PEPTIDE DERIVATIVES OF alpha -MSH AND THEIR APPLICATION

0001PEPTIDE DERIVATIVES OF oC-MSH AND THEIR APPLICATION

0002The present invention notably has peptide derivatives of α- MSH ("Melanocyte Stimulating Hormone"), presented in Lipoyl-Peptides form.

0003A -MSH and its counterparts have been the subject of numerous publications, or even therapeutic trials, without this resulting in the production of a medicament.

0004The main reason is the extreme ubiquity of α-MSH depending on the doses administered and the routes of administration.

0005In addition to the absence of dose / effect relationship (which is the case with many peptide hormones), seriously complicates its therapeutic use.

0006The cellular receptors for α-MSH and its counterparts are of the G type and the intra-cellular transduction takes place according to the cycle of the cyclic AMP. The activation of these cellular receptors is substantially inversely proportional to the doses of peptide hormones accepted by the structures of the membrane receptor.

0007The present invention is based on the search for peptide structures specifically oriented towards anti-allergic and anti-inflammatory activities on the one hand, and activators of melanogenesis on the other hand to the exclusion of any other pharmacological effect in particular at the level of the system central nervous system, like the peripheral nervous system.

0008More particularly, the present invention relates to a compound comprising a peptide sequence comprising at least one sequence of

00094 amino acids originating from α-MSH, the amino acids being in natural form or not, said sequence being conjugated with thioctic acid or a derivative of this acid, in the form of the corresponding salts, esters or amides.

0010The sequence of α-MSH is as follows:

0011N-acetyl - Ser - Tyr - Ser - Met - Glu - His -

0012Phe - Arg - Trp - Gly - Lys - Pro - Val - NH₂ In this definition, as in the following, the amino acids can be in D, L or D, L form and the non-natural forms of the amino acids correspond to derivatives, in particular substituted. Thioctic acid or α-lipoic acid can be in the oxidized form:

0013<img file="WO9508564A1_D0001.tif" />

0014or in the form of a dihydrolipoic derivative:

SH SH

II CH2-CH2-CH-CH2-CH2-CH2-CH2-COOH

0017Among the derivatives of this acid, mention should be made of the N-lysine derivative of the oxidized or dihydro form.

0018One of the main objects of the present invention is the therapeutic application by the topical (cutaneous) route of the above derivatives.

0019These compounds are molecules capable of crossing the skin barrier, and of presenting the peptide fraction to cellular receptors, by inducing a biological response of the dose / effect relationship type.

0020These low molecular weight peptides, whose amino acid sequences have been modified, are linked in the form of salts, esters or amides to active biochemical groups, playing a crucial role in the tricarboxylic cycle (at the level Mitochondria in particular). These cofactors are lipoic or thioctic acids in oxidized or reduced form and their Lipoyl-Lysine derivatives, which are naturally covalently linked to the polypeptide chains of the cellular enzymatic system.

0021More specifically, the present invention relates to peptides of 4 to 6 amino acids linked in the form of Lipoyl-Peptides and Lipoyl-Lysyl-Peptides with anti-allergic, anti-inflammatory activity and activator of melanogenesis. The compounds according to the invention preferably have the peptide sequence comprising at least the following sequence:

0022- His - Phe - Arg - in which Phe represents phenylalanine or a halogen derivative of phenylalanine, the amino acids possibly being in D, L or D, L form, and in particular they may be compounds having the formula:

0023[Up] X - His - Phe - Arg - Y in which

0024Iip is thioctic acid or one of its derivatives, X is Glu, OH or NH2,

0025Y is Trp - Gly - OH,

0026Trp - Gly - NH2 Trp - NH2 Trp - OH Phe is homo Phe or p-fluoro Phe, the amino acids being in D, L or D, L form

0027The invention particularly relates to the following compounds:

0028I [(DL) Iip] Glu - His - D.homoPhe - Arg - Trp - Gly - NH2 II [(DH Up] Glu - His - D.homoPhe - Arg - Trp - Gly - NH2

0029III [(DL) Up] Glu - His - ParaFluoroPhe - Arg - Trp - Gly - NH2

0030IV [(DL) Up] His - D.homoPhe - Arg - Trp - NH2

0031V [N.Upoyl-Lysine] Glu - His - D.homoPhe - Arg - Trp - Gly - NH2 VI [N.lipoyl-Lysine] His - D.homoPhe - Arg - Trp - Gly ~ NH2 VI I [N.lipoyl -Lysine] His - D.homoPhe - Arg - Trp - NH2 as well as the derivatives of these molecules in the form of ester salts or amides.

0032In the formulas, the position of thioctic acid corresponds to the ester or amide derivative, the acid fraction of thioctic acid providing the bond.

0033The amino acid sequences mentioned above can be natural amino acid sequences or non-natural amino acid sequences. Likewise in certain cases it is possible that some of these amino acids comprise functions, for example they are glycosylated and / or sulphated.

0034It should be understood that all of these forms are covered by the present description. The present invention also relates to pharmaceutical or dermo-cosmetic compositions comprising a compound as defined above.

0035The pharmaceutical compositions can be in form which can be administered orally or parenterally, intraperitoneally or injectable in particular, but preferably in form which can be administered by external topical route.

0036These compositions can be, in particular, in the form of a cream, spray or lotion for example and include known excipients and possibly other active ingredients.

0037The compounds according to the present invention are useful in particular in the prevention and treatment of allergies and inflammations.

0038Peptide III, whose sequence has in position 3 the amino acid paraFluoro Phenyl, is particularly oriented towards an anti-allergic and anti-inflammatory activity, by immunosuppression of Monokines (IL1, IL6, α TNF).

