OA17207A - Use of spider venoms for skin whitening/depigmenting and composition comprising spider venoms molecules or synthetic analogs. - Google Patents

Abstract

The present invention relates to the use of spider venom, molecules or synthetic analogs thereof for skin whitening. The invention also relates to compositions comprising spider venom, molecules or synthetic analogs thereof for skin 5 whitening/depigmenting. The invention finally relates to a non-therapeutic method for whitening human skin comprising topically applying an effective amount on said human skin of the composition of the invention.

Description

FIELD OF THE INVENTION

The présent Invention relates to the use of spider venoms for skin whitenlng/depigmenting and to compositions comprising spider venoms molécules or synthetic analogs.

BACKGROUND OF THE INVENTION

Melanogenesis is the scientiflc term to refer to skin pigmentation. Although melanogenesis is necessary as a mechanism of defense against UV radiation, a disruption of this process may cause an abnormal accumulation of melanin called hyperpigmentation, including melisma and senile lentigines.

The évolution of the melanogenesis pathway takes place in the epidermis, in particular in dendritic cells, which Internet with kératinocytes: mélanocytes. These latter contain spécifie organelles called melanosomes: site of the synthesis of different pigments (melanîns), responslble for our spécifie skin color, /.e. phototype.

Among black people, hyperpigmentation is visualized by a spotted and uneven skin. It may be the resuit of immune system activity in response to Inflammation, infection and/or healing but also of daily use of topical products containing substances lightenlng, now banned in cosmetics, such as hydroquinone and/or topical »

cortlcosterolds.

In order to reduce these dermatoses, understanding the mechanlsms of melanogenesis is essentlal. Thus, It will be easler to visualize where the potential inhibitor will a et to stop and/or reduce its production of melanin.

The melanogenic pathway Is a complex process that allows the production of two different skin pigments: black-brown eumelanlns and yellow to reddish pheomelanins. Tyroslnase Is the key enzyme required for melanin production. Her first fonction is the hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step of this process. Tyroslnase allows the oxidatlon of DOPA to DOPAquInone. Then, two pathways can be chosen.

In the absence of thlol compounds (cysteine) (Biack population), DOPAquInone ls oxidized spontaneously to dopachrome, a red Intermediate product. This latter can cydize spontaneously to give 5,6-dihydroxylndole (DHI), a biack insoluble molécule, or be converted into 5,6-dihydroxyindole-2-carboxylic-acid (DHICA) in the presence of a second enzyme: Tyrosinase-Related-Protein 2 (TRP-2). This second Intermediate must be in the presence of a DHICA oxidase activlty to be converted into DHICA-melanins. This activity ls ported by Tyrosinase-Related-Protein 1 (TRP-1).

Using skln bleaching cosmetlcs has been a social practice for about 30 years in the biack female population. Today, this concept is spreading on men in certain countrles in central Africa. Currently, 60% of biack African women admit to use skin lightening products in order to obtain a dearer and more uniform complexion. This practice, initlated by the media, may not only cause significant dermatological complications but also systemlc complications in the long run. About 70% of users hâve skin problems such as keloid acné, trophic disorders, hyperpigmentation, etc. These further complications, foremost dermatologie in the first time, corne from the toxlc activity of the compounds présent in lightener products such as hydroquinone or mercury dérivatives.

Currently, in France and in Africa, the most active substances used are hydroquinone, often at high concentrations exceeding 4% and topical corticosteroids with strong activity, such as dobetasol proplonate at 0.05%, which is one of the most potent topical corticosteroids. These products are used in the form of creams (hydroquinone or steroids), gels (corticosteroids) or milk (hydroquinone). The amount of active substance is often indicated but may be imprécise. The use of mercuray dérivatives, previously widespread seems to be more limited today. They may be used in the form of soaps called antiseptie*.

A prospective, descriptive study has been performed over a 6-month period in Sénégal Including 86 female patients with a mean âge of 29-34 years-old (range 16-49 yearsold). The break-down by skin-bleaching products showed that topical corticosteroids were the most frequently used (78%), followed by hydroquinone (56%), products based on vegetable extracts (31,7%), caustic products (8,5%) and finally, products of unknown composition (41,4%). Two components or more are frequently combined (86.5%).

Of the 19 types of complications listed, dyschromia, including hyperpigmentation of the joints, was dearly the most common. This remains a signlficant stigma associated with artificial depigmentation, with 85.4% sensitlvity. Striae atrophicae (72%) and skin atrophy (52.4%) were also very common, testifying to the very frequent use of corticosteroids.

Moreover, since several years, évidences demonstrating that there are links between the production of reactive oxygen species (ROS) and melanin overproduction hâve been well described in the literature. Indeed, UV stimulation induces melanin production which In turn provokes ROS or H2O2 release in the skin leading to skin aging. Some flavonoid compounds are also known for their properties able to both inhibit melanin and ROS production (For review·. GILLBRO & OLSSON, Int. J. Cosmet. Sd., vol.33(3):210-21, 2011) Thus, the Identification of tyrosinase or TRP-1 Inhibltors may hâve an Interest for treating skin aging.

Thus, there remains a need in the art for substances and cosmetic compositions able to reguiate the melanogenesis pathway without side effect for black and mixed skin (phototype IV-VI).

