PEPTIDES FOR USE IN THE PREVENTIVE AND CURIAL TREATMENT OF ALOPECIA

Description

[0001] The object of the present invention relates to a new family of peptide compounds as well as their synthesis processes, their use to reduce hair loss and stimulate hair growth. Introduction

[0002] Appearance is a significant social factor in our time. Hair has strong symbolic value; it plays a major role in the image a person has of themselves and presents to others. Hair loss is a real problem experienced by many as a disability. For this reason, hair is the focus of attention in the hair care sector, which represents a quarter of the global cosmetics market. Consequently, there is a constant search for effective active ingredients without side effects to eliminate or reduce hair loss (alopecia).

[0003] Today in France, more than 10 million people are affected by abnormal hair loss. It affects almost two out of three men and one out of five women (generally after menopause). The causes of abnormal hair loss can be varied: aging of the hair shaft, diseases such as cancer or lupus, hormonal changes, stress, certain medications, or nutritional deficiencies.

[0004] Human hair comprises approximately 100,000 individual hairs. Each hair is born, lives, and then dies, only to be replaced by a new growth. Every single hair is produced by the hair follicle, an autonomous skin appendage with its own hormonal control, its own cycle, and a complex, stable structure. ( Bernard BA, "The Revealed Life of the Human Hair Follicle." Médecine / Sciences 2006;22:138-43 ).The follicles serve as a "reservoir" of stem cells capable of giving rise to all the cell lines necessary to reconstitute the follicles themselves, the epidermis and the sebaceous glands.

[0005] Hair, which has a lifespan of two to seven years, does not grow continuously but according to a cyclical rhythm that can vary depending on the individual, their age, and the seasons. The hair cycle is composed of three phases: the anagen phase, or growth phase (which lasts three to five years); the catagen phase (which lasts one to two weeks), the resting period; and the telogen phase, during which the hair dies. Following this last phase, which results in hair loss, the follicle regenerates through a process of neomorphogenesis from a reservoir of stem cells and initiates a new anagen phase.

[0006] In normal hair, which is constantly renewing itself, approximately 85% of the follicles are in the growth phase, 2% in the resting phase, and more than 10% in the shedding phase. Over a lifetime, there are about twenty cycles, never all in the same phase (asynchronous), which allows us to maintain healthy hair. However, repeated damage, poor diet, illness and stress, as well as pollution, can disrupt the hair growth cycle.

[0007] Under normal physiological conditions, we lose between 50 and 100 hairs each day, having reached the telogen phase. Beyond this number, hair loss is considered excessive and should be treated. 'alopeciaHair loss is a term used to describe hair loss affecting all or part of the scalp, leaving the skin partially or completely bare. It affects approximately 20% of men starting at age 20, and this percentage increases by roughly 10% every 10 years. Thus, after age 50, slightly more than half of men experience some degree of baldness. While abnormal hair loss is generally more common in men, this condition can also affect some women and become a significant aesthetic concern.

[0008] Congenital, acquired, localized, diffuse, acute, or chronic, there are various forms of alopecia, with diverse causes and mechanisms. Depending on the origin of the baldness, hair may regrow more or less easily. There is no one-size-fits-all treatment; each case of alopecia is complex and requires appropriate treatment.

[0009] Androgenetic alopecia is the most common form of hair loss and the leading cause of baldness in both men and women. It results in a progressive and permanent decrease in both the quality and quantity of hair. Androgenetic alopecia stems from an oversensitivity of hair follicles to dihydrotestosterone (DHT), a male hormone produced from testosterone. DHT shortens the hair growth phase (anagen), and consequently, the number of possible cycles for the hair follicle is reduced. The hair eventually disappears; this is referred to as hair miniaturization.

[0010] Oral administration of antiandrogens such as finasteride (Propecia®), which blocks 5-alpha reductase, an enzyme responsible for converting testosterone to DHT in hair follicles, reduces hair loss and stimulates regrowth. However, this product, which was once highly promising, is contraindicated in women, and recent warnings have been issued regarding its use in men. Indeed, the results of a clinical study suggest a risk of developing a severe form of prostate cancer in patients taking this medication. ( Lynn R et al., “Therapeutic hotline. Treatment of androgenic alopecia with finasteride may result in a high grade prostate cancer in patients: fact or fiction?" Dermatol Ther. 2010;23:544 . Lebdal S et al., High-grade prostate cancer and finasteride. BIU Int. 2010; 105:456 ).