0039The dermo-cosmetic compositions are preferably in the form of a solution, lotion, emulsion or cream which can be used in particular as an accelerator for tanning the skin without exposure to ultraviolet rays.

0040Peptides II and TV whose sequences have in position 3 and 2 the amino acid D.homo Phényl are particularly oriented towards a stimulation of the processes of melanogenesis and activation of Tyrosinase.

0041The peptide sequences according to the present invention can be obtained by any of the methods known to those skilled in the art, in particular by methods of chemical synthesis in which thioctic acid can be integrated. In any event, taking into account the small size of Peptides, chemical synthesis is entirely possible and allows very pure products to be obtained. Example 1

0042Synthesis of compound 1

00431 - [(DL) Up] Glu - His - D.homoPhe - Arg - Trp - Gly - NH2

0044The synthesis is carried out by the Merrifield method in solid phase using an MBHA resin and as a protective group FMOC (fluorenyl methoxy carbonyl).

0045The amino acid derivatives used are:

0046FMOC Gly OH,

0047FMOC Arg (Tos) OH, FMOC Trp OH,

0048FMOC D.homo.Phe OH,

0049FMOC His (Trt) OH

0050FMOC Glu chex) OH which is coupled using BOP as a coupling agent. Each amino acid is used in excess (x2) as well as the BOP (x2) and each coupling is repeated 2 times.

0051Upoic acid is similarly coupled, the FMOC group is eliminated at each step with piperidine (20% in dimethylf ormamide). The final deprotection is carried out in two stages: a) Trifluoroacetic acid (2 times x 5 minutes) b) anhydrous hydrofluoric acid / p-Cresol 95.5 (45 minutes)

0052The crude compound obtained with a yield of 78% is taken up in a water / acetic acid mixture 95.5% and is then lyophylized. Compound 1 obtained has a purity of 82% (HPLC).

0053100 mg of this compound are purified by HPLC using a Cl 8 column

005465 mg of pure product are obtained.

0055Retention time = 17.04 minutes under the following conditions:

0056- column C8 (250 mm x 5 mm), UV detection 210 nm - phosphate buffer solvent Triethylamine - acetonitrile 10-60% in 1 5 minutes - flow rate 1.4 ml / minute.

0057Amino acid analysis:

0058Glu 1.02 - Gly 1.01 - His 1.00 - Arg 0.94 - D.homo.Phe 1.03 - Tryptophan has not been determined because it degrades during acid hydrolysis.

0059Mass spectrum (FAB +) 1018.4 / 1126.5

0060Examples 2 and 3

0061By replacing DL lipoic acid with DH lipoic acid, compound 2 is obtained: [DH Up] Glu - His -D.homoPhe - Arg - Trp - Gly ~ NH₂.

0062By replacing FMOC D.homoPhe - OH by FMOC ParafluoroPhe - OH, we obtain compound 3:

0063[(DL) Lip] Glu - His - ParaFluoroPhe - Arg - Trp - Gly - NH₂.

0064Example 4

0065Synthesis of Compound 4 The procedure is as in Example 1 to prepare:

0066[(DL) Up] His D.homoPhe Arg Trp NH2

0067Physical constants ...

0068F - 130 ° c - Retention time 19.72 minutes

0069Column Cl 8 "Nucleosil" - UV - 279 nm Solvent TFA - 0.1% Acetonitrile 75/20 flow rate - 1.2 ml / minute

0070Amino acid analysis

0071His = 0.80 - Dh- Phe 1.11 Arg = 0.96

0072Trp = not dosed - Chemical mass - 887.18

0073Examples 5 to 7

0074By operating as above, we obtain: compound 5 [N.Upoyl-Lysine] Glu - His - D.homoPhe - Arg - Trp ~ Gly - NH2 compound 6 [N.Upoyl-Lysine] His - D.homoPhe - Arg - Trp - Gly - NH2 compound 7 [N.Upoyl-Lysine] His - D.homoPhe - Arg - Trp - NH2 Example 8

0075Study of the immunosuppressive activity of the compound 4 derivative on the inhibition of the synthesis of Interleukin 1 "IL1" in mice. The secretion of IL1 is induced by parenteral administration of "LPS" lipopolysaccharides in BALB / C mice.

0076The release of bacterial lipopolysaccharides during microbial infections, strongly induces the synthesis and the release in the organism of the monokines Interleukins "1 IL1" Interleukin 6 "IL6" and of "Tumor - Necrosis - factor" α TNF which are responsible for the violent reactions inflammatory including septic shock.

0077Equipment

0078Compound 4 (P-IV) is dissolved in physiological saline. It is quantitatively measured by spectrophotometry at 280 nm (assay of Tryptophan) which allows precise dosing of "P-IV" in the solutions which are stored at -25 ° C.