SUMMARYOFTHE INVENTION

The inventors hâve focused on finding novel tyrosinase inhibltors to level the DOPA oxldase activity of tyrosinase and the DHICA oxidase activity of TRP-1. They hâve surprlsingly found that some venoms, particularly spider venoms, contain this DOPA oxidase-inhibiting activity and DHICA oxidase-lnhibiting activity. In addition, the inventors hâve isolated from a venom of spider a molécule having both DOPA oxidaseinhibiting activity and DHICA-oxidase Inhibiting activity.

Now, these spider venoms activées were not known nor suggested In the prior art. Almost, venoms from bee were disclosed In patent application KR 2010-2264 as cosmetic agent including skin whitening activity.

A first object of the invention relates to a topical composition comprising spider venoms, molécules or synthetics analogs thereof.

Another object of the Invention relates to the use of spider venoms, molécules or synthetics analogs thereof as a skin whitenlng/deplgmenting cosmetlc agent.

Another object of the Invention relates to a composition comprising spider venoms, molécules or synthetics analogs thereof as defined in the Invention for treating and/or preventing hyperpigmentation such as melasma, chloasma, lentlgines, vitiligo, postInfîammatory hyperpigmentation due to an abrasion, a burn, a scar, a dermatosis, a contact allergy, naevi, hyperpigmentation with a genetic determinism, hyperpigmentation of metabolic or drug origin, melanomas or any other hyperpigmentary lésions.

Finally, the Invention relates to a non-therapeutic method for whitenlng/deplgmenting human skin comprising the step of topically applying an effective amount on said human skin of spider venoms, molécules or synthetics analogs thereof as defined In the invention.

BRIEF DESCRIPTION OFTHE DRAWINGS

Figure 2 shows the screening of 16 different spider venoms.

Figure 3 shows the dose-response effect s of 5 active spider venoms.

Figure 9 shows the dose-response effect of Argiope Lobata venom on DOPA oxidase activity.

Figure 10 shows the Inhibiting activity of Argiotoxin on the DOPA oxidase activity.

Figure 11 shows the Inhibitory mechanism of Argiotoxin on DOPA oxidase activity thanks to Lineweaver-Burk représentation.

Figure 12 shows the percentage of Inhibition of the DOPA oxidase activity depending on the concentration of 2,4-DHPAA.

Figure 13 shows the absorbance and thus inhibiting activity of Argiotoxin on DHICA oxidase activity.

Figure 14 shows the Argiotoxin inhibiting activity on melanogenesis in a cell-based assay.

Figure 15 compares the potency of 2,4-DHPA compound and Kojlc Acid on the melanogenic cell-based assay.Figure 16 shows the regulatory effect of Argiotoxin compared to Kojic acid on both Tyroslnase and TRP1 expression.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the Invention relates to a topical composition comprising spider venoms, molécules or synthetics analogs thereof.

As used herein, the term topical composition refers to a composition that is applied externally to any part of the body exduding mucous membranes such as the eyes, mouth, and $o on. The topical composition may, therefore, be applied to any part of the body excluding mucous membranes such as the eyes, mouth, and so on. However, the topical composition of the Invention may also be incorporated into sponges, swabs, pads and or wipes, which are the used to apply the topical composition to any part of the body excluding mucous membranes such as the eyes, mouth, and so on.

As used herein, the term spider venom refers to molécules produced by spiders and injected into their victims by the means of a bite, sting or other sharp body feature, to kill or paralyze them. Such molécules of the venoms comprise but are not limited to neurotoxins, cytolysins and hemolysins.

As used herein, the term molécule refers to a compound purified from the venom of a spider (e.g. by HPLC) orto a compound présent In a spider venom extract.

The term extract as used herein refers to a substance extracted to a natural product, regardless of the extraction method or the composition of the Ingrédients. For example, It Includes one obtained by extracting soluble ingrédients from a natural product using water or an organic solvent, or one obtained by extracting only spécifie ingrédients, such as oil, from a natural product.

As used herein, the term synthetic analog refers to any chemical or biological compound derived from an active molécule of venom.

As used herein, the term analog refers to a chemical compound that is structurally simiiar to another but dlffers slightly in composition. Thus an analog is a compound that is simiiar to or comparable in fonction and appearance to the reference compound.

Another object of the Invention relates to the use of spîder venoms, molécules or synthetlcs analogs thereof as a skin whitening/depigmenting cosmetic agent

As used herein, the term skin whitening agent refers to any compound or substance that hâve the effect of altering the pigment of the skin as long as the agent has antityrosinase activity and/or anti-melanogenesis activity.

As used herein, the term depigmenting or depigmentation refers to the réduction of the pigmentation of the skin that already exîsts and/or also to the prévention of any additional pigmentation greater to the natural pigmentation. For example, depigmentation 1$ obtained by reducing the formation or rate of formation of melanin. In a preferred embodiment, the spider venoms are chosen among venoms of spiders belonging to the genus Lycosa, Argiope or Araneus, preferably Arglape or Araneus. Arglape labata is a species of spider belonging to the family of Araneidae. It has a wide distribution encompassing the whole Africa and stretchlng to southern Europe and Into Asla.

Arglape bruennlchi is a species of spider belonging to the family of Araneidae. Its distribution encompasses central and northern Europe, northern Africa and parts of Asia.

Araneus tartaricus Is a species of spider belonging to the family of Araneidae. It Is found in western Asia.

Araneus camutus Is a species of spider belonging to the family of Araneidae. It is predominantly found in Europe, North of America and western Asia.

Lycasa Singariensis Is a species of spider belonging to the family of Lycosldae. It is predominantly found in central Europe.ln a preferred embodiment, the spider venom of the Invention Is the venom of Argiape Labata.

