Quantifying Skin Relaxation and Well-Being C. Mas Chamberlin, O. Peschard, Ph. Mondon and K. Lintner

Sederma, Le Permy-en- Yvelines, France

ith two quotes, Professor Misery pointed to a new way oflookuigat ourbody's envelope.' He said, "Skin w

and brain are embryological and nostalgie brothers " He also said, "Skin is a peripherally spread out nemus system "

Skin is more than an assembly of several layers of cells (corneocytes, keratinocytes, fibroblasts, etc.). Skinis aliving sensory receptor of environmental stimuli, directiy con- nected to the centrai nervous system. Skin is aiso often the mirror of our mentai state of mind Many emotions are foremost seen or felt at the level of the skiu. Fear, stress and happiness manifest themselves in paling (vasoconstriction), sweating, goose bumps, raised hairs, blusbing (vasodilata- tion)%r radiance and blisshù glow.

Even surprise, consternation oranger translate into reac- tions that affect the skin: raising the eyebrows and fmwning lead eventually to expressionlines, the deep wrinkles on the face. Although this laner phenomenon is an indirect effect, caused by repeated muscle contraction, it has attracted mudirecent attentionwiththeaimoftreatingdeep wrinWes with muscle relaxing agents.

Mental welibeing andphysicai relaxation go handin hand, complemenung each other. Eodogenous molecules such as endoiphs and enkephaüns mediate some of these effects in the brain, and also now in the skin, as ths article shows.

Skin and Nervous Systern

The rehtionships between the mind and skin are cur- rentiy seen in a new light from the discoveries made in skin physiology, ernbryogenesis and neurc-immunology. The nervous system and the skui share a commonembryological origin since both organs denve from differentiation of the ectoblast, one of the three primary germ layers of the embryo Dunng the devefopment of the embryo, the ner- vous system sends out neuronal prolongations to innervate the skin In so doing, it is guided hy Merkel's cells (the epidermal neuro.endocrine cells) and stimulated by ü nerve growth factor (NGF)

Besicles the anitomical links (embryology), tiiere Xe hinctionül links brtween bram and skin V:~sdil:ihtion,

sebaceous excretiou, sweating and pi- loerectton are regulated by vanous ce- rebral neuromediators

The converseisalso tnle. Initsroleas a systemfor the simefiance of environ- mental changes (heat, humidity, etc) and perception of that environment (body M t s , recogniuon of the non-self and objects, touch, etc.), the skingener- ates stimuli that are transmined to the iiervous system via mediators

It has not been possible so far to tie the anatomical description of the van- ous dermal and epidermai nerve end- ings to their preeise functious, hence the di!%culty of describing the sensory nervous system of the skin. This is hir- ther complieated by the fact chat a given stimulus activates several Npes of fiber concomtanrly

From reading the literature on neuroperception and the skin, it will be readiiy understoodthat itisverydifîïcult to base a sensory, "neurocosmetic" ac- tivity on a simple mechanism

However, with appropnately perti- nent protocols andwellorganized clini. cal trials, it is possible to document cosmetic effects on the skinthat clearly ure related to ne=-perception at the cutaneous level

Neuropeptides

Molecules that medidte events and actiwties in Our organism fali lnto sev- en1 classes of chemicül nature Among these classes are the steroids, the amines, the peptides and a group of virious other substance5 Peptides ap

Kfy worck

skin, n m sysiem, narzppndes, iV-acery(-Tyr-Arg- hexndecylesrw

Abshact

Skzn m1axahon and zuell-bezg can be lneaszlred by pantilatiue nzethods, and moduhted by a Tyr-Arg neumpepttde wed in top~cutcosmetzc preparatiom

pear to play a particularly iniportaiit role in the modulation of neural activity and signaling: neurotensiii, substance P, enkephaliii and endorpliiii are but a few of those identlfied in tlus field

Aiiumberof biologically active pep- tidesfound in the organism are, in fact, fragments of bigger precursors Oiie of these is pro-opiomelanocortin (TOMC), which is secreted hy a num- ber of epithelial cells, including keratinocytes. The POMC sequence contains, for instance, the a-MSH mol- ecule (melanine sümulating hormone), the ACTH molecule (adrenocortico- tropic hormone), and p.endorpliin, whicli is a sleep inducing opioid type peptide. And within the endorphin sequence, the 5-amino acid peptide enkephalinisfound, wluchwas sbown to be apowerful, endogenous, analge- sic peptide in the brain, binding as it does to the morphine receptor.14

Furthermore, somewhat later, the dipeptide Tyrosyl-Arginine (Tyr-Arg) was discovered (at the University of Kyoto, hence its scientific name 'Kyotorphin'). It has powerful analge- sic activity in the hrain.&' The mecha- nism of action was shown to involve the release of enkephalins in the bram, which block the pain signal in similar ways to morphine.