0079"LPS" lipopolysaccharides:

0080Extracts of Escherichia Coli serotype 0127 - B8 - "Ref. SIGMA - L3137" are used at a concentration of 6 mg / ml in physiological saline. Animals:

0081BALB / C mice - females - 5 weeks old from IFFA - CREDO subjected to a photoperiod of 12 hours of light per 24 hours of water and food ad libitum

0082Methods Two techniques have been developed to carry out this assay:

0083- one based on a 5-day treatment used to demonstrate the anti-inflammatory activity of PIV (called "SHEEHAN" technique)

0084- the other based on a single treatment, therefore much faster will be used to control the activity of PIV ("DAYNES" technique)

00851- SHEEHAN technique a) dosage

0086Three dosages are being studied: - 1 ng of PIN in 0.2 ml of physiological saline (i.e. 5 ng / ml)

0087- 0.5 ng of PIN in 0.2 ml of physiological saline (i.e. 2.5 ng / ml)

0088- 0.1 ng of PIV in 0.2 ml of physiological saline (i.e. 0.5 ng / ml) b) Treatment of animals It was carried out on mice divided into five batches of 5 animals:

0089- a negative control batch

0090- a positive control batch

0091- three PIV lots (one per dosage)

0092The animals in the control lots receive an injection of 0.2 ml of physiological saline by the subcutaneous route, the animals in the PIV lots receive an injection of the product at the various dosages by the subcutaneous route. This treatment takes place for 5 days. c) Induction of ILl α

0093Forty-eight hours after treatment with PIV, a sublethal dose of LPS (1.2 mg in 0.2 ml of physiological saline) is injected intraperitoneally into each animal of the PIV lots and of the positive control lot. d) Obtaining sera

0094One hundred eighty minutes later all the mice are punctured by the retroorbital sinuses on dry tubes. As soon as the blood coagulates the clot is detached. The blood is centrifuged for 10 minutes at 1800 g at a temperature of 10 ° C.

0095The sera, once removed, are frozen at -25 ° C. 2- DAYΝES technique a) Posology Three dosages are studied

0096- 40 μg of PPvt in 0.2 ml of physiological saline - i.e. 200 μg / ml

0097- 25 μg of PIN in 0.2 ml of physiological saline - i.e. 125 μg / ml

0098- 10 μg of Pr in 0.2 ml of physiological saline - ie 50 μg / ml b) Treatment of animals II was carried out on mice divided into 5 batches of 5 animals

0099- a negative control batch

0100- a positive control batch

0101- three PIV batches (1 per dosage) The animals in the control batches receive an injection of 0.2 ml of physiological saline by the intravenous route.

0102The animals in the PIV batches receive an injection of the product at the various doses intravenously. (Caudal vein) c) Induction of ILl α

0103Immediately afterwards, a sublethal dose of LPS (1.2 mg and 0.2 ml of physiological saline) is injected intraperitoneally into each animal in the PP lots and in the positive control lots, d) Obtaining the sera One hundred and eighty minutes later all the mice are punctured by the retroorbital sinuses on dry tubes. As soon as the blood coagulates the clot is detached. The blood is centrifuged for 10 minutes at 1800 g at a temperature of 10 ° C. Once collected, the sera are frozen at - 25 ° C. 3- Determination of ILl α Interleukin 1 α contained in these sera is dosed 24 hours after collection with the ELISA IL1 α murine assay kit (ref. 1900 00 GENZYME)

0104- Quick description of the Kit: the method used for this assay is of the sandwich type. We use a first monoclonal antibody against murine IL1 α

0105The samples are then deposited, and a second biotinylated anti IL1 α antibody is made to act.

0106The revelation takes place with avidin coupled to peroxidase. The colored reaction uses tetramethylbenzidine (TMB) Reading takes place at 450 nm on a MULTISKAN plate reader

MCC 340 MKII (TITERTEK)

0108The results obtained by the SHEENAN method are collated in Table 1 below:

0109Table 1

VA THEIR POU RC ENTA GE OF PIV ACTIVITY BY MEANS IN NE DOSAGE

0111DU TESTIMON POSITI F 200 μg / ml 125 μg / ml 50 μg / ml (in pgtml)

0112STUDY 1,250 -94% -50% -82%

0113STUDY 2,230 -50% -46% -31%

0114STUDY 3,200 -62% -37% -47%

0115STUDY 4,430 -62% -49% -76%

0116STUDY 5 430 -38% -62% -58% The average decreases in IL 1 α obtained by dosage, are of the order of 55% (60% to 200 μg / ml, 50% to 125 μg / ml and 50% at 50 μg / ml) for all five studies.

0117All the results obtained confirm the anti-inflammatory activity of PP, through its inhibitory action on IL1α synthesis.

0118Indeed, with the two methodologies used, there is a decrease in the synthesis of Interleukin 1 α of at least 50% compared to the control.

0119This anti-inflammatory activity was supplemented by a study of the immunosuppressive activity of PIV vis-à-vis Tumor Necrosis Factor α.

0120The results obtained (-40%) confirm the suppressive activity of PIV on the synthesis of Interleukins IL 1 and α INF.

0121There is also a protection of the treated animals having received a lethal dose of LPS: this protection resulting in a delay in the highly significant mortality rate. EXAMPLE 9 Inhibition of the Activity of TNF α by the Peptide PIV

0122Material and method

0123The DAYNES techniques described in Example 8 are used. DAYNES technique a) Dosage

0124Three dosages are under study

0125- 40 μg of PTV in 0.2 ml of physiological saline - i.e. 200 μg / ml

0126- 25 μg of PrV in 0.2 ml of physiological saline - either of 125 μg / ml - 10 μg of PPV in 0.2 ml of physiological saline - or of 50 μg / ml b) Treatment of animals

0127It was performed on mice divided into 5 lots of 5 animals

0128- a negative control batch

0129- one positive control batch - three PPV batches (1 per dosage)

0130The animals in the control lots receive an injection of 0.2 ml of physiological saline by the intravenous route. The animals in the PIV batches receive an injection of the product at the various doses intravenously. (Caudal vein) c) Induction of TNF α

0131Immediately after the treatment of the mice, a sublethal dose of LPS (1.2 mg in 0.2 ml of physiological saline) is injected intraperitoneally into each animal in the batches and positive control. d) Obtaining sera

0132Ninety minutes later all the mice are punctured by the retroorbital sinuses on dry tubes. As soon as the blood coagulates the clot is detached. The blood is centrifuged for 10 minutes at 10 ° C at 1800 g. Once collected, the sera are frozen at - 25 ° C.