In a preferred embodiment, the spider venom molécule or synthetic analog of the invention Is represented by formula (1).

R1-R2-R3-R4-R5-R6 (1) wherein

RI Is an H or an aromatic such as (OHhCcHj- CH2-C(=O)- or tyrosine;

• R2 is -NH - CH[(CH₂)„- C(=O) -NH₂] - C(=O) -, with n=l or 2, preferably n=l (Le. R2 is asparagine);

• R3 ls -NH- (CH₂)_n—NH-, with n' is an integer between 1 and 7, preferably between 1 and 6, and most preferably n'=5;

• R4 ls absent or is - (CH₂)_n—NH-, with n ls an Integer between 1 and 7, preferably n=3 or 4, and still preferably n=3;

• R5 ls absent or ls - (CH₂)_n—NH-, with n' ls an Integer between 1 and 7, preferably n'=3 or 4, and still preferably n'=3;

• R6 ls an arginine residue; and • RI, R2, R3, R5 or R6 can be modify so that each peptide link RI - R2, R2 - R3 or R5 - R6 may be Independently replaced by a bond selected In the group comprising -CH2-CH2-, -CH = CH-, -C(=O)-CH2-, -CH2-S-, -CH2-NH, CH2-O-, -CH (OH)-CH2-, or-CH2-SO-.

In another preferred embodiment, the spider venom molécule ls the represented by formula (2)

Ho

Said compound of formula (2) called Argiotoxin-636, also known as argioplne, ls a polyamine Isolated from the venom of A. lobata. (CHEMBL1098240, CHEBI 724404).

In a preferred embodiment, the use ofthe whitening/deplgmentlng cosmetlc agent of the invention is for preventing and/or treating photo-lnduced or chronologie signs of aglng ofthe skin.

As used herein, the term photo-lnduced signs of aging refers to the extrlnsic aging of the skin, caused by the sun and more particularly by ultraviolet rays, which induce an increase in free radicals and oxidatlve stress in the dermis.

As used herein, the term chronologie signs of aging refers to intrinsic aging of the skin, caused by genetic and metabolic and leadlng to a progressive atrophy and degeneratlon ofthe dermis, hypodermls and support structures of the skin.

Another object of the invention relates to a composition comprising spider venoms, molécule or synthetic analog thereof as defined in the Invention for treating and/or preventing hyperpigmentation such as meiasma, chloasma, lentigines, vitillgo, postinflammatory hyperpigmentation due to an abrasion, a burn, a scar, a dermatosis, a contact allergy, naevi, hyperpigmentation with a genetic determinism, hyperpigmentation of metabolic or drug origin, melanomas or any other hyperpigmentary lestons.

As used herein, the term treating means to cure an already présent disease state or condition in a patient or subject. Treating can also include arresting the development of a disease state or condition, and relieving or ameliorating, i.e. causing régression of the disease state or condition.

The term preventing, as used herein, means to completely or almost completely stop a disease state or condition from occurring in a patient or subject, especially when the patient or subject is predisposed to such or at risk of contracting a disease state or condition. Preventing can also include arresting the development of a disease state or condition.

As used herein, the term hyperpigmentation refers to a range of skin disorders caused by an increased production of melanin and results in localized areas of increase skin pigmentation. Hyperpigmentation can refer to régional hyperpigmentation due to melanocytic hyperactivity, such as idiopathic melasma, to localized hyperpigmentation due to benign melanocytic hyperactivity and prolifération, such as senescent pigmentary blemishes (senile ientigo), and to accidentai hyperpigmentation, such as photo-sensitization or cicatriciel hyperpigmentation, and for the treatment of certain leukodermias, such as vitiiigo.

Finally, the invention relates to a non-therapeutic method for whitening/depigmenting human skin comprising the step of topically applying an effective amount on said human skin of spider venoms, molécules or synthetic analogs thereof as defined in the invention.

As used herein, the term effective amount of a composition comprising an active agent means a sufficient amount of said composition to provide the desired effect.

As used herein, the term therapeutic effect refers to the inhibition of the abnormal condition. The term therapeutic effect also refers to the inhibition of factors causing or contributlng to the abnormal condition. A therapeutic effect reiieves to some extent one or more of the symptoms of the abnormal condition.

The foiiowings experlments are offered to iiiustrate embodiments of the invention and should not be viewed as limiting the scope of the invention.

EXAMPLES

I. MATERIAL AND METHODS

1) In vitro experlments

a) HPLC fractionation of venoms

Solvents were purchased from Carlo Erba (Val de Reuii, France) and venoms are obtained thanks to LATOXAN (Valence, France).

To Identify the active molécules, the venoms were splitted by high pressure liquid chromatography (HPLC) In twenty fractions. HPLC was performed on a model 1100 from Hewlett Packard, the eluate absorbance was recorded at a wavelenght of 214 nm and a reversed phase column (Eurospher 100 or 300-5 C18,120*16 mm) was used. The filtered venom solutions were manually Injected . The solvent system (mobile phase) was: buffer A = 0.1% TFA in water and buffer B = 0.08% TFA in Acetonitrile 90 %/ water; the flow rate was 4 mL/mln of 0 to 60 % of buffer B in 60 min. Then, ail HPLC fractions were tested to iocalize the inhibition of the DOPA oxidase activity. Lyophilized active fractions of venoms were then purified, the method used was the same as previously described.

b) Mushroom tyroslnase assay

Mushroom (Agarlcus bisporus) tyroslnase (SiGMA-ALDRICH, T3824) has both DHiCA oxidase and DOPA oxidase activity (SUGUMARAN et al, Pigment Cell Res., vo!.12(2), p:118-25,1999).

