn. C. Bruno and G Fabriz1
35
Rote of top1col glycolic ocid ond phosphotidilcolme linole1c ocid-rich in the pothogenesis of acne.
Table I
TEWL MEASUREMENTS OF ACNE AFFECTED PATIENTS TREATED
BY PHOSPHATIDYLCHOLINE-CREAM GLYCOLIC ACID ENRICHED
o 22.3±7.2 24.5±8.I
2 20.4±6.8 18.6±7.6
4 15.2±7.0 12.0±3.5
6 14.8±6.5 11.2±2.7
8 12.1±4.8 11.5±3.2
10 11.8±3. l 11.6±2.9
12 11.7±3.5 11.8±2.5
Ali p values are not significant as to groups and highly significant (p<0.005) as to baseline values.
36
P Morgont1. A Agosf1nt, C Bruno ond G Fobnz1
n = 20 - t = 22 •e - RH = 50%
30
25
-
.e 20
~
~
........
-
N
E 15
~
O>
'-"'
.....J
sw 10 --------
I-
5
o
o 4 6 8 10 12
WEEKS
1--ACTIVE (Cream A)--VEHICLE (Cream B)I
All p value are not significant as to groups and highly significant (p<0.005) as to baseline value
Fig I
pooled. Collections from the right and the left The obtained resuls are shown in Fig. 2 and
areas were analyzed separately. Ali solvents Tab.II.
used were chromatography grade. Tota) lipids
were exstracted from the 3C plastic foil (1 cm 2) Colonies of propionibacterium
using ethyl ether methanol (2: I) for 3 h. at acnes
room temperature according to Folch et al. (13). The evaluation of P. acnes was carried out ac-
The solvent was dried under nitrogen and the li- cording to the method of Williamson and Klig-
pids were redissolved in chloroform (2: 1) accor- man (16). Samples were taken at lst day and af-
ding to Cavina et al. (14). Lipid fractions were ter 2, 4, 6, 8, I Oand 12 weeks of treatment.
chromatographilly separated into their indivi- Before taking the sample, both cheeks of the
duai lipid classes. subjects were cleaned by using a sterile gauze sa-
Fatty acids were then converted to their methyl turated with a 0.1 % sterile solution of Triton x-
esters according to Stoffel et al. (1 5). Oleic and 100, followed by a further cleaning with sterile
linoleic acid methyl esters and squalene were distilled water and finally rubbed with a gauze
quantitatively identified by using internal stan- saturated with hexane for 30 seconds. Tue clean-
dards (Sigma Chemical). sed skin was protected with a porous sterile pla-
37
Rote of topica/ glycolic acid and phosphatidi/coline linoleic acid·rich in the pathogenesis of acne.
4,0
e: 3,5
o
;
-
...ca
Q)
CJ
e:
e:
3,0
2,5
2,0
o 1,5
CJ
o~ 1,0
0,5
2 4 6 8 10 12
weeks
IO Squalene • Linoleic Acid I
All p values are highly significant (p < 0.005) as to baseline values
Fig2
Table Il
TOPICAL APPLICATION EFFECT OF PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON LINOLEIC ACID AND SQUALENE
CONCENTRATIONS OF ACNE-AFFECTED SKIN.
n=20 T=22°C RH=50%
o 3.8±0.2 1.2±0.1
2 3.2±0. 1 2±0.l
4 2.7±0.3 2.9±0.2
6 2±0.l 3.2±0. l
8 1.2±0.1 3.4±0.2
IO 1±0.1 3.7±0.1
12 0.9±0.l 3.9±0.2
All p values are significant (p<0.05) both as to weeks and to baseline values.
38
P. Morganti, A. Agostini, C. Bruno and G.Fabrizi
~
z
o
a:
o
o::
c..
u.
o
z
o
j::
(.)
::>
e
w
o::
~
VEHICLE (CREAM B) ACTIVE (CREAM A)
WEEKS 4 8 12 4 8 12
All p values are highly significant (p< 0.005) as to baseline and groups
Fig3
Table lii
TOPICAL APPLICATI ON EFFECT OF A PHOSPHATIDYLCHOLINE-CREAM
GLYCOLIC ACID ENRICHED ON QUANTITATIVE P. ACNES COUNTS
n=20
o 4 8 12
All p values are significant (p<0.01) as to groups and highly significant (p<0.005) as to baseline values.