The doserelationship between brain and skin outlinedabove, the identifica- tion of enkephalins in dose proximity to cutaneous nerve cells, and the fact that keratinocytes are capable of syn- thesizing met-enkephalin precursors (F'OMC) led us to investigate the topi- caluse of an appropriately derived Tyr- Arg peptide, namely N-acetyl-Tyr-Arg- Iiexadecyiester (NATAH). This chemi- cal derivation is necessary to ensure sufficient skin penetration and bioavailabilrty.

In Vitro Studies

Endorphin precursor rekase: Does this particular peptide sequence show shilar activity on skin cells as it does in the mammaliaii brai? Human norma1 keratinocytes in culture were exposed to mcreasing levels of the NATAH peptide Meanwhde tlie pro-

I I I F+r.e 1. Inmase oser basefisina of POMC-nrRN.4 refease mit/> t l ~

addition of N A T M Ko b w a n k e m t i m q e s in nrltlrre

duction of POMC (endorphin andmet-enkepl1&precursor molecule) via the activation of the comesponding gene was assayed quantitatively by RTPCR technique. Figure 1 shows the effect of low levels of the peptide NATAH on tliis activation: adosedependent stimulationis clearly observed.

The phorbol ester PMA (negative control) strongly iiiliib- its this gene expression, as we expected based on the published literature! It is thus suggested that the natural dipeptide TyArg is able to stimulate in the skin the release of opiateprecursors, justasit doesinthe b r a i Keratinocytes were already known to synthesize POMC after W irradia- tions Furthermore, met-enkephalin peptides have been lo- cated hy ELISA techniques in close proximity to epidermal neuronal ce Il^.^

Myobiast rehation.The clinical trials described later show diat the transmission of heat, chemical stiiiging and mechanical stresses is reduced hy topical application of NATAH, leadmg to decreased sensitivity of the skui nerve cells. IfindeedPOMCissynthesized by the keratinocytes and enzymatic breakdown ofPOMC to endorphins andenkepha- lins occurs in the s k i , we can ask: does the NATAH peptide also lnteract diiectly with the nerve celle7 Do tlie released enkephalin peptides mterfere with the neuronal transmis sion? And if the heat-, pain- and mechano-receptor fibers are affected by the peptides, what would occur at the junction of nerves and muscles? We studied these questions hy using the protocol tliat nerves and muscle ceils can be cocultured in vitro @igure 2) '1

It is thus possible to observe the regular, rhythnuc con- traction of muscle cells under the microscope and to study inhibition or stmulation of neural-muscuiar mteractions A decrease in frequency and/or a total hlockage of muscle contractLon are indications of synaptic inhibition. WeU- known controls such as mbungarotoxin (positive control) serve as benchmarks.

The incubation of tllis nerve-muscle mode1 with non- cytotoxic amounts of NATAH peptide for 5 and 120 min- utes respectrvely shows (Table 1) a small trend of immedt-

Vol 119. No 1010ctober Po04

Figure 2. Newe and m s c l e co-ntlhrre (corrrfesy 867: Bso Ezpern'se Technologies, France)

Table 1.NATAH-modulated decrease In muscle contraction frequency

NATAH NATAH

Negative PusIlive (1 ppm) 11 ppml nintml oontrol aiier 5 min aller 2 hr

Fibsr 1 O Block >20% Block Rber 2 O Block Block Fiber 3 Block

ate (5 min) contractile frequency decrease and a later (120 min) total blockage of muscle activity (three separate trials). The effect is however, reversible and shows that no permanent damage occurs to the nerves, muscles or their junction. This time delay of 2 hours is not fully understood at present, but might be an indication that the NATAH peptide triggers the release of fufther neuromediators, a process that takes more time than immediate binding to a receptor on the neuromuscular junction

Although repeatedly performed(n=3), this semi-quantita- tive experiment does not allow for statistical analysis.

S>cnrmary:Tosummarize theinvitro stud~es, it cm be said that the dipeptide Tyr-Arg, whose analgesic properties had been descnbed in brain re~earch,~' also stimulates endorph precursor release in skm cells and slows or blocks neur* muscular interaction. The transformation of the dipeptide into the more lipophüic and bioavaüable NATAH bas neither decreased its activity nor introduced toxic side effects; it was thus an ideal candidate for clinical utals of cosmetic usage.

Chnical Trials

The NATAH peptide was formulated at 1% (w/w) into a hydrogiycolic excipient' and incorporated into skjn care

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WARM HOT VERY HOT MAXIMUM

1 10 m T2ho"n TOLERATED

1 I Figure 3. NATAH-nlodzrlated change ir r lent pevception

lotions at the level of 300 ppm. Tlirçe

independent, veliicle-coiitrolled experi-

ments were carried out, testing for tlie

skiii's sensitivity agaiiist lieat,"' cheiiii-

cal stinging" and nieclia~iical stress

(abrasion)."

