PEPTIDES OF USE IN THE PREVENTIVE AND CURATIVE TREATMENT OF ALOPECIA

Abstract

The object of the present invention relates to a novel family of peptide conjugates of formula (I):

A-X.sub.1—X.sub.2-Pro-Ala-X—B   (I) wherein: A represents a hydrogen atom, a C.sub.6 to C.sub.20 acyl group or a cholesterol residue; X.sub.1 represents a covalent bond, an alanine or a proline; X.sub.2 represents an arginine, a lysine, or an alanine; X represents a lysine, an alanine, or a phenylalanine; and B represents a hydroxyl or an amine; or one of its preferentially pharmaceutically, dermatologically or cosmetically acceptable salts, as well as their synthesis processes and their uses for reducing hair loss and stimulating hair growth.

Claims

1.-16. canceled

17. A method for the treatment of alopecia in a person in need thereof, said method comprising administering to said person a peptide conjugate of formula (I):
A-X.sub.1—X.sub.2-Pro-Ala-X—B   (I) wherein: A represents a hydrogen atom, a C.sub.6 to C.sub.20 acyl group or a cholesterol residue; X.sub.1 represents a covalent bond, an alanine or a proline; X.sub.2 represents an arginine, a lysine, or an alanine; X represents a lysine, an alanine, or a phenylalanine; and B represents a hydroxyl or an amine; or one of its preferentially pharmaceutically, dermatologically, or cosmetically acceptable salts, wherein the peptide conjugate is not Palm-Ala-Ala-Pro-Ala-Lys-NH.sub.2 (SEQ ID NO:6), Palm being a palmitoyl group.

18. The method according to claim 17, wherein group A represents a hydrogen atom, a C.sub.10 to C.sub.18 acyl group, or a cholesterol residue according to formula (II): ##STR00005## wherein: Y represents the carbon atom of the ester bond between the cholesterol residue and X.sub.1.

19. The method according to claim 17, wherein the salt is an acid addition salt.

20. The method according to claim 17, wherein X.sub.1 is an alanine or a covalent bond, and X.sub.2 is an arginine or an alanine.

21. The method according to claim 17, wherein the peptide conjugate of formula (I) is selected from: TABLE-US-00006 (SEQ ID NO: 1) H-Ala-Arg-Pro-Ala-Lys-OH, (SEQ ID NO: 2) Palm-Ala-Arg-Pro-Ala-Lys-OH, (SEQ ID NO: 3) Palm-Ala-Arg-Pro-Ala-Lys-NH.sub.2, (SEQ ID NO: 4) Palm-Ala-Arg-Pro-Ala-Ala-NH.sub.2, (SEQ ID NO: 7) Chol-Ala-Arg-Pro-Ala-Lys-NH.sub.2, (SEQ ID NO: 8) H-Arg-Pro-Ala-Lys-OH, and (SEQ ID NO: 9) Palm-Arg-Pro-Ala-Lys-OH, and wherein Palm represents a palm itoyl group and Chol represents a cholesterol residue.

22. The method according to claim 17, wherein the peptide conjugate of formula (I) is used in combination with another dermatologically-active ingredient.

23. The method of claim 22, wherein said dermatologically-active ingredient is minoxidil, a nicotinic acid ester, a 5α-reductase inhibitor, another peptide with dermatological activity, or combinations thereof.

24. The method according to claim 17, wherein the alopecia is selected from androgenetic, congenital, acquired, localized, diffuse, acute, and chronic alopecia.

25. The method according to claim 18, wherein said C.sub.10 to C.sub.18 acyl group is a palmitoyl group.

26. A pharmaceutical, dermatological, or cosmetic composition comprising at least one peptide conjugate of formula (I):
A-X.sub.1—X.sub.2-Pro-Ala-X—B   (I) wherein: A represents a hydrogen atom, a C.sub.6 to C.sub.20 acyl group or a cholesterol residue; X.sub.1 represents a covalent bond, an alanine or a proline; X.sub.2 represents an arginine, a lysine, or an alanine; X represents a lysine, an alanine, or a phenylalanine; and B represents a hydroxyl or an amine; or one of its preferentially pharmaceutically, dermatologically, or cosmetically acceptable salts, wherein the peptide conjugate is not Palm-Ala-Ala-Pro-Ala-Lys-NH.sub.2 (SEQ ID NO:6), Palm being a palmitoyl group.