0133Determination of TNF α

0134The TNF α contained in these sera is assayed 24 hours after collection with the Murine TNF α ELISA assay kit (ref. 1509-00 GENZYME) - Quick description of the Kit: the method used for this assay is of the sandwich type.

0135A first monoclonal antibody against murine TNF α is used.

0136The samples are then deposited and a second anti TNF α goat antibody is made to act. The revelation takes place with a peroxidase-labeled donkey anti-immunoglobulin donkey antibody.

0137The colored reaction uses o-phenylene diamine (OPD).

0138Reading takes place at 492 nm with a plate reader (TITERTEK

0139MKII MCC 340) The results obtained are collated in Table 2 below:

0140Table 2

VALUE PERCENTAGE OF PIV ACTIVITY BY

0142AVERAGE DOSAGE OF WITNESS POSITI F 200 μg / ml 125 μg / ml 50 μg / ml (in pgtml)

0143STUDY 1 2600 -41% -26% -31%

0144STUDY 2 4690 -65% -53% -38%

0145STUDY 3 4,690 -52% -45% -42%

0146STUDY 4 4040 -30% -41% -41%

0147STUDY 5 4 360 -42% -34% -31% This methodology confirms the average results obtained previously.

0148Indeed, average decreases in TNF α obtained by dosage, are of the order of 40% (46% at 200 μg / ml, 40% at 125 μg / ml and 37% at 50 μg / ml) for the whole of the five studies.

0149Example 10

0150Study of the Antiallergic activity of Peptide compounds, by the contact hypersensitivity test to Dinitrofluorobenzene (DNFB).

0151Material and method

0152Compound 3 "PIII" Lots 1 - 2 - 3

0153Compound 4 "PIV" Lots 4 - 5 - 6

0154C57 BL / 6JICO female mice, 5 weeks old, are divided into ten batches of ten animals (five per cage), with free access to water and food, subjected to a photoperiod of twelve hours of light per twenty- four hours.

0155The animals of lots I to 6 receive daily and by topical application on the shaved skin of the back, the products to be studied, dissolved in propylene glycol, at a constant volume of 50 μl and this for five consecutive days.

0156Dosage per mouse and per day

0157PIII - Lots 1 - 2 - 3 - 2.5 μg - 0.5 μg - 0.1 μg - mouse / day

0158PIV - Lots 4 - 5 - 6 - 2.5 μg - 0.5 μg - 0.1 μg - mouse / day The animals in lot X serve as controls (they receive only,

015950 μl of propylene glycol by topical application for five days).

0160On the fifth day, thirty minutes after the last application, all the mice are sensitized with 25 μl of DNFB (2.4 - Dinitro - 1

0161FluoroBenzene (FLUKA CHEMIKA PURUM 97% lot no. 33820890) 2% in a 4 for 1 mixture of acetone (SDS ref 0510) and triolein (FLUKA) applied topically to the dorsal region of shaved skin.

0162On the sixth day, a new sensitization is carried out by applying DNFB. On the eleventh day, the thickness of the ears of the mice, is measured using a micrometer (ROCH 0 to 25 mm at 1/100 mm) in order to obtain basic values, then the ears are stimulated by application topical of 20 μl of DNFB at 0.8 96.

0163On the twelfth day, the thickness of the ears is again measured.

0164The average value of the thickening of the ears is established for the mice of the lots which have received the products to be studied, as well as for the mice of the control lot. The percentage of possible deletion will be expressed by the

0165following calculation: _jclQQ

T

0167The results are collated in the following Tables 3 to 6:

0168<img file="WO9508564A1_D0002.tif" />

0169Table 3 Results PIII

LOTS NUMBER AWARENESS STIMULATION AVERAGE RESULTS PIII MOUSE

0171LEFT EAR RIGHT EAR% INCREASE% INCREASE

L0T1 10 EAR BACK 19.21 19.21

0173PIII (S1) DNFB 2% DNFB 0.8%

01742.5 ng / mice day

LOT 2 10 EAR BACK 19.81 19.81

JA

0177P III (SI) DNFB 2% DNFB 0.8%

01780.5 ng / souristjour

LOT 3 10 BACK EARS 17.71 17.71

0180P lil (SI) DNFB 2% DNFB 0.8%

01810.1 μg / mouse / day

LOTX 10 BACK EARS 58.92 58.92

0183Witnesses DNFB 2% DNFB 0.8%

0184Table 4 PIV Results

LOTS NUMBER AWARENESS STIMULATION AVERAGE RESULTS P IV OF MOUSE

0186LEFT EAR RIGHT EAR% INCREASE% INCREASE

LOT 4 1 0 EAR BACK 17.99 17.99

0188P IV (S2) DNFB 2% DNFB 0.8%

01892.5 μg / mouse] our

LOT 5 10 EAR BACK 18.03 18.03

0191P IV (S2) DNFB 2% DNFB 0.8% C / l

01920.5 μg / mouse /] our

LOT 6 1 0 BACK EARS 20.35 20.35

0194P IV (S2) DNFB 2% DNFB 0.8%

01950.1 μg / mouse] our

LOTX 1 0 BACK EARS 58.92 58.92

0197Witnesses DNFB 2% DNFB 0.8%

0198Table 5 Results PIII

0199Percentage of suppression of "DNFB" contact hypersensitivity

0200% deletion compared to the Witness Lot

0201T - E according to the formula: X "i 1n unu

T

LOT 1 (S1) LOT 2 (S1) LOT 3 (S1)