[0011] On the other hand, Minoxidil (Rogaine®) and Aminexil®, two topical solutions applied to the scalp, are also effective in treating alopecia. Their anti-hair loss properties are associated with the stimulation of microcirculation, which ensures the health of our hair. ( Mecklenburg L et al., “Active hair growth (anagen) is associated with angiogenesis. » J Invest Dermatol. 2000;114:909 . Lachgar S et al., “Minoxidil upregulates the expression of vascular endothelial growth factor in human hair dermal papilla cells.” Br J Dermatol. 1998;138:407 ).

[0012] However, these two products are only useful in the early stages of alopecia, and their effectiveness remains limited because hair loss resumes as soon as treatment is stopped. Furthermore, daily use is likely the cause of adverse side effects observed in patients using them long-term, such as localized skin reactions or systemic effects. In addition, the response rate is less than 50%.

[0013] The current hair care market offers numerous treatments to strengthen hair fibers and slow excessive hair loss. However, in general, they are insufficiently effective, primarily because they often only address one of the causes directly involved in alopecia. There are also purely cosmetic products containing agents designed to add volume to thinning hair. These agents, however, do not stimulate growth. They can, however, give the impression of fuller hair by coating the hair shaft, thus increasing its diameter. These products offer only a temporary solution, as they wash out after each shampoo.

[0014] Thus, targeted research aimed at identifying new compounds that can suppress or reduce alopecia is a focus for researchers. One frequently explored avenue is the search for new molecules capable of influencing the expression of growth factors that regulate hair growth.

[0015] Indeed, hair follicle homeostasis and hair growth remain under the control of numerous growth factors. This is why a procedure performed in medical offices, which consists of injecting autologous platelet extracts into the scalp, allows for an improvement in the condition of the hair. ( Li et al., “Autologous platelet-rich plasma: a potential therapeutic tool for promoting hair growth.” » Dermatol Surg. 2012;38:1040 ).Platelet extracts, due to their richness in growth factors (VEGF, PDGF, bFGF and HGF), regenerate, slow down hair loss and promote hair regrowth.

[0016] Among the factors involved in regulating the hair cycle, platelet-derived growth factor (PDGF) is paramount. Platelet-Derived Growth Factor PDGF is a cytokine that plays a key role in hair growth and in the activation of the follicular stem cell. ( Karlsson et al., “Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle. » Development. 1999;126:2611 . Tomita et al., “PDGF isoforms induce and maintain anagen phase of murine hair follicles.” J Dermatol Sci. 2006;43:105 ).

[0017] Indeed, PDGF-induced follicular cell proliferation is 100 times faster than that observed for other cellular targets of this factor. It has been shown that PDGF production by adipocyte precursors present at the base of hair follicles is essential under physiological conditions to initiate the anagen phase. ( Festa et al., “Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling. » Cell. 2011;146:761 ). Indeed, when hair dies, the layer of fat in the scalp shrinks. The development of adipose tissue producing PDGF has proven necessary for hair regeneration.

[0018] A recent study highlighted the role of the epidermis in the secretion of factors responsible for adipogenesis: IGF1, BMP2, and BMP6. These factors, by stimulating adipocyte growth, induce the production of PDGF, which in turn acts on the follicle to activate hair growth. ( Donati et al., “Epidermal Wnt / β-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors. » Proc Natl Acad Sci US A. 2014;111:E1501 ). There is therefore an overall physiological synchronization between the adipose tissue and the follicle, with an initiating signal that comes from the epidermis.

[0019] This discovery of the source of signals that trigger hair growth could lead to the development of new active ingredients for treating baldness. Furthermore, compounds that stimulate PDGF production are potential candidates for further research in the field of alopecia.

[0020] The role of vascular endothelial growth factor (VEGF, Vascular Endothelial Growth Factor The role of VEGF in scalp homeostasis is also widely described. Indeed, it is well known that good blood flow to the scalp is essential for preserving hair and that the length of the anagen phase of hair depends on the presence of VEGF.