27. The composition according to claim 26, further characterized in that it is a composition for topical use.

28. The composition according to claim 26, wherein said composition comprises the peptide conjugate of formula (I) in the form of enantiomers and/or of diastereoisomers.

29. The composition according to claim 26, characterized in that it is a composition for the scalp.

30. The composition according to claim 26, wherein said composition is a lotion, a serum, a shampoo, a spray, a gel, or a cream.

31. The composition according to claim 30, wherein said shampoo is a medicated shampoo.

32. The composition according to claim 26, wherein the concentration of the peptide conjugate of formula (I) ranges between 10-9 and 10-4 mole/litre of composition.

Description

DETAILED DESCRIPTION

[0079] The object of the present invention thus relates to a peptide conjugate of formula (I) as defined herein, characterized in that group A represents a hydrogen atom, a C.sub.10 to C.sub.20 acyl group, preferentially a palmitoyl C16 acyl group or a cholesterol residue according to formula (II):

##STR00004##

[0080] wherein: [0081] Y represents the carbon atom of the ester bond between the cholesterol residue and X.sub.1.

[0082] Furthermore, the object of the present invention relates to a peptide conjugate of formula (I) as defined herein, characterized in that group A represents a hydrogen atom, a palmitoyl group or a cholesterol residue.

[0083] When A is a cholesterol residue, it is understood that group Y of formula (II) above represents the carbon atom of the ester bond formed between the cholesterol and X.sub.1.

[0084] The object of the present invention further relates to a peptide conjugate of formula (I) as defined herein, characterized in that the salt is an acid addition salt, wherein the acid is for example hydrochloric acid, trifluoroacetic acid and/or acetic acid. Preferably, the acid is selected so as to increase the lipophilicity of the peptide conjugate of formula (I) and thus to help it penetrate the skin.

[0085] The object of the present invention further relates to a peptide conjugate of formula (I) as defined herein, characterized in that X.sub.1 is an alanine or a covalent bond, and X.sub.2 is an arginine or an alanine.

[0086] The object of the present invention advantageously relates to a peptide conjugate of formula (I) as defined herein, characterized in that it is selected from: [0087] (1) H-Ala-Arg-Pro-Ala-Lys-OH, [0088] (2) Palm-Ala-Arg-Pro-Ala-Lys-OH, [0089] (3) Palm-Ala-Arg-Pro-Ala-Lys-NH.sub.2, [0090] (4) Palm-Ala-Arg-Pro-Ala-Ala-NH.sub.2, [0091] (5) Palm-Ala-Arg-Ala-Ala-Lys-NH.sub.2, [0092] (6) Palm-Ala-Ala-Pro-Ala-Lys-NH.sub.2, [0093] (7) Chol-Ala-Arg-Pro-Ala-Lys-NH.sub.2, [0094] (8) H-Arg-Pro-Ala-Lys-OH, or [0095] (9) Palm-Arg-Pro-Ala-Lys-OH,
wherein [0096] Palm represents a palmitoyl group and [0097] Chol represents the cholesterol residue.

[0098] Preferably, the object of the present invention relates to a peptide conjugate of formula (I) for use in the treatment or the prevention of alopecia, characterized in that the alopecia is selected from androgenetic, congenital, acquired, localized, diffuse, acute or chronic alopecia.

[0099] The object of the present invention further relates to a pharmaceutical, dermatological or cosmetic composition comprising at least one peptide conjugate of formula (I) as defined herein further characterized in that it is a composition for topical use.

[0100] The object of the present invention also relates to a composition as defined above characterized in that said composition comprises a peptide conjugate of formula (I) in the form of enantiomers and/or of diastereoisomers, preferentially the amino acids of the peptide conjugate being of L or D form exclusively (i.e., higher than a 95/5% ratio) or of L/D form (i.e., 50/50%±10%).