02042.5 μg / mouse / day 0.5 μg / mouse / day 0.1 μg / mouse / day

EAR EAR EAR EAR EAR EAR LEFT RIGHT LEFT RIGHT LEFT RIGHT

0206-67,40 -67,40 -66,38 -66,38 -69,94 -69,94

0207Table 6 PIV Results

0208Percentage of suppression of "DNFB" contact hypersensitivity

0209% deletion compared to the Witness Lot

0210according to the formula: x 100

T

LOT 4 (S2) LOT 5 (S2) LOT 6 (S2)

02132.5 μg / mouse / day 0.5 μg / mouse / day 0.1 μg / mouse / day

EAR EAR EAR EAR EAR EAR LEFT RIGHT LEFT RIGHT LEFT RIGHT

0215-69.47 -69.47 -69.40 -69.40 -65.46 -65.46 PIII and PIV suppress highly significantly - "67.90% and 68.11" the induced skin hypersensitivity reaction by administration of DiNitroFluoroBenzene under the experimental conditions described above.

0216Example 1 1

0217Study of the anti-inflammatory activity of PIII and PIV

02181) Aim of the study:

0219Demonstration of the inhibitory effect of peptides PIII and PIV on the production by fibroblasts stimulated by IL 1, of a metabolite of arachidonic acid, PGE2.

02202) Methods:

02212-1 Culture of embryonic pulmonary fibroplasts of human origin: We have chosen to use the ATCC strain MRC5 maintained in the laboratory on a continuous basis and already tested by Cannon et al. (J. Immunol., 1986).

0222After subculturing, the fibroplasts are cultured in microwells (24-well plate of 2 cm 2, Falcon). The innoculum is 30,000 cells per well; the nutrient medium used is composed of RPMI 1640 (90%) and decomplemented fetal calf serum (SVF, 10%). The medium is changed every two days until the cells meet.

02232-2 Experimentation

0224The single-cell layers thus obtained are washed and then preincubated for 24 hours in fresh medium containing only 1% of FCS. The substances to be tested are added to the medium at different concentrations between 10-6 and 10-4 M. After 20 minutes of incubation in the presence of these compounds, the recombinant human IL1 (Tebu, France) at the concentration of 5, 2.5 or 0.5 ng / ml of medium is brought into contact with cells for 18 hours.

0225At the end of this incubation, the supernatants are removed and frozen at -80 ° C until analysis. The single-cell layers are fixed with methanol. 2-3 PGE2 dosage

0226The RIA assay is carried out according to the method described by Dray et al. (Europ. J. Invest. 1975). 3 series of duplicate experiments were carried out for each concentration of product studied and for the controls. The results are expressed in pg / μg of DNA. The inhibitory effect is expressed as a percentage relative to the controls.

0227In order to avoid any possible error due to counting in optical microscopy, the DNA was assayed by fluorimetric method according to the protocol described by Brunk et al. (Analytical Biochem., 1979).

0228Table 7 below brings together a 1st series of tests on the MRC5 strain

0229Table 7

0230PIII action

0231PIII PGE2 samples pg / μg DNA% inhibition

0232IL1: 2.5 ng 389 ± 60

0233+ PIII 10-⁴ M 179 ± 50 53

0234+ PIII 10 "⁵ M 232 ± 45 40

0235+ PIII 10-⁶ M 292 ± 68 24

0236+ PIII 10 "⁷ M 364 ± 68 6

0237+ PIII 10-⁸ M 335 ± 50 13

0238+ PIII 10 "⁹ M 320 ± 43 17

0239IL1: 0.5 ng 288 ± 25

0240+ PIII 10-⁴ M 113 ± 25 60

0241+ PIII 10 "⁵ M 176 ± 37 38

0242+ PIII 10-⁶ M 231 ± 55 1 9

0243+ PIII 10-⁷ M 249 ± 60 13

0244+ PIII 10-⁸ M 258 ± 35 10

0245+ PIII 10-⁹ M 364 ± 56 no inhibition A good PIII inhibiting action is observed with dose effect ratio. This action does not depend on the IL1 concentration. No action is observed from 10 -7M in both cases.

0246Table 8 PIV action

0247PIV samples PGE2 pg / mg DNA% inhibition

0248IL1: 5 ng 7576 ± 310

0249+ PIV10-⁴ M 2964 ± 184 60

0250+ PIV10-⁵ M 4580 ± 230 38

0251+ PIV 10-⁶ M 4900 ± 340 34

0252+ PIV10 "⁷ M 6150 ± 365 17

0253+ PIV 10-⁸ M 6142 ± 255 17

0254+ PIV 10-⁹ M 5047 ± 279 32

0255PIII and PIV have an inhibitory effect on the action of IL1 and PGE2.

0256Very significant this action could be linked to a blockage of receptors.

0257Example 12

0258Study of the action of P III and PVI on the melanogenesis of the mouse after topical application in the form of dermal cream (Warren technique). Material and method

02591) Material

0260- Compound 3 PIII

0261[(DL) Up] - Glu - His - ParaFPhe - Arg - Trp - Gly - NH2

0262- Compound 6 PIV [N.Iipoyl - Lysine] - His - D.homoPhe - Arg - Trp - Gly - NH2

0263- Dermal excipient n ° 66 Lano - vaseline - Ac

0264Petroleum jelly Pharmacopoeia

0265Purified Ethoxyle Lanolin beeswax

PEG - 200

0267Dermal formulas

0268Peptides PIII and PVI incorporated at a rate of 5% in the dermal excipient n ° 66 Animals

0269DBA / 2 IFFA - CREDO mice, 5 weeks old

02702) Methods

0271• A dorsal tonsure is demarcated on each animal after depilation

0272• The products to be tested are applied by massage at the rate of 2 applications per day, 50 μl of each preparation for 5 days

0273• Two days after the last application, the animals are sacrificed and fractions of approximately 50 mg of treated skin are removed.