[0021] It has been shown that VEGF, a potent pro-angiogenic factor, stimulates the formation of the vascular network at the level of the hair follicle and nourishes the root throughout the anagen phase. This is crucial for the proper functioning of the hair cycle. ( Yano et al., “Control of hair growth and follicle size by VEGF-mediated angiogenesis. » J Clin Invest. 2001;107:409 ). The demonstration of local VEGF production by dermal papilla cells corroborates the contribution of this cytokine to hair growth ( Lachgar et al., “Vascular endothelial growth factor is an autocrine growth factor for hair dermal papilla cells., Vascular endothelial growth factor is an autocrine growth factor for hair dermal papilla cells. » J Invest Dermatol. 1996;106:17-23 ).A sharp decrease in VEGF levels in people experiencing hair loss confirms the importance of this cytokine in preserving our hair health. ( Goldman et al., “Loss of vascular endothelial growth factor in human alopecia hair follicles. » J. Invest Dermatol. 1995;104:18 ).

[0022] Therefore, stimulating VEGF production at the follicular level is one of the pathways promising to stimulate hair growth and ensure its good health ( Gnann et al., “Hematological and hepatic effects of vascular epidermal growth factor (VEGF) used to stimulate hair growth in an animal model. » BMC Dermatology 2013;13:15 ).

[0023] Hepatocyte Growth Factor (HGF, Hepatocyte Growth Factor HGF is a multifunctional cytokine also involved in the control of follicular activity. The stimulatory effect of HGF on hair growth is widely demonstrated.( Jindo et al., « Hepatocyte growth factor scatter factor stimulates hair growth of mouse vibrissae in organ culture. » J Invest Dermatol. 1994;103(3):306 . Shimaoka et al., "Hepatocyte growth factor / scatter factor expressed in follicular papilla cells stimulates human hair growth in vitro." J Cell Physiol. 1995;165:333 . Lee et al., "Hepatocyte growth factor (HGF) activator expressed in hair follicles is involved in in vitro HGF-dependent hair follicle elongation." J Dermatol Sci. 2001;25:156 ). De plus, le HGF induit l'expression du VEGF par les kératinocytes et de cette manière contribue au processus de l'angiogenèse ( Gille et al., « Hepatocyte Growth Factor / Scatter Factor (HGF / SF) Induces Vascular Permeability Factor (VPF / VEGF) Expression by Cultured Keratinocytes. » J Invest Dermatol. 1998;11:1160 . Gerristen et al., HGF and VEGF : a dynamic duo. Circulation Res. 2005;96:272 ).

[0024] On the other hand, in the same way as for VEGF, HGF is produced locally by the cells of the dermal papilla. ( Shimaoka et al., “Dermal papilla cells express hepatocyte growth factor. » J Dermatol Sci. 1994;7 Suppl:S79 ).

[0025] Keratinocyte growth factor (KGF / FGF7, Keratinocyte Growth Factor KGF also belongs to the family of cytokines that oversee normal hair growth. KGF is directly involved in the production of keratinocytes by the matrix and strengthens keratin cohesion, thus stimulating hair growth, the majority of which is made up of keratinocytes. ( Danilenko et al., “Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development and differentiation: normalization of the nu / nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia.” Am J Pathol. 1995;147(1):145 .Guo et al., “Keratinocyte growth factor is required for hair development but not for wound healing.” Genes Dev. 1996;10:165 ).

[0026] A multitude of other growth factors are also involved in controlling the hair cycle. Among them are stem cell growth factor (SCF or kit-ligand), Stem Cell Factor ) ( Randall et al., “Stem cell factor / c-Kit signaling in normal and androgenetic alopecia hair follicles.” » J Endocrinol. 2008;197:11 ), nerve growth factor (NGF, Nerve Growth Factor) ( Zhou et al., “Dynamic changes in nerve growth factor and substance P in the murine hair cycle induced by depilation.” Dermatol. 2006;33:833 ), as well as insulin-like growth factor 1 (IGF1, Insulin Growth Factor 1) ( (Ahn et al., Effect of IGF-1 on hair growth is related to the anti-apoptotic effect of IGF-1 and up-regulation of PDGF-A and PDGF-B. Ann Dermatol. 2012;24:26 . Su et al., Insulin-like growth factor 1 and hair growth. Dermatol Online J. 1999;5:1 . Li et al., “Exogenous IGF-1 promotes hair growth by stimulating cell proliferation and down regulating TGF-B1 in C57BL / 6 mice in vivo.” Growth Horm IGF Res. 2014;24:89 ).