The effect of different venoms, synthetic molécules and peptides on the tyroslnase activity was determined.

1. Quantification of DOPA oxidase inhibiting activity by spectrophotometry

The effect of different spider venoms on the DOPA oxidase activity was measured spectrophotometrically. The DOPA oxidase activity was determined using L-DOPA as substrate at 0.2 mM. The amount of DOPA chrome (red pigment) formed was measured against blank at 475 nm. The percentage of DOPA oxidase inhibition was obtained from:

Inhibition percentage = [(A-B)/(C-D)*100]

With:

• A : Absorbance at 475 nm of the reaction medium + inhibitor sample solution with enzyme • B : Absorbance at 475 nm of the reaction medium + inhibitor sample solution without enzyme (blank) • C : Absorbance at 475 nm of the reaction medium enzyme without inhibitor sample solution • D : Absorbance at 475 nm of the reaction medium without both enzyme and Inhibitor sample solution

L-DOPA and mushroom tyrosinase (Agaricus Bisporus, T3824), were obtained from Sigma Aldrlch (Saint Quentin Fallavler, France).

The inhibition of DOPA oxidase activity using mushroom tyrosinase was assayed in vitro as previously described by Elmer-Rico E Mojica et al. (CitPhilippIne Journal of Crop Science, vol.30(l), p:47-51, 2005) with some modifications. Solutions of 1 mg/ml of mushroom tyrosinase in PBS buffer solution (137 mM NaCI, 2.7 mM KCI, 10 mM Na₂HPO₄, and adjust the pH to 6.8), and 0.4 mM L-dihydroxyphenylalanine (L-DOPA) were prepared.

For the DOPA activity assay : 500 pL of the L-DOPA solution at 0.4 mM Is mixed with different volumes according to the different natural inhibitors at 10 mg/mL, and 5 pL of the mushroom tyrosinase solution (added last to Initlate the enzymatic reaction and must be kept In Ice). The final volume is 1 mL In Phosphate buffer saline (PBS) solution, pH 6.B. The absorbance Is measured at 475 nm every minute for 15 min using a UV-VIS spectrophotometer BIOMATE 5*. The blank used was the sample without the enzyme solution.

To make the venom solution at 10 mg/mL, 10 mg of each venom were weighted and solubilized in 1 mL of PBS buffer solution. The solution was filtered on a 0.45 pm filter. The Llneweaver-Burk assay

Experimental conditions of this assay were cited above. L-DOPA was used at different concentrations (to 0.1 mM from 0.4 mM), the enzyme is the mushroom tyrosinase at 25 Units. Each point has been done in duplicate.

The DOPAchrome formation was determlned by spectrophotometry at 475 nm.

II. Quantification of DHiCA oxidase-inhibiting activlty by MBTH assay

DHICA ls used as substrate at 0.25 mM. MBTH was obtained from Alfa Aesar and DHICA was prepared according to WAKAMATSU & ITO,(Anal. Biochem., voi.170(2), p:335-40, 1988). Here, the MBTH method was used. This method consists to visuaiize the production of hydrazone-quinone adduct by spectrophotometry. The prlnclple is that MBTH captures the indole-qulnone which was generated by the oxldation of dihydroxyindole compounds. DHICA ls converted to indole-5,6-quinone-2-carboxyiic acid by the DHICA oxidase activity of mushroom tyrosinase, the indole product is trapped by MBTH and the complex formation can be detected at 492 nm (OLIVARES et al, Biochem. J., vol.354, p:131-139,2001).

c) The DHICA oxidase Inhibiting activity using mushroom tyrosinase was assayed

In vitro. The procedure is the same as described by WINDER & HARRIS ( Eur. J. Biochem., vol,198(2), p:317-26, 1991) for DOPA oxidase with mlnor modifications.Purification ofthe fractions

The same HPLC method as described above is used. The HPLC-MS used was the LC2010A HT Liquid Chromatograph (SHIMADZU, Marne la Vallée, France). Two different wavelengths, at 214 and 280 nm were used.

d) Molecular characterization by liquid chromatography/mass spectrometry

The mass analysis was carried out on shimadzu LCMS-2010 EV HPLC System. The mass ofthe interest molécule was determlned usingthe followingformula:

m _ Mproduct + (z * Madduct) z z

Adducts products are: m/z + H* (1 g/moi), or m/z + Na⁺ (23 g/moi), or m/z + K* (39.1 g/mol).

The amino add analysis

In addition to mass spectrometry, the identity of the Argiotoxln was assessed by an amino add analysis (AAA) after add hydrolysis [6N HCl, 72h, 110*CJ. Ali purified argiotoxln batches were quantlfied by thls AAA method.