0274• Each sample is dried by lyophilization and weighed

0275• The skin fragments are then subjected to an enzymatic hydrolysis by protease K for 72 hours at 45 ° C.

0276(Protease K Merck - 24.568)

0277• 500 μl of Na2CO3 and 20 μl of 35% H202 are added to the hydrolysates obtained.

0278• Incubated for 30 minutes at 80 ° C. • After cooling, 200 μl of chloroform / methanol (2 vol / 1 vol) are added to each sample

0279• Centrifuge at 10,000 g

0280• The aqueous phase (200 μl) is distributed in the wells of a "NUNC" microplate

0281Determination of Melanin

0282• The melanin assay is performed at 405 nm using a Multiskan - Titertek - MCC - 340, compared to a standard melanin range Sigma M. 8631

0283Results

0284They are expressed as a percentage of melanin compared to the excipients considered as controls

0285I - Cream P III - 5 10-5 M PIII for 50 μl of cream

0286II - PVI cream - 5 10-5 M PVI for 50 μl of cream

0287III - Excipient 66 - as control T Average of the results over 15 dosages

0288Samples% of Melanin in mg

0289I Cream PIII 4 mg% melanin

0290II PVI cream 71 m% melanin

0291III Excipient 66 - Control 0 mg% melanin

0292Table 9

0293Percentage of stimulation

0294QE = average amount of melanin per mg of skin - Sample

0295QJ = average amount of melanin per mg of skin - Control

0296% stimulation ₌ _2__L_Q_ χ 100

QT

0298The dermal cream containing PVI administered topically, highly induces the synthesis of Melanin in the epidermis (+ 71%).

0299This activity in accordance with the object of the present invention is linked to the chemical structure of PVI which contains N.Lipoyl - Lysine and D.homoPhenyl groups.

0300The dermal cream whose formula contains PIII administered topically, has no activity on the stimulation of Melanogenesis

0301Epidermally, this absence of activity is linked to the presence in the PIII structure of the "para.FIuoroPhenyl" group in accordance with the subject of the present invention.

0302These results were confirmed by a study on Melanogenesis according to the "CLOUDMAN" technique by melanocyte culture in vitro, using CLOUDMAN melanoma cells in rats. All the results obtained are consistent with the subject of the present invention, the objective of which is the production of peptide derivatives active by the Topical route and whose anti allergic and anti inflammatory activities can be separated from the activity on melanogenesis according to the applications envisaged.

0303Example 13

0304Comparative study of the activity of Lipoyl-Peptides and their peptide structures, on contact hypersensitivity to DNFB Material and method

0305The method used is that described above in Example 10. Hypersensitivity of contact induced in mice by Dinitrofluorobenzene - "DNBF"

0306Products studied Lot I: (DL) - Up - Glu - His - D.homoPhe - Arg - Trp - Gly - NH2 Lot II: H - Glu - His - D.homoPhe - Arg - Trp - Gly - NH2 Lot III: Control = Upoic acid (DL)

0307Solvent: propylene Glycol previously used as a control in Example 10.

0308A measurement of the activity of the witness will be carried out for each experiment.

0309Experimentation and dosage Lot I: Doses used 1 = 1 μg / mouse / day

03102 = 10 μg / mouse / day 3 = 100 μg / mouse / day Lot II: Doses used 4 = 1 ng / mouse / day

03115 = 10 ng / mouse / day 6 = 100 ng / mouse / day Control: dose used "DL-Upoïc-Acide" = 10 ng per mouse and per day.

0312On the fifth day, thirty minutes after the last application, all the mice are sensitized with 25 μl of DNFB (2.4-Dinitro-l

0313FluoroBenzene (FLUKA CHEMIKA PURUM 97% lot no. 33820890) 2% in a 4 for 1 mixture of acetone (SDS ref. 05510) and triolein (FLUKA) applied topically to the dorsal region of shaved skin.

0314On the sixth day, a new sensitization is carried out by applying DNFB

0315On the eleventh day, the thickness of the ears of the mice, is measured using a micrometer (ROCH 0 to 25 mm at 1/100 mm) in order to obtain basic values, then the ears are stimulated by application topical with 20 μl of 0.8% DNFB.

0316On the twelfth day, the thickness of the ears is again measured.