Heat: Twenty-one paiielists were

selected and briefly trainrd to recog- Figure 4. Lie-dctector sigrial. the ORD widlli ispvopoi-lionnl to

nizefour levels (warm, hot, ver). hot, sti.ess

maximum tolerated, as a function of

ski11 temperature) of heat sensation

applied instrumeiitallyb to tlle back sive effect of the cream. Changes at the NATAH-treated at sites treated witll a cream that sites, showedp values of 4 0 5 , <0.01, <0.01 and <0.01, colitained 300 ppni NATAH or a pla- respectively, for the four levels. cebo (the cream veliicle). The tein- The skiti had become less sensitive to heat tiirough the peratures of al1 palielists' respoiises action of NATAH, presumably on the iierve celis. were recorded before treatinent aiid Cl~enzical stinging: Capsaicin, the spice molecule from averaged for eacli level at the two cliili pepper, dissolved in alcohol, is a powerful irritant and treatineiit sites, aiid agail1 two Ilours inducer of uiipleasant sensation of prickling on the skin. after applicatioii of the tested prod- However, sensitivities of differeiit persons Vary widely. uct or placebo. 011 a selected paie1 of respondents, an etliaiiolic solution

Figure 3 shows tlie modulatioii of of capsaiciri was applied dose to tlie ear lobe, tlieii the tlie heat perceptioii. A sigilificant iil- stingiiig iiltensily was evaluated by each paiielist over 60 crease il1 the tenlperature 1t.vels de- niin~~tes and scored. Aftcr a rest period, the veliicle or the scribed as warm, ]lot, v ~ r y hot 2nd NATAH lotion was applied to the same sites, and 30 minutes iiiaxilnulii tolerated was obsensed tn.0 later again capsaiciil solution was dabbed ta the s h . The lionrs afterapplication. Statistically sig- scores of perceived stingiiig intensity decliiied @<0.05), on nifiCalit differel~ces he tw~ei i NATA- tlie average, by 30.50% ontlie NATAH treated sites, coinpared treated and placeho siteswereobtaiiied to the control (vellicle) 1 1

for the tliree levels hot, ver). hot and Mecl~naical si>-ess: Since the inventioii of the lie- liiaxiliiu~ii toleratçd VI<O.O~). A trend. dçtector it is wrll hiiowii tliat cmotions subtly infl~ieiice altliouglinotst;~tisric;illysignlficant,n~as phe~ionle~i ;~ tliat cari be measured. ~l~~ fol. pumived witli pcrsisteiice of tlie cf- lowiiig çxperiiiiçnL was tlilis set up in collaboration with fcct 1111 to 4 liours. Spin Coiitrol, aproduct çfficacy testing compaiiy inTours,

<:li:iiiges ai placebo sites nrere liot France. aiid Dr. Arnaud Aiil~crt, I'li.D., of the University of sigiiificant (/1>0.05); tlie small in- TOUES, France. creases are prohahly due 10 the occlu- Eighteçii vol~intççrs wcre selçcted for their ability to

- discrimin:ite diffcrriit degrecs of sa11dp:iper text~ire by \<,i,.\irli<.r,ii /inll>(../iiiiii l*~rriiico,, l j r i , , .

1: r,,,,<<, , ?,,,>, ,vr , , , , ,v<, ,< , , y ,,v*,r<,<, iflit c . , , <,, ,,,.,,, t l l ~ i r f i ng~ r l i l )~ . To cr?:itr an "ernt~ti«nal stress," an i)<'iiiiiiiiii. invrstigatur passed saiidl~apçr <>ver one liaiid of eacli

dçtçctiii- prohc' recorded tlic clcc-

iroclçriiial respoiisc :ilid Oliiiiic He-

s lxinx 1I)lir:itioii (ORD) sigiial (Fig~

tire 4), wliicli is ~~ri>pr~rtioii:ll to the

iiitçiisity oltlie çiiiotioii. Afier çstab-

lisliiiig the h;iselinç for encli volun-

teer, eitlirr veliicle or the NATAI-I

lotion was applied to the "test" liaiid,

and tlie lie-detector sigiial was agaiii

1 haur 2 houri 3 hourr O

-5 - s c; -10 O .- - m 'L -15 5

-20

- . were asked grade degree of 1. L Misery, Pioceedings. XXéme Journées Europeennes de

DermocoçmBtoiogie, Lyon, No" 2000 discomfort 011 an analog scale (data 2. L Misery. ~ e i ~ u e de Meikei ei systeme neuro-cutan~, ~ 8 t h ~ i o 1 4 4 849

iiot sliown) and observed the same (1996) 3. M Ban et al, The nurnber and distribution of merkel cels in iudimentary

decrease in skin reactivity on tlie ~ o i v d a ~ t ~ l v . Dermatoloo~202!11 31 (2001)

ineasured.