In vivo experlments al Reagents

Venoms were kindly provided by LATOXAN (Valence, France). Ceil culture media, trypsin-EDTA, penidliin/streptomydn were purchased from GIBCO (Saint Aubin, France). Alpha-Melanocyte Stimulatlng Hormone (α-MSH), 3,4-dihydoxypheniialanine (L-DOPA) and Kojlc Add were purchased from SIGMA-ALDRICH (Saint Quentln-Falavier, France) and ALFA AESAR (Ward Hili, Mass., USA) respectively.

b) Cell culture

B16F10 were obtained from the Amerlcan Type Culture Collection (ATCC CRL6475, Manassas, VA, USA). Cells were cultured in Dulbecco's Modifiée! Eagle Medium (DMEM) supplemented by 10% heat-inactivated foetal bovine sérum (FBS, LONZA Itd., Basel, Switzerland) and penidliin/streptomydn (50 pg/ml) in a humidified atmosphère containing 5% CO₂ at 37 ‘C.

c) Cell vlabllity assay

The évaluation of B16F10 cells survival relies on the réduction of the tétrazolium ring of water soluble MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide ; Sigma Aldrich) Into Insoluble purple formazan crystals by the mitochondrial succlnate dehydrogenase enzyme. The production of formazan directly reflects the number of iiving or dead cells. B16F10 cells were seeded at 25000 per well In a 96-well plate and Incubated In standard culture condition. After 24 h of growth, the supernatant was removed and replace by fresh culture medium containing various concentration of DHPA, Argiotoxln or Kojic Add for 6 h at 37 *C. Then, the supernatant was removed and replace by 50 μΙ of 0.5 mg/ml of MTT solution. The cells were exposed to MTT for 2 h. Finally, each well was washed by PBS1X and the crystals contained In welis are solubilized by adding 100 μΙ of DMSO. After briefly shaked the plate, the absorbance Is measured at 600 nm.

d) Measurement of melanin content In B16F10 melanoma cells

Détermination of the extracellular melanin Is consldered as an index of melanogenesls. B16F10 cells were cultured In 24-well plate (5.10⁴ cells/well) in a phénol red free medium and stimulated with 100 nM of a*MSH. Then, 24 h later, cells were Incubated for 48 h with Increasing concentration of DHPA (from 0 to 250 μΜ), Argiotoxln (from 0 to 42.1 μΜ) or Kojic Acid (0 to 100 μΜ) as a control. To quantity the amount of melanin released in the medium, the absorbance of 200 μΙ of supernatant was read at 405 nm. The values were normalized by the measurement of total protein level présent in each well.

e) Protein level détermination

Cells were washed with PBS1X and dissolved in 200 μΙ of NaOH IN for 1 h at 60’C. The lysate was then centrlfuged and the protein content determined by the Folin Lowry method, using the DC protein Assay kit from BIO-RAD (Marnes-îa-Coquette, France). The absorbance is read at 475 nm.

f) Western Blot Analysis

Cells were seeded at 1.10⁶ cells/dish and cultivated in π-dish as previously described. After trypsination, the pelled were homogenized in a solution containing Sodium Dodecyl Sulphate (SDS) and were boiled 5 min at 96*C to dénaturé the proteins. Protein extracts were separated via a 10% SDS-PAGE for 1 h at 200 V and transferred onto a polyvinylidene difluoride membrane (GE healthcare Europe, Vélizyvillacoublay, France). The membranes were blocked ovemight with 5% non-fat skim milk In a Trls-buffered saline solution containing 0.1% Tween. Membranes were incubated for 1 h at 37’C with spécifie antibody against tyrosinase, TRP1 or M!TF (C-19; G-17; N-15,1:200 for Tyrosinase and TRP1 and 1:100 for MITF, Santa Cruz, Heidelberg, Germany) and actin as a control (C-ll, 1:200, Santa Cruz). Membranes were Incubated with secondary antibody coupled to horseradish peroxidase (HRP) for 2 h at room température (anti-goat 1: 2500 for tyrosinase, TRP1 and MITF (ab 97120, Abcam, Paris, France) and antl-rabblt 1:2500 for actin (NA 934V, GE Healthcare)). The antibody staining was revealed by a chemiluminescent HRP substrate (Immobiion Western Kit,

Milipore, Molsheim, France). Pre-stalned protein markers were used for molecular weight.

g) Statistica! analysis

Statistical significance was demonstrated by the Student's t-test. The test Is performed on expériences done In triplicate, quadruplicate or conducted at least 3 times independently. Values of p < 0.05 were considered to be statistlcally significant and marked by one asterisk. Two asterlsks reflect that p Is less than 0.01.

II. RESULTS

1) In vitro assays

a) Venoms screening

Sixty eight venoms extracted from different species and families of venomous animais such as snakes, scorpions, spiders, etc., hâve been screened using UV spectrometry. The objective is to show the potentiai inhibiting activity of these different venoms on DOPA oxidase activity ofthe mushroom tyrosinase.

Venom 21, a spider venom, has a DOPA oxidase Inhibiting activity on the mushroom tyrosinase.. Venom 21 shows the most Interesting DOPA oxldase-lnhibitlng activity among the 68 venoms tested.

b) Spider venoms screening

From this first screening, it was found that the most active venom was the Argiope Lobata venom (venom 21). A second screening was performed, with only spider venoms. Each initial venom solution Is at 10 mg/mL, and 50 pL of them hâve been tested.

The figure 2 shows the results obtained with 16 spider venoms. For venoms having an Inhlbitory effect, a dose-response was realized (Figure 3). Many venoms from different spider families are able to Inhibit the DOPA oxydase activity. Here, Argiope Lobata, Araneus Tartaricus, Araneus Cornutus, Lycosa Singoriensis and Argiope Bruennichi are cited for their inhibltory properties.