0317The average value of the thickening of the ears is established for the mice from the batches which have received the products to be studied, as well as for the mice from the control batch. The percentage of possible deletion will be expressed by the calculation

0318next:: IJ._xι_∞ Table 10

MOUSE: HYPERSENSITIVITY OF CONTACT SUMMARY TABLE LOT I

LOTS NUMBER AWARENESS STIMULATION RESULTS: AVERAGE

MOUSE

0322Left ear Right ear

0323Average% Average% Increase Increase

0324Lot 1: Back Ears

0325100 10 DNFB 2% DNFB 0.8% 11.66 11.66 ng / mouse / day

0326Lot I: Back Ears 10 10 DNFB 2% DNFB 0.8% 12.95 13.01 ng / mouse / day

0327Lot I: Back Ears 1 10 DNFB 2% DNFB 0.8% 13.86 13.86 ng / mouse / day

0328Back Ears

0329Witnesses 10 DNFB 2% DNFB 0.8% 56.34 56.75

0330Table 11

MOUSE LOT I HYPERSENSITIVITY OF CONTACT DNFB

0332% DELETION FROM THE WITNESS LOT

0333ACCORDING TO THE FORMULA: - - * 100

T

0335Lot 1: Lot I: Lot I: 100 ng / mouse / day 10 ng / mice / day 1 ng / mouse / day

0336Ear Ear Ear Ear Ear Ear left right left right right left right

0337-79.30 -79.45 -77.01 -76.90 -75.40 -75.58 Table 12

MOUSE: CONTACT HYPERSENSITIVITY - DNFB SUMMARY TABLE LOT II

LOTS NUMBER AWARENESS STIMULATION RESULTS: AVERAGE

MOUSE

0341Left ear Right ear

0342Average% Average% Increase Increase

0343Lot II: Back Ears 1 10 DNFB 2% DNFB 0.8% 37.07 37.07 ng / βouriβ / day

0344Lot II: Back Ears 10 10 DNFB 2% DNFB 0.8% 34.84 34.84 ng / βouriβ / day

0345Lot II: Doβ Ears 100 10 DNFB 2% DNFB 0.8% 43.10 43.10 ng / mouse / day

0346Back Ears

0347LOT 10 DNFB 2% DNFB 0.8% 58.92 58.92 WITNESSES

0348Table 13

MOUSE: CONTACT HYPERSENSITIVITY - DNFB

0350% DELETION FROM THE WITNESS LOT

0351ACCORDING TO THE FORMULA: - - * 100

T

0353Lot II: Lot II: Lot II: 1 ng / βouriβ / day 10 ng / mouse / day 100 ng / mouse / day

0354Ear Ear Ear Ear Ear Ear left right left right right left right

035537.08 37.08 40.83 40.87 27.15 27.15 Results

0356Lot I - Lipoyl-Peptide - I

035779% suppression of cutaneous hypersensitivity is observed at the dose of 100 ng / mouse / day. There is also a clear dose / effect relationship between doses of 1 ng - 10 ng and 100 ng.

0358Lot II - Peptide alone

035940% suppression of cutaneous hypersensitivity is observed at a dose of 10 ng / mouse / day. For the 1 ng and 100 ng doses, the suppressive response is of the order of 37.08 and 27.15%, without a dose / effect relationship being observed.

0360Control batch - Upoic acid only

0361No suppressive effect of cutaneous hypersensitivity is observed by administration of Upoic acid, in solution in Propylene - Glycol.

0362Administered topically and under the experimental conditions described, the "Upoyl Peptide" structure highly induces the suppression of skin hypersensitivity in mice.

0363Example 14 Embryo - Toxicology

0364Qualitative and quantitative analysis of the effects of P IV on melanocyte differentiation in in vitro culture. Sample to be analyzed - compound 4 = PIV Biological material Embryonic cells isolated in in vitro culture in a defined, purely saline medium (standard culture conditions: 2 ml of medium / culture).

0365The embryonic territory is removed (from the Pleurodele embryo and from the Axolotl embryo) from the first stage of melanogenesis, immediately after melanocyte induction.

0366At this early stage of embryonic development, the neural bead, which has just individualized, contains all of the melanoblastic precursors. The cultivation of the cells of the neural bead and the underlying mesoderm makes it possible to cocultivate (under completely identical conditions) the melanoblasts with various other cellular precursors belonging to various categories: myoblast neuroblasts, epidermal cells, fibroplasts, mesenchymal cells.

0367This allows a comparative kinetic study, in living organisms, of the effects of PIV on melanocytes and various other cell types.

0368Duration of culture (primary culture): 2 to 3 weeks at 20 ° C.

0369Cell differentiation appears morphologically from the 3rd day and is almost complete at 8-10 days of culture.

0370Treatments 1 ° Dose / response tests

0371A range of PIV concentrations was analyzed:

03720.01 μg / ml; 0.1 μg / ml; 1 μg / ml; and 10 μg / ml

03730.01 μg / ml: no effect

03741 μg / ml: optimal effect on melanocyte differentiation (according to the criteria indicated in "Results")

0375The 2 doses are therefore used for this work: 0.1 and 1 μg / ml.

03762 ° Treatments

03771 only treatment when seeding cells in culture. After their attachment (3 days at 20 ° C) the medium is replaced by fresh medium without the Peptide PIV

03781 treatment during seeding and a second treatment during the change of environment.

0379Results

0380Qualitative effects 1 °) Stimulating effect of PIV on melanogenesis

03812) This effect is not accompanied by cytotoxicity phenomena. 3 °) The other cultivated cell categories are not affected, their morphology and their differentiation take place normally, except those of neurons which present shorter neuritic prolongations and in denser network than in the controls. It should be noted that these neurons come from the neural crest (neural bead) and are therefore the neurons at the origin of the peripheral nervous system. 4) No toxic effect is manifested, all the cells, other than the melanoblasts, differentiate and evolve like the untreated control cells.

0382Quantitative analyzes The following effect of PIV on melanogenesis results not only in an increase in the size of the melanocytes (Cf. plate I and

0383II) but also:

03845) by an increase in their number;

03856) by an increase in the quantity of biosynthesized melanin. A - Stimulating effect of PIV on melanogenesis

03861 °) It is already important to emphasize that the stimulating effects described below manifest themselves in a similar way after 2 treatments or a single treatment (in this case, the cells are maintained in the presence of PIV for only 2 first days of culture, when they are still morphologically undifferentiated melanoblasts.