[0101] The object of the present invention further relates to a composition as defined above characterized in that it is a composition for the scalp.

[0102] The object of the present invention thus relates to a composition as defined above characterized in that said composition is selected from a lotion, a serum, a shampoo, such as a medicated shampoo, a spray, a gel or a cream such as a medicated cream.

[0103] The object of the present invention relates to a composition as defined above characterized in that said composition comprises at least one peptide of formula (I) as described above at a concentration ranging between 10.sup.−9 and 10.sup.−4 mole/litre of composition, preferably between 10.sup.−7 and 10.sup.−5 mole/litre of composition.

[0104] The object of the present invention thus relates to the cosmetic use of a composition as defined above for reducing hair loss and/or stimulating hair growth.

[0105] The object of the present invention also relates to a composition as defined above, as medicinal product.

[0106] The object of the present invention preferentially relates to a composition as defined above for dermatological use optionally comprising in combination with the peptide conjugate of formula (I) according to the invention, another preferentially dermatological active ingredient such as those defined above.

[0107] The object of the present invention further relates to a composition as defined above for use in the treatment or the prevention of alopecia.

[0108] The object of the present invention relates to a composition as defined above for its use above, characterized in that the alopecia is selected from androgenetic, congenital, acquired, localized, diffuse, acute or chronic alopecia.

EXAMPLES

[0109] The following examples illustrate the present invention but in no way limit its scope.

[0110] I. Synthesis

[0111] Solid phase peptide synthesis, according to the following examples, was carried out according to a Fmoc/tBu strategy.

[0112] A known amount of resin is introduced into a reactor then coupling and deprotection cycles are repeated until the desired peptide is obtained.

[0113] The peptide is then cleaved from the resin then isolated by precipitation then purified.

[0114] The peptides were synthesized on Rink-amide resin. The couplings were carried out with HBTU as coupling agent and DIEA as base.

[0115] At the end of the synthesis, side chain cleavage and deprotection were carried out according to the following protocol for Rink-amide resin.

[0116] A. Cleavage from the resin:

[0117] Cleavage from the Rink-amide resin is accomplished with TFA in a proportion of about 10 mL/g resin for 1 h.

[0118] After 1 hour, the resin is filtered then rinsed with DCM. The filtrate is then concentrated under vacuum and the peptide is isolated by precipitation in ether then centrifuged.

[0119] The supernatant is then removed, then the pellet is dried under vacuum.

[0120] The final compounds were characterized by analytical HPLC and LC/MS mass spectrometry.

[0121] Yields were calculated based on the initial loading of the resin.

[0122] B. Acylation of the peptides:

[0123] P1: Palmitovlation of the peptides

[0124] Place DCM in the reactor containing the supported peptide (the peptide must be deprotected on the N-terminal side) and allow the resin to swell for 15 min.

[0125] Dissolve 3 equivalents of Palm-Cl (equivalents based on resin loading) in DCM. Next empty the reactor and add Palm-Cl dissolved in DCM then stir for 3h.

[0126] Wash the resin and dry under vacuum.

[0127] P2: Grafting of cholesterol onto the peptides

[0128] Place DCM in the reactor containing the supported peptide (the peptide must be deprotected on the N-terminal side) and allow the resin to swell for 15 min. Solubilize 3 equivalents of cholesterol (equivalents based on resin loading) in the form of cholesterol chloroformiate in DCM.

[0129] Add Cholesteryl-Cl (a synonym for cholesterol chloroformiate) dissolved in DCM then stir for 3 h.

[0130] Wash the resin and dry under vacuum.