Argiope Lobata, Araneus Tartaricus, Araneus Cornutus, Lycosa Singoriensis and Argiope Bruennichi venoms show an important DOPA oxidase-inhiblting activity because they hâve the lowest absorbance, compared to other spider venoms (Figure 2). Argiope Lobata (50 pL) has an absorbance around 0.1 and an inhibiting activity of DOPA oxidase around 90.9%. Araneus Tartaricus (50 μι) has an absorbance around 0.2 and an inhîbiting activity of DOPA oxidase around 74.5%. Aranaeus Cornutus (50 μΙ) has an absorbance around 0.3 and an Inhîbiting activity of DOPA oxldase around 41.0%. Lycosa Singoriensis (50 μι) has an absorbance around 0,250 and an inhîbiting activity of DOPA oxidase around 56.3%. Arglope Bruennlchl (50 μι) has an absorbance around 0.175 and an inhîbiting activity of DOPA oxidase around 76.5% (Figure 3).

Due to Its higher potency among the active splder venoms, the venom extracted from Arglope Lobata has been further studied for its activity on meianogenesls processes.

c) Fractionation, HPLC and mass spectrometry analysis of Arglope Lobata venom

Fractionation of Arglope Lobatavenom

From an initial venom solution of Arglope Lobata venom at 7 mg/mL, a fractionation In twenty équivalent fractions has been reallzed to localize this DOPA oxidase inhibition activity. These fractions are always tested using UV spectrophotometry. 40 μί of each fraction were lyophilized then taken in 100 μί of PBS. Only 20 μί of this latter solution is used for the spectrophotometrlc assay.

The G8 fraction exhibits the lowest absorbance (about 0.25) and thus the highest inhîbiting activity of DOPA oxidase activity (35.6%).

These results show that the DOPA oxidase-lnhibiting activity Is localized in fraction G8 of Arglope Lobata venom.

The purification of the fraction 68 (data not shown) of the Arglope Lobata venom was done by Alliance HPLC to Isoiate the active molécules and characterlze them by mass spectrometry.

Sub-fractlon 5 exhibits the lowest absorbance (between 0.3 and 0.4) and the highest Inhîbiting activity (between 40 and 44%). The DOPA oxidase-Inhlbltlng activity of the fraction G8 appears to be locallzed In the sub-fractlon 5.

Molecular characterlzatlon by liquid chromatograohy/mass spectrometry

Using the LCMS-2010 EV Liquid Chromatograph mass spectrometer, and thanks to the following formula:

m _ Mproduct + (z -2010 EV Ll z z the mass ofthe interest moiecule has been determined.

Adducts products are: m/z + H* (1 g/mol), or m/z + Na* (23 g/mol), or m/z + K* (39.1 g/mol).

Here, m/z » 637 has been obtained which was Isolated firstly by Grishln et al., 1989, Toxlcan. It corresponds to M_ARGiotoxin + H*. The presence of adduct product with m/z at 319, corresponds to the arglotoxln mass loaded 2 times with H * (m/z = 638) and divlded by 2 (638/2 = 319). This chromatogram which Is assoclated to the mass spectrum, confirms that Argiotoxin-636 is the correct moiecule.

The amino add analysis

To confirm the Arglotoxln structure, an amino acid analysis of different batches, has been realized, thanks to IBS laboratory (Grenoble, France). Ail the batches were quantified by thls method.

The amino acid analysis was managed as illustrated with batch 1.

The amino acid reference used is the N-Leu.

N-Leu loaded : 38904 picomol. / N-Leu read : 9831 plcomol.

Sample mass weight of Arglotoxln solution: 60.8 mg.

Initial volume sample: 5 mL

A.A.	Picomol	Res. %	1.	A.A./MO	It	Round
Asp/Asn	3941	46.81		0.97		1
Arg	4216	50.08		1.03		1
Others (Insignifiant)	262	3.11		0.06

Calculation :

(3941 + 4216) + 2 = 4080 Nb pmol/AA

4080m(38904ol/AA) x (10004ol/A) = 265.55 nmol/g

The concentration of the Argiotoxin solution 1 is 171.13 pg/mL or 269.1 μΜ.

Thanks to the mass characterization and the amino acid analysis, the Argiotoxin structure was confirmed. This structure is comparable to the one described by GRISHIN et al.(Toxicon, vol.27(5), p:541-549,1989). Argiotoxin is the interest molécule and will be evaluated for Its potency on DOPA and DHICA oxidase activities on mushroom tyrosinase.

d) DOPA oxldase-inhlbiting activity of V21

I. Half maximal Inhlbltory concentration et Argiope Lobata venom

From a solution of venomat 10 mg/mL, a half maximal Inhibitory concentration (ICso) measurement has been realized.

Figure 9 shows the ICso curve of Argiope Lobata venom. This curve shows that 500 pg/mL of Argiope Lobata venom inhibits 88% of the DOPA oxidase activity and 10 pg/mL of the venom Inhibits 5.6% of DOPA oxidase activity. A concentration of 62.5 pg/mL of the venom inhibits 50% of DOPA oxidase activity.

A dose-response curve with kojtc acid has been realized to use as reference. It permits to correlate DOPA oxidase-inhibiting activity with another Inhibitor (Data not shown).

ii. Argiotoxin inhibitory effect on DOPA oxidase activity

Argiotoxin Inhibitory effect on DOPA oxidase activity was also assessed on the mushroom tyrosinase. From an argiotoxin solution at 190.8 pM, the ICso was determined.