0387It is therefore not necessary, in order to have optimal stimulation, to maintain these cells in the presence of PIV throughout the period of their differentiation.

0388The stimulating effect of PIV manifested itself in exactly the same way on the embryonic cells of Pleurodeles like Axolotl.

03892) The phenotypic differentiation of the melanocytes appears more quickly in the treated cultures (from the 2nd day after seeding) than in the control cultures.

03903 °) The stimulation of melanogenesis relates to the size of the melanocytes which increases by a factor x 4 and even x 5 after 8 days of culture for treatment with 1 μg / ml.

03914 °) The stimulating effect of PIV also relates to the quantity of biosynthesized melanin.

0392Already the only observation, on alive, of the treated melanocytes makes it possible to note that they are much darker, in spite of their larger size, than the control melanocytes.

0393We have developed two dosages of melanin:

0394- by HPLC with electrochemical detection

0395- by spectrophotometry (by adaptation of the method of TOMITA et al., 1990) at 470 nm. The development of these assays was carried out using standard melanin solutions (Sigma, ref. M 8631) in order to define the comparative sensitivity and the minimum detection threshold by either method. The μg is the only minimum which can be measured by spectrophotometry, the ng is the lower threshold, which can be measured by HPLC.

0396It should however be noted that these two techniques, in particular the spectrophotometric assay, can be used since, for our cell cultures, 15 embryonic explants associated in culture are sufficient to allow quantification (in spectrophotometry).

0397Table 14

0398Cu ltures Cultures treated control Stimulation (15 explants) (15 explants)

03991 era 830 ng / explant 2,120 ng / explant x 2.5

04002nd experiment 950 ng / explant 2,150 ng / explant x 2.3

04013rd experiment 1820 ng / explant 2710ng / explant x 1.5

04024th experiment 1913 ng / explant 4,782 ng / explant 2.5

0403It should be noted that the treated melanocytes show no apparent sign of cytotoxicity. In addition, they have a lifespan equivalent to that of control melanocytes in vitro.

0404B - Effects of piv on the other co-cultured cell categories Ciliated and non-ciliated epidermal cells, mesenchymal cells, fibroblasts: - no particular cell proliferation

0405- no stimulating effect on their size, their spreading ...

0406- no cytotoxic effect

0407- no delay or acceleration in their differentiation, nor their behavior in vitro.

0408Neuroblasts (embryonic origin of the SNP):

0409- no particular stimulating effect on the number of neurons, nor on the size of the cell bodies (Plate II, A).

0410Effect on the morphology of the neuritic network: Overall, the neuritic network in the treated cultures consists of shorter and more dispersed neurites than the control neurites. This observation should be analyzed in more detail. All the observations and quantifications were carried out on several experimental series in order to perfectly control the reproducibility of the phenomena described.

0411Example 15

0412Effects of Peptides PI and PIV on embryonic cells in in vitro culture ... "Melanocytes - Neurons - Muscle Cells and Fibroblasts ..."

0413Material and methods

0414The experimental conditions used are as follows:

0415- dose used 1 μg / ml (defined by dose / response tests)

0416- 1 single treatment during seeding of the cells or several successive treatments (renewal of the medium every 48 hours)

0417- cultivation time: 6 to 8 days

0418- quantification

04191 ) Melanocyte counting

04202) Determination of melanin by HPLC and / or by spectrophotometry - Daily observation of cultures

0421- Ultrastructure in transmission electron microscopy. Stimulating effects of PI and PIV on melanogenesis

0422The PI and PIV treatment of melanocyte precursor embryonic cells stimulates their differentiation both on a qualitative and a quantitative level. The number of melanocytes increases at least by a factor of 2 per culture.

0423The quantity of biosynthesized melanin per cell also increases by an equivalent factor (1.4 ng / cell) after eight days of culture.

0424Preliminary confocal microscopy study trials with the SAMBA 2005 system from TITN - ALCATEL show that it will be possible to analyze the melanin particles (melanosomes) themselves.

0425The peptide formulas PI and PrV stimulate the differentiation of melanoblasts. It is important to underline the following remarkable result: they do not stimulate the precursor embryonic cells not yet determined in the melanoblastic pathway. They are therefore not inducers and only target the melanoblast: no effect on the precursor cells; stimulating effect on already induced melanoblasts). Analysis of the cytotoxicity of PI and PIV

0426- Neuroblasts

0427The two peptide formulas studied have no effect, toxic or stimulating, on neuroblasts or neurons.

0428The size of the cell bodies, the length and the number of neurites, the ultrastructure and finally the lifespan of the neurons treated are equivalent to those of the control neurons.

0429- Glial cells (astrocytes) No toxic effect is observed on astroglial cells treated with PI and PIV which differentiate normally and without delay compared to control astrocytes.

0430- Myoblasts

0431Here again no toxic effect is detected even at an ultrastructural level.

0432Myoblasts spread and differentiate without delay and without anomaly compared to myoblasts -control. - Fibroblasts, mesenchyme

0433Similar results are obtained: no toxic effect is observed.

0434All of the cytology and biochemistry work on the PI and PIN effects on melanogenesis on the one hand and on the differentiation of various other cell categories on the other, demonstrate that:

0435- PI and PIV stimulate melanogenesis both qualitatively and quantitatively.

0436- PI and PIV have no stimulating effect on the other cell types studied: neurons, astrocytes, muscle cells, fibroplasts, ...

0437- PI and PIV have no cytotoxic effect on these various cell categories which differentiate and evolve quite normally.