[0131] Synthesis of peptide (3) Palm-Ala-Arg-Pro-Ala-Lys-NH.sub.2

[0132] The peptide was palmitoylated according to protocol P1 then the peptide was cleaved from the resin and was purified by preparative HPLC. Yield: 40%; Purity: 93%; LCMS: m/z 779.7 [M+H].sup.+

[0133] Synthesis of peptide (7) Chol-Ala-Arg-Pro-Ala-Lys-NH.sub.2

[0134] The cholesterol was grafted according to protocol P2 then the peptide was cleaved from the resin and was purified by preparative HPLC. Yield: 50%; Purity: 97%; LCMS: m/z 953.8 [M+H].sup.+

[0135] Synthesis of peptide (5) Palm-Ala-Arg-Ala-Ala-Lys-NH.sub.2

[0136] The peptide was palmitoylated according to protocol P1 then the peptide was cleaved from the resin and was purified by preparative HPLC. Yield: 60%; Purity: 93%; LCMS: m/z 753.6 [M+H].sup.+

[0137] Synthesis of peptide (6) Palm-Ala-Ala-Pro-Ala-Lys-NH.sub.2

[0138] The peptide was palmitoylated according to protocol P1 then the peptide was cleaved from the resin and was purified by preparative HPLC. Yield: 75%; Purity: 99%; LCMS: m/z 694.6 [M+H].sup.+

[0139] Synthesis of peptide (9) Palm-Arg-Pro-Ala-Lys-OH

[0140] The peptide was palmitoylated according to protocol P1 then the peptide was cleaved from the resin and was purified by preparative HPLC. Yield: 60%; Purity: 99%; LCMS: m/z 709.6 [M+H].sup.+

[0141] The peptides whose use is the object of the invention were subjected to pharmacological tests for purposes of showing their anti-hair-loss and hair regrowth activity.

[0142] II. Evaluation of the Biological Activity of 9 Peptides

[0143] 1) Study of the effect of the peptides on the proliferation and on the viability of cells forming part of the human hair follicle

[0144] Cell proliferation and viability were evaluated in vitro with a kit from Promega, CellTiter-Blue® Cell Viability Assay, for estimating the number of living cells present in the culture. This kit is based on detection, by measurement of fluorescence, of conversion of the dye, resazurin, into a fluorescent product (resorufin) by living and thus metabolically active cells.

[0145] The effect of the peptides tested, (1) and (9), at 4 different concentrations (10.sup.−5, 10.sup.−7, 10.sup.−9 and 10.sup.−11M) was examined on five cell types: immortalized human skin keratinocytes (HaCaT), human hair follicular keratinocytes (HHFK), normal human dermal fibroblasts (NHDF), hair follicle dermal papilla cells (HFDPC), and normal human epidermal melanocytes (NHEM) exposed to the molecule studied for 72 hours.

[0146] The results obtained show that all the peptides studied at the 4 concentrations tested have no toxicity with respect to the four cell types used in this experiment.

[0147] In addition, mild stimulation of proliferation of NHDF by (6) at 10.sup.−5M (+11%) and of HaCaT by (1) at 10.sup.−5M (+15%) was observed.

[0148] 2) Quantitative PCR (TLDA) analysis of expression of genes encoding the protein factors potentially involved in male androgenetic alopecia

[0149] To study the effect of 9 peptides on these genes, HFDPC were treated for 24 hours with the molecules tested at 10.sup.−7M and 10.sup.−9M concentrations.

[0150] RNA was extracted and purified from cell pellets using the RNA XS Kit (Macherey-Nagel). RNA quantification was carried out with a NanoDrop device and sample quality was verified on a Bioanalyzer with RNA 6000 Nano chips (Agilent). cDNA was synthesized from 300 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTech). Quality-control qPCR assays of the cDNA were carried out in microplates on the reference gene, GAPDH, and the SIRT1 gene.

[0151] Final qPCR experiments were carried out in a 1 μl volume on microfluidic cards (TaqMan Low Density Arrays, LifeTech) and the ABI 7900HT qPCR device. Amplification reactions were performed in duplicate using 1 to 2 ng of cDNA per qPCR reaction and TaqMan Universal Master Mix II (LifeTech).

[0152] Ct values were obtained using the RQ Manager software. For analyses of qPCR results, Ct values were limited to 35 cycles and the duplicate assays are averaged. The GeneXPro software was used for the studies of stability and of selection of the best reference genes. Expression variations are calculated by the relative quantification method and are expressed as percent increase in expression of the gene of interest relative to the untreated controls.