Figure 10 shows the dose-response curve of Argiotoxin on the DOPA oxidase activity. Argiotoxin display an ICso at 8.56 pM.

iii. Inhibitory mechanism of Argiotoxin on DOPA oxidase activity

Figure 11 represents the Lineweaver-Burk représentation, a graphical interprétation allowing the détermination of enzymatic parameters like the Michaelis constant (Km) characterizing characterizing the affinity between an enzyme and its substrate. The figure 11 shows that In the presence oflncreasing concentrations of Argiotoxin, the Km and the Vmax for the mushroom tyrosinase are varying, reflecting that Argiotoxin displays a mix Inhibition. Thus, Argiotoxin Is a non-competitive Inhibitor and Its blnding site differs from the substrate one.

e) Calibration of 2,4-DHPAA

The 2,4-DHPAA is the aromatic portion of arg!otox!ne-636.

Purification of synthesized 2,4-DHPAA was done by HPLC and a calibration curve was obtained by UV spectrometry. The software which has been used is RÈGRESSI. Several known concentrations of DHPAA -l.e. between 0 to 1 mg/mL- were obtained and stocked.

Table 1 : Results of the calibration assay of 2,4-DHPAA

[DHPAA] mg/mL	Absorbances (DO)
0.05	0.063
0.1	0.153
0.15	0248
0.2	0.34
0.25	0.421
0.3	0511
0.35	0.617
0.5	0.652
1	1.672

f) Détermination of 1C_M of 2,4-DHPAA for the DOPA oxidase activity

Figure 12 shows the percentage of inhibition of the DOPA oxidase activity depending on the concentration of 2,4-DHPAA. This figure suggests that mushroom tyroslnase has two different active sites for the l-DOPA substrate with two different affinities. So, the 2,4-DHPAA would be a compétitive inhibitor. Here, the activity is lower than for the whole Argiotoxin compound and around 6 mM. This resuit reflects that the entlre activity of the argiotoxin compound seems to be not only restricted to the head part of the compound.

g) Argiotoxin effect on DHICA oxidase activity

The mushroom tyrosinase has both the DOPA and the DHICA oxidase activities (SUGUMARAN et al, Pigment Cell Res., voi,12(2), p:llB-25, 1999). The DHICA oxidase activity 1s spedfically required for the biosynthesls of black/brown pigment. The abllity of Arglotoxin to Inhibits this activity is evaluated thank to the mushroom tyrosinase.

A dose-dependent inhibition is visible up to 100 μΜ of Argiotoxin (Figure 14). The experimental conditions are the same as previously mentioned. DHICA was used as substrate at 0.25 mM.

Figure 13 shows that Argiotoxin inhibits the DHICA oxidase activity of mushroom tyrosinase in a dose-dependent manner up to 75 μΜ.

2} In vivo experiments

Before going further in the évaluation of the potential use of argiotoxin as a skin bleaching agent, its cytotoxlc potency is evaluated at the maximal concentration (42.1 μΜ) displaying an effect In vivo. Argiotoxin présents neither cell death nor cytotoxlclty at 42.1 μΜ respectively evaluated by the MTT or the LDH cell-based assays (Data not shown).

a) Melanogenesis assay

B16F10 are murine melanoma cells, a cell iine able to produce melanin and consldered as an accurate mode! to assess melanogenesis inhibition. Cells were seeded at 5.10⁴ cells/weil in a P24 well plate and stimuiated by 100 nM of α-MSH, the melaninstimulating hormone. 24 h later, cells were treated by Increasing concentration of kojic acid as a control (from 0 to 250 μΜ) and from 0 to 42.1 μΜ for argiotoxin. 4B h later, according to SIEGRIST & EBERLE (Analytlcal Blochemistry, vol,159(l), p:191-7, 19B6), the amount of melanin reieased In the medium was spectrophotometrically measured at 405 nm and normaiised to the protein content.

The argiotoxin is also able to biock the melanogenesis in B16F10 in a dose-dependent manner and présent an ICsq around 0.01 μΜ (Figure 14).

The head part of the argiotoxin which Is Identified as the DHPA was also tested on this cellular assay to evaiuate whether the ex-vivo activity of Argiotoxin relies on the activity of this known compound, The protocol was the same as previously described for the argiotoxin. As a control, kojic acid was used in the same range of concentration.

Both compounds show a dose-response profile (Figure 15). DHPA présents an IC50 comparable to the kojic acid and around 5 μΜ. The presence of the DHPA group In the argiotoxln could only partlally explain the high potency of the argiotoxin. Indeed, argiotoxin présents a 100 fold higher activity on this cellular assay suggestlng that the activity of the argiotoxin is not concentrate in the head part of the molécule.

b) Western blot analysis

To explore the mechanism of action of Argiotoxin In melanogenesis processes, we assess its ability to regulate the protein expression of two enzymes, the tyrosinase and TRP1 which Is Implicated in the biosynthesis of black/brown pigments specifically. Western blot analyses were performed on B16F10 cells treated as prevlously described.

Figure 16 shows that as expected aMSH stimulation enhances the expression of tyrosinase and TRP1. Kojic add treatment falntly decreases the expression of murine tyrosinase or TRP1 protein. Contrary to the reference inhibitor, Argiotoxin display a slgnlficant Inhibition of the expression of both proteins. Argiotoxin reduces significantly the expression of tyrosinase and TRP1 proteins suggestlng that the compound may directly Interact with these enzymes to dégradé them or interfère with their biosynthesis pathway.

The inhibition of TRP1 expression by Argiotoxin highlights the fact that this compound may hâve a different mechanism of action compared to classic Inhibitors like kojic acid and confions Its relevance In cosmetic to develop spécifie skln-bleaching products targeting phototypes V and VI.