39
Rote of topica/ glycolic acid and phosphatidilcoline linoleic acid-rich in the pathogenesis of acne.
stie gauze, so as to maintain normai evaporative (cream A) resulted much more effective than
activity. After one hour a cylinder of sterile glass the vehicle (cream B). The vehicle also (cream
(internal area 3.8 cm2) with hollow base was ap- B) reduces the presence of corinebacterium ac-
plied to the area. Into said cylinder 1 ml of sterile nes of about 50% after 12 weeks of treatment.
solution of 0.1 % Triton x-100 in a pH 7.9 pho- This unexpected result is to be ascribed proba-
sphate buffer was introduced. Having cleaned the bly to the antioxidant activity typical of the soya
area with a teflon spatula for one minute, two phospholipids, that is Iikely to interfere with the
successive samples of liquid were taken. The survival and the development of corineabacte-
samples thus obtained were diluted a number of rium acnes.
times with a solution of 0.05% Triton x-100, im- According to what we verified (18) it has also to
mediately set in culture with a solution of O. I % be pointed out that by adding to the vehicle only
Tween 80 and incubated anaerobically for 7 days. the glycolic acid buffered to pH 4.5 it was ob-
The colonies of P. acnes were determined accor- tained a further decrease of the whole bacterial
ding to Mc Finley et al (17). The obtained mean charge, apart from the well-known bactericide
results are shown in Table ill and Fig. 3. activity carried out by both the salicilic acid and
clorexidine. It has been verified experimentally
Statistica/ analysis that the glycolic acid alone buffered wi th spe-
Differences between means were calculated cial mixtures of aminoacids increases of a
using the Student's test. Statistica[ correlations further about 30% the bacteriostatic activity ty-
between percent values of squal ene and linoleic pical of the used phosphatidylcholine.
acid and absolute tota[ amount recovery of li-
pids were calculated using the Pearson correla- Finally, as it was expected, it was possible to
tion coefficient (Statistica! Analysis System, verify a considerable decrease of TEWL in ali
SAS, North Caroline, USA). patients treated by both the vehicle and the acti-
ve cream (Fig. 1 and Tab. I).
RESULTS ANO DISCUSSION Considering the res ults obtained by our pre-
vious study, the results obtained by Ghyczy et
According to data obtained from Ghyczy et al al. (I O, l l) and the data obtained wi th this work
(I O), and as it can be seen in the fig. 2 and Tab. it seems possible to assert that phospholipidic
Il, the squalene concentration decreases drasti- creams particularly rich in linoleic acid can be
cally since the second week of treatment, while used as new cosmeceutical means adj uvant in
at the same time it can be noted a regular in- the acne juvenilis therapy.
crease of the Iinoleic acid present in the stratum These emulsions have, moreover, proved to be
corneum lipids. It seems that this activity can be even more active when the right proportion of
ascribed exclusively to the phospholipidic vehi- glycolic acid, properly buffered with aminoa-
cle rich in linoleic acid. cids, is added to them.
The data obtained from the cutaneous areas
treated with the cream B (vehicle) compared
with the co rrespo nding areas of th e same
subject treated with the cream A (active), resul- Author address:
ted almost similar and are therefore reported as P. Morganti, PhD
the average of a unique treatment (Fig. 2 and Via Innocenzo Xl, 41
Tab. li). 00165 Roma ltaly
With regard to the presence of corinebacterium E-mail=mavi@colosseum.it
acnes (Fig. 3 and Tab. III), the active cream URL=http://www.colosseum.it/st 81 Jmavi
40
P. Morganti, A Agostini, C. Bruno and G.Fabriz1
REFERENCES
1) Kligman AM (1974) An overview of acne J. l nvest. Dermatol. 62:268
2) Fortrom L (1980) The influence of sex hormones on acne Acta Derm. Venereol 89 (sup):27
3) Wu SF, Kinder BN, Trunnell T N and Fulton J E (1988) Role of anxiety and anger in acne
patients: a relationship with the severity of the disorer J. Am. Acad. Dermatol. 18:325-333
4) Pochi PE and Strauss JJ (1964) Sebum production, casual sebum levels, titratable acid ity of
sebum, and urinary fractional 17-ketosteroid excretion in males with acne.
J . l nvest. Dermatol. 43:383-388.
5) Kellum RE (1968) Acne vu lgaris: studies in pathogenesis: relative irri tancy of free fatty ac ids
from C2 to Cl 6 Arch. Dermato! 97 :722
6) Kellum RE (1979) Free fatty acids hypothesis In : Frank SB (ed) Acne: Update fo r the pratitioner
Yorke Medicai Books. N, pp 65-73
7) Williams ML (1991) Lipids in normai and pathological desquamation In: Advances in li pid
research voi 24 (Elias, Havel, Small Edtrs) Acad. Press, NY, p 237.
8) Dowining DT, Stewart ME, Wertz PW and Strauss JS (1986) Essential fatty acids and acne
J. Am. Acad. Dermato/. 14:22 1-25
9) Morello AM Dowining DT and STRauss J S (1976) Octadecadienoic acids in the skin surface
lipids of acne patients and norma! subjects. J. !nvest Dermatol 66: 319-323
1 O) Ghyczy M, Nissen HP and Biltz H (1986) The treatment of acne vulgaris by phosphatidyl-
chol ine from soybeans, with an high conteni of lino leic acid J. App/. Cosmetol. 14: 137-145
11) Morganti P, Randazzo SD, Giardina A, Bruno C and Tiberi L (1997) Effect of phospatidyl-
choline linoleic-rich and glycolic acid in acne vu lgaris J. Appl. Cosmetol., 15, 21
12) Cardillo A and Morganti P. (1994) Fast and non-invas ive method for assess ing skin hydration.