[0153] The results, presented in Table 1, show that (1), (2), (3), (5), (6) and (9) stimulate several genes whose expression is reduced in subjects characterized by alopecia. The intensity of the effect varies as a function of the structure and of the concentration of the peptide tested.

TABLE-US-00001 TABLE 1 qPCR analysis of the in vitro effect of the peptides (10.sup.−5M) on modification of expression of genes encoding growth factors involved in hair growth % stimulation Formulae KGF HGF PDGF VEGF IGF 1 BMP 2 1 H-Ala-Arg-Pro-Ala-Lys-OH 14% 32% (10.sup.−7)  26% (10.sup.−9) 2 Palm-Ala-Arg-Pro-Ala-His-OH 71%  29% 11% 136% (10.sup.−9)  3 Palm-Ala-Arg-Pro-Ala-Lys-NH.sub.2 83% 4 Palm-Ala-Arg-Pro-Ala-Ala-NH.sub.2 5 Palm-Ala-Arg-Ala-Ala-Lys-NH.sub.2 10% 29% (10.sup.−7) 16% (10.sup.−9) 6 Palm-Ala-Ala-Pro-Ala-Lys-NH.sub.2 24% 94% (10.sup.−9) 116% (10.sup.−9) 7 Chol-Ala-Arg-Pro-Ala-Lys-NH.sub.2 49% 8 H-Arg-Pro-Lys-OH 14% 9 Palm-Arg-Pro-Ala-Lys-OH 156%  304% 93% Study performed on HFDPC

[0154] 3) Study of the effect of the peptides on secretion of growth factors involved in regulation of the hair cycle

[0155] Considering the fact that hair growth is controlled by numerous growth factors whose role is well established, the capacity of the peptides to induce hair cells to secrete these factors was evaluated. Furthermore, it would be interesting to know if these peptides have an effect on secretion of protein factors encoded by genes suppressed in alopecia and whose expression was increased following treatments with the compounds tested.

[0156] NHDF, HFDPC, HHFK and NHEM cells were treated with the peptides studied for 48 h. At the end of the treatment, cell supernatants were collected and stored at −80° C. until analysis. The factors of interest secreted into the supernatant were assayed by ELISA® (RED) or BioPlex® (BioRad®).

[0157] The results obtained (Table 2) demonstrated a stimulatory effect of the peptides at concentrations of 10.sup.−7M and 10.sup.−5M on secretion of several growth factors involved in hair growth, such as PDGF, KGF, VEGF, HGF, SCF, NGF. In addition, treatment of cells forming part of the hair follicle led to an increase in secretion of protein factors encoded by genes whose expression is reduced in subjects developing alopecia (BMP2). (6) as well as (1) show the most significant activity.

[0158] The results are expressed as percent increase in the concentration of the factor present in the supernatant relative to the untreated control. The intensity of the effect varies as a function of the concentration of the peptide studied and of the cell type treated.

TABLE-US-00002 TABLE 2 In vitro effect of the peptides on secretion by hair follicle cells of factors involved in hair growth Peptide [M] HFDPC NHDF HHFK NHEM (6) 10.sup.−7 PDGF 38% NGF 12% HGF 52% VEGF 15% KGF 13% VGF 407% SCF 9% NGF 10% 10.sup.−5 PDGF 15% VEGF 40%, NT NT KGF 7% 275% VEGF 18% NGF 51% BMP2 44% SCF 8% (1) 10.sup.−7 PDGF 31% VEGF 4% KGF 8% VEGF 9% 10.sup.−9 PDGF 23% VEGF5% KGF 25% HGF 18% VEGF 4% NGF 13% BMP2 22% (5) 10.sup.−7 PDGF 55% HGF 21% HGF 104% VEGF 5% HGF 8% 10.sup.−5 PDGF 39% VEGF 12% NT NT NGF 15% (3) 10.sup.−7 KGF 53% NT VEGF 9% 10.sup.−9 PDGF 39% VEGF 48% KGF 45% HGF 5% HGF 33% NGF 89% NGF 6% (7) 10.sup.−7 NT HGF 104% SCF 10% NGF 11% 10.sup.−9 NT NT (4) 10.sup.−7 PDGF 8% HGF 188% VEGF 9% SCF 6% NGF 12% 10.sup.−9 NT HGF 95% SCF 8% NGF 18% (9) 10.sup.−7 PDGF 117% SCF 8% HGF 94% SCF 11% 10.sup.−5 PDGF 62% VEGF 24% VEGF 10% (2) 10.sup.−7 HGF 565% HGF 13% HGF 28% SCF 37% 10.sup.−5 HGF 403% SCF 24% (8) 10.sup.−7 HGF 129% 10.sup.−5 HGF 26% HGF 8% NT not tested