[0027] It should be noted that an increase in the synthesis of at least one of the factors described above following treatment with an active ingredient should result in improved hair growth.

[0028] Alopecia is described as a "polygenic" phenomenon because the modulation of several genes is involved in hair loss. Furthermore, genetic predispositions are recognized as one of the factors influencing the onset of baldness (van der Steen P, Traupe H, Happle R, Boezeman J, Strater R, Hamm H. "The genetic risk for alopecia areata in first-degree relatives of severely affected patients. An estimate." Acta Derm Venereol. 1992;72:373). Indeed, one-third of men experience baldness before the age of 45, a hereditary phenomenon dependent on a combination of genetic factors.

[0029] Several documents disclose peptides containing a maximum of 5 amino acids and beginning with a hydrogen atom, including the peptides H-ARPAK-OH (see EP 2 894 160 A1, US 2005 / 080015 and GB 2 387 386), H-RPAK-OH (EP 2 894 160 A1), and H-ARPA(D-)K-NH2 (WO 00 / 52147). Reimer et al. (Journal of Biological Chemistry, 283, 48, 2008, pp. 33375-33383) also described the peptide Ac-AAPAAKK-NH2. However, this peptide contains more than 5 amino acids and begins with an acetyl group (-CO-CH3). None of these documents, however, disclose the use of such compounds for the treatment of alopecia.

[0030] This invention describes a stimulatory effect of a new family of peptides on the growth of hair maintained in survival ex vivo.These data combined with the inducing effects of these peptides on the proliferation of cells that are part of the human hair follicle as well as on the production of growth factors involved in the hair cycle lead us to propose a preparation, containing at least one of these peptides which would make it possible to suppress or reduce alopecia, and in particular to induce or stimulate hair growth and / or increase its density and / or decrease its loss. Summary of the invention

[0031] The object of the present invention thus relates to a peptide conjugate of formula (I): AX1-X2-Pro-Ala-XB (I) in which: A represents an acyl group at C6 to C20 or a cholesterol residue; X1 represents a covalent bond, an alanine or a proline; X2 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 salts, preferably pharmaceutically, dermatologically or cosmetically acceptable.

[0032] The object of the present invention further relates to a method for manufacturing the peptide conjugate of formula (I) above, characterized in that said method comprises the following successive steps: a. synthesis, at least partially on a solid support, of the peptide strand of formula HX 1-X 2-Pro-Ala-XB, in which X 1 , X 2 , X are as defined above and (whose reactive groups) are suitably protected; B is the solid support or a suitably protected OH or NH 2 group; b. grafting of group A to the peptide strand of step (a) by acylation reaction (when it is desired that A not be a hydrogen atom), preferably by the use of an acyl activated in the presence of a base; c. deprotection of the protected amino acids possibly in conjunction with cleavage of the peptide from the resin, when B is a solid support; d. possible purification of the peptide conjugate of formula (I) obtained, for example by HPLC; and e. recovery of the product of formula (I).

[0033] Furthermore, the object of the present invention relates to a cosmetic use of a peptide conjugate of formula (I) as defined herein to reduce hair loss and / or stimulate hair growth.

[0034] Another object of the present invention relates to a pharmaceutical, cosmetic or dermatological treatment method to combat alopecia, and / or to stimulate hair growth, comprising administering to a patient a peptide conjugate of formula (I) as defined herein.

[0035] More specifically, the object of the present invention relates to a treatment method as described above, characterized in that administration is by applying to the scalp a composition comprising at least one peptide as described above. Advantageously, the object of the present invention relates to a treatment method as described above, characterized in that at least one peptide as described above is combined with another dermatological active ingredient, for example, one that enhances hair regrowth activity and has been described for this activity.

[0036] Among these compounds, we can mention: Minoxidil, nicotinic acid esters, 5α-reductase inhibitors, or another peptide with dermatological activity.

[0037] In particular, it can be cited as an example of peptides with dermatological activities that can be associated with peptides according to the present invention, such as those found in PCT application WO97 / 18239 or WO2005 / 009456 for these particular embodiments.

[0038] The object of the present invention also relates to a peptide conjugate of formula (I) as defined herein, as a drug.

[0039] The object of the present invention thus relates to a peptide conjugate of formula (I) as defined herein, for its dermatological use possibly in association with another preferably dermatological active principle, such as those improving activity on regrowth and having been described for this activity, more particularly those mentioned above (