J.A ppl. Cosmetol. 12: 11- 16
13) Folch J. et al.(1957), J. Biol. Chemistry, 226, 497
14) Cavina G. et al.(1965), Ann. lst. Super. Sanità, 1, 566
15) Stoffel A. et al.(1959), Ann. Chem. 31, 407
16) Williamson P, Kligman AM (1965) A new method for the quantitative investigation of
cutaneous bacteria J . !nvest. Dermatol. 45:498
17) McFinley K, Welster G, Leyder J (1965) Regional variations in cutaneous propionibacteria
J. App/. Env. Micro 45 :998
18) Morganti P. (1996), unpublished data
41
Bookreview
DERMATOLOGIC SURGERY
Principles and practice - Il Edition
It took long for o ur journal to review the second editon of this very interesting text of "Dermatolo-
gie Surgery" by Roeni gk & Roenigk's, as the board wanted it to be read by both dermatologists and
plastic surgeons, to whom it is essentially dedicated.
I personally devoted many hours to read this reall y complete text of clinica! diagnoses and updated
therapies, ranging from the detailed description and treatment of the always neglected wh ite popular
lesions called Milia, particularly frequent in the skin of Asian peoples, to the different techn iques
for the surgical management of skin tumours.
No topic of a dermo-surgical interest is left aside by the "directors" of this interesting text, written
by many experts of the academic world.
In fac t, it begins by describing the preparation of the skin before any treatment and the various sur-
gical tools required, without omitting the important matter of the infotmed consent by the patient
who asks for the treament. For legai purposes, in fact, the patient must be informed of ali indicaiions
and contraindications of the proposed treatment, and of any alternative treatment options.
Two entire chapters are devoted to the techniques and problems of an effective locai anaesthesia
with reference to methodologies and chemical compounds, which will have to be carefully evalua-
ted fo r the required quickness and duration of action, also considering any systemi c toxicity. A wide
space is also dedicated to ali the precautions and special proceeding the physician wi ll have to com-
ply with before and after treatment to prevent any complications, including careful evaluation and
psychogical support for the patient before and soon after a surgical operation.
The book also describes the attitude to be adopted in the various emergencies which might occur du-
ring a surgical operation, ranging from fainting to convulsions and allergie reactions, up to a cardiac
arrest, although extreme ly rare in dermatologie office surgery. Two inte resting c hapters dea! with
wound healing and wound dressing, which very clearly describe the physiological and biochemical
aspect of wound fornrntion and healing, as well as ali methodologies adopted or to be adopted to try
to avoid the formation of the unaesthetic keloids.
This first part of the book, consisting of ten chapters, ends with an eleventh chapter which explains
the various complications arising during any cutaneous surgery.
Another seven chapters thoroughl y describe standard procedu res, ranging from an accurate analysis
of how to correctly execute a skin biopsy, to the various suturing and electroepilation techniques, up
to the methods fo r the cryosurgical treatment of c utaneous lesions, of which it outlines the referring
contraindications.
Many chapters dea! with reparative surgery of the various cutaneous areas such as ear, lips, nose or
cutaneous appendages such as nails and hair, of which the different grafting techniques are skilfull y
Book rev1ew
described, from single hair grafting to rotation flap, up to the scalp expansion method.
No technique, whether old or modem , is ever casuall y described. Al! possible treatments, from
simple cosmetic treatments, such as camouflage, to seriou s surgical operations, such as the remo-
val of a malignant melanoma, are always explained and dealt with in a very accurate, simple and
professional way. Nothing is ever accidental, and no interesting subject of dermatologie surgery
has been forgotten.
Laser techniques, cover as much as six chapter which, starting from the basic physical laws, descri -
be with full details both the operating basis of the various types of lasers and the treatment of telan-
giectasias or wrinkles, or the difficult elimination of tattoos. Obviously, it also deals with the diffe-
rent techniques for reducing face wrinkles' depth, such as collagen injections or the use of non-col-
lagen-based fi ller materi als, or the techniques used to eli minate fat pads, such as liposuction. Wide
space is left to the various chemical peelings used in cosmetic dermatologie surgery, such as,for
example, trichloroacetic acid, phenol or the more recent g lycolic ac id. Forali treatments, the techni-
ques and referring contraindications are always included. Certainl y, this review does not full y show
the interest this book can raise in readers, whether medicai students or generai practitioners, derma-
tologists or plastic surgeons. This is not a common plastic surgery book, but a real " bible" for refe-
rence before any plastic surgery operation, both cosmetic and reparative. Therefore, this second edi-
tion of "Dermatologie Surgery" by Roenigk & Roenigk's should not be missing in the library of ali
those who deal with cosmetic dermatology in a broad sense, as the science conceming the beauty of
the human body, whether non-physicians, such as biologists, chemists, cosmetologists and physiolo-
gists, or generai practitioners and dermatology, plastic surgery, pediatry or ginecology specialists.
f'roçrJ:'ml/M foxnrwcfealt«Jnolo;in,
-
fil . ~ ; - -~ ·~- ·~
LA LINEA ANTIAGING
CON ACIDO GLICOLICO H ATTIVATO"
PER NORMALIZZARE
IL TURNOVER CUTANEO