[0159] III. Study of the Stimulatory Activity of (1), (6) and (9) on the Growth of Isolated Hair Ex Vivo, and on Associated Morphological Changes

[0160] This study was carried out on microdissected hairs from scalp surgery specimens from women aged 52 to 71 years. Isolated hairs were kept alive for 11 to 12 days in Williams medium, under conventional cell culture conditions (37° C., 5% CO.sub.2). The peptide is added to the culture medium at two concentrations: 10.sup.−7 M and 10.sup.−9M, every 2 days. minoxidil (2,4-diamino 6-piperidinopyrimidine 3-oxide) sulphate was used as positive control at a concentration of 10.sup.−5 M.

[0161] To evaluate the effect of (1), (6) or (9) on hair growth, the hairs were photographed and their lengths measured using image analysis software. Thus, the growth of the hairs between D0 and D11/D12 could be calculated and compared with that of the untreated control. At the end of the treatments (D11 or D12), the hairs were collected and frozen or fixed for immunolabeling and histological staining.

[0162] The results obtained, summarized in Table 3, show that peptides (1) and (6), and to a lesser extent (9), stimulate hair growth after 11 to 12 days of treatment ex vivo.

[0163] The growth increase with (1) at 10.sup.−9M is close to that obtained after treatment with minoxidil sulphate (+48% at D11). The growth increase with (6) at 10.sup.−9M is higher than that obtained after treatment with minoxidil sulphate (+28% at D12).

[0164] In addition, these three peptides stimulate expression of follicular markers involved in regulation of stem cells (CK15, CK19), in follicular growth (IGF1), and in anchoring of the hair to the extracellular matrix (collagen IV, laminin-5).

TABLE-US-00003 TABLE 3 Ex vivo effect of 9 peptides on hair growth Vs Control (1) (6) (9) 10.sup.−7M 10.sup.−9M 10.sup.−7M 10.sup.−9M 10.sup.−7M 10.sup.−9M Growth at No +40% +28% +38%# No +5% D 11/D 12 effect effect Follicular         markers IGF1 CK19 CK19 CK19 Laminin-5 CD34 CK15 CK15 Coll IV Coll IV   increase; #Student's t-test with p < 0.1.

[0165] This invention relates to hair growth stimulants containing as active ingredient synthetic peptides of the general formula A-X.sub.1—X.sub.2-Pro-Ala-X—B. As a whole, the observations obtained under the experimental conditions of this study identify these peptides as activators of the gene expression suppressed in alopecia as well as stimulators of production of the growth factors essential for hair growth.

[0166] Ex vivo evaluation of biological activity demonstrates their hair growth-inducing effect. The peptides studied induce the set of changes described at concentrations varying between 10.sup.−9M and 10.sup.−5M. Thus, the preparation according to the invention can be effectively employed as preparation for external use, such as a pharmaceutical or cosmetic product for promoting hair growth and/or for increasing hair density and/or for reducing hair loss.

[0167] The following formulation examples further illustrate the present invention: Example 1: lotion comprising peptide (1)

TABLE-US-00004 In g Peptide (1) 5.10.sup.−6 95° ethanol 20 Propylene glycol 10 Water, preservatives qs 100

EXAMPLE 2

Lotion Comprising Peptide (9)

[0168]

TABLE-US-00005 In g Peptide 10.sup.−5 Water 81 Keltrol T 0.5 Techpolymer MB—4° C. 1 Sepigel 305 0.5 Silicone Oil 140 2 Butylene Glycol 5