III. Synthetic analogs whitening activity

1) Synthetic analogs

The argiotoxin synthetic analogs are listed In table I In reference to formula (1).

Table I

Ν’	RI	R2	R3	R4	R5	R6
1	Tyr	Asparagine (Asn)	n'=7	absent	absent	Arginine (Arg)
2	(οη)αη3-οηη:(=ο)-	Asn	n'=5	n=6	absent	Arg
3	H	Asn	n’=7	absent	absent	Arg
4	H	Asn	n'=6	absent	absent	Arg
5	H	Asn	n’=5	absent	absent	Arg
6	H	Asn	n’=7	n=7	absent	Arg
8	H	Asn	n’=7	n=6	absent	Arg
9	H	Asn	n’=7	n=5	absent	Arg
10	H	Asn	n'=6	n=6	absent	Arg
12	H	Asn	n'=6	n=5	absent	Arg
13	H	Asn	n'=6	n=4	absent	Arg
14	H	Asn	n'=6	n”=7	absent	Arg
15	H	Asn	n'=5	n”=6	absent	Arg
16	H	Asn	n'=5	n”=5	absent	Arg
17	H	Asn	n’=7	n=3	n'=3	Arg
18	H	Asn	n'=6	n”=4	n'=3	Arg
19	H	Asn	n'=6	n=3	n'=4	Arg
20	H	Asn	n'=6	n=3	n'=3	Arg
21	H	Asn	n'=5	n=4	n'=3	Arg
22	H	Asn	n'=5	n=3	n”'=4	Arg
23	H	Asn	n'=5	n=3	n'=3	Arg
24	H	Asn	n'=4	n=5	n'=3	Arg
25	H	Asn	n'=4	n=3	n =5	Arg
26	H	Asn	n'=4	n=3	n'=3	Arg
27	H	Asn	n'=3	n=4	n”'=4	Arg
28	H	Asn	n'=3	n”=5	n”'=3	Arg

29	H	Asn	n'=2	n=5	n'=4	Arg
30	H	Asn	n'=2	n=6	n'=3	Arg

2) Inhibition of DOPA and DHiCA oxidase activities

The abovementioned analogs synthetics are tested at different concentration on the mushroom tyrosinase as disclosed previously. Argiotoxin is used as positive 5 control.

) In vivo experimen t

The analogs synthetics having an ICso Inferior to 10 μΜ for DOPA and/or DHiCA activities are tested on melanogenesis as described previously. Again, Argiotoxin is used as a positive control.

Claims (10)

1. A toplcal composition comprising spider venoms, molécules or synthetic analogs thereof.

2. Use of spider venoms, molécules or synthetic analogs thereof as a skin whitening/depigmenting cosmetic agent.

3. The use according to claim 2, wherein spider venoms are chosen among venoms of splders belonging to the genus Lycosa, Arglope or Araneus.

4. The use according to any one of daims 2 or 3, wherein spider venoms are chosen among Arglope Lobata, Arglope Bruennichl, Araneus tartaricus, Araneus Cornutus, and Lycosa slngariensls venoms.

5. The use according to any one of claims 2 to 4, wherein the spider venom is the venom of Arglope Lobata.

6. The use according to claim 2, wherein the spider venom molecuie or synthetic analog is represented by formula (1):

R1-R2-R3-R4-R5-R6 (1) wherein:

• RI is H or an aromatic such as (OH)₂C₆H₃- CHr-ClOJ- or Tyr;

• R2 is -NH - CH[(CH₂)_n- C(=O) -NH₂] - C(=O) -, with n=l or 2, preferably n=l;

• R3 ls -NH- (CH₂)_n—NH-, with n' ls an integer between 1 and 7, preferably between 1 and 6, and most preferably n'=5;

• R4 ls absent or is- (CH₂)_n—NH-, with n is an Integer between 1 and 7, preferably n=3 or 4, and still preferably n=3;

• R5 ls absent or is - (CH₂)_n—NH-, with n' is an integer between 1 and 7, preferably n^w'=3 or 4, and still preferably n'=3;

• R6 is an arginine residue; and • RI, R2, R3, R5 or R6 can be modify so that each peptide lïnk RI - R2, R2 - R3 or R5 - R6 may be independently replaced by a bond selected in the group comprising -CH2-CH2-, -CH = CH-, -C(=O)-CH2-, -CH2-S-, -CH2NH, -CH2-0-, -CH (0HJ-CH2-, or -CH2-SO-.

7. The use according to claim 6, wherein the spider venom molécule Is represented by the formula (2):

nh₂ (2).

8. The use according to any one of claims 2 to 7, wherein said whitenlng/ depigmentlng cosmetic agent is for preventing and/or treating photoInduced or chronologie signs of aging of the skin.

9. A composition comprising spider venoms, molécules or synthetic analogs thereof as defined in any one of claims 1 to 8 for treating and/or preventing hyperpigmentations such as melasma, chloasma, lentigines, vitiligo, postInflammatory hyperpigmentations due to an abrasion, a burn, a scar, a dermatosis, a contact allergy; naevi, hyperpigmentations with a genetic determinism, hyperpigmentations of metabolic or drug origin, melanomas or any other hyperpigmentary lésions.

10. A non-therapeutic method for whitenlng/deplgmenting human skin comprising the step of topically applying an effective amount on said human skin of spider venoms, molécules or synthetic analogs thereof as defined in any one of daims 1 to 7.