Figure 5 shows that for a duratioii of

.. . . . trrated sites (p<0.05 at the T=lh 4. T E ' W I S O ~ et ai. Absence of arterra barorelex modulation of skin

çympathelcactlvityandsweatiatedurngwhole-body heatingin humans, point in inter-treatment coinparison) J Physiol536 615 (2001) Objective and subjective results tlius 5 . M Campero. Slowiy conductng aflerents activated by innocuous ow

followed the same variation, demon- temperature in human skn, J PhysioI535(Pl 3) 855 (Sep 2001) 6. H Shiorni et a l Morphne4ke anageçia by a new dipeptde. EuiopeanJ

stratiiig the reality of tlie observed Phaimacol 55 109 (1 9791

skiii calming. 7. H Shlomi et a Mechaniçm of Kyotorphin induced release ol Met- Enkephalin fiom Guinea pi9 striatum and spinal co i d Biain Reçeamh

-25 Placebo 300 ppm peptide

221 161 (1981) C O ~ C ~ U S ~ O ~ 8 E Schauei et ai. Pioopomeianacorln-deiived peptides are synthetised

and reeaçed by human keratnocytes. J Clin lnvesl 93 2258 (1994) "Wçu-bçing%" "CocoOiiin~"aiid"Zeii" 9. SStandeieta1,Localizatonoimcro~opodreceptar IAansensory nerve

more tlian 2 Iiours, the skin's sensitiv- Figure 5. NATAH-r>iodulated change in Ol>,nic respotrsc duratio>z

ity ta tlie uiipleasant feeling of sand u,if,,

paper abrasion NaS lowered. This was

signitïcaiitly(p<0.05) different froiii tlie

smauer~laceboeffectalidpersistedfor sliots or surgery. Of course, tlie compouiid aiid its use is 3 Ilours. Placebo were Ilot entirel)' cosmetic. Neitlier drug claini iior medical (anal-

(p'0.05). Of gesic) activity is implied. Furtlieriiiore, coinbiiiatioiis of NATAH-treated sites were significaiitlJ' this niolecule witli inany other active iiigredieiits in skiii

'Ower T2h or hair care are possible and desirable. (p=0.011). Differeiices between treat-

melits are sigilificant for the Tl11 point R ~ ~ ~ O ~ , , ~ ~ I , J , , o ~ a r ~ or.prri-t sjtl3is rrr-tic~e ir r f r i c i~p io l i i b i t cd

@=0.044).

This instrumental fiiidiiig of low-

ered skin sensitivity paper Address correspondence Io C. Mas Chamberlin. cl0 Editor Cosmetics &

abrasioii correlated well witli the sub- Toiletriesmagarine, 362 South Schmaie Road Carol Stream, iL 60188-2787 USA,

iective scoriiiz bv tlievolunteers who

are tlie catcliwords of Our time. "To be

a hundle of iierves" ("avoir les nerfs a

flçur de 11~211" in FI-eiicli) is oot. Relax- ;ilioii <iiiiiiiid ;iiid body, of s h i rracthity

aiid inoscle twitcliing, they al1 go to-

getlirr. "Frçliiig goad" aiid "looking

good" c;iiiiirit he dissociated.

TIir iiçuro~>çptidr tliai is the basis

of NATAH dçlivers, by cosnietic

iiiç:iiis, tlicsc activitirs and beiiefits

to ilir ci~iisuiiirr withoiit tliç iiiidesir-

;iblc- sidr cffccis (;ilid cost) (if drugs.

ilbers in human skin. Regul Pept 1 lO(1) 75-83 (Dec 2002) 10. OC Yeornans et a Nociceptlve respanses ta high and low rates of

~ O X \ O U S cutaneous heafing are medated by diierent nociptors. Pain 68(1) 1 (1996)

11 KLntnerandOPeschard. Bologicallyactive peptides framaaboratory benchcurosly toa Iunctiona çkincare product /nt JCosmSci22207- 21 8 (20001

12. J Sudheçh. lnves!igaIion nt0 the eilects of musc and meditatton on gaivanic skin response ITBM~RTBM 21 158 (2000)

13 R Steinschnetder et al. Reconstrucled eadermis with sençorv nevrons,

01 enkephalin the passible endogenous ligand for the morphine receptoi LrieSci 161121 1753~1758 (Jun 15. 1975)

GT

Vol 119, No. 1010ctober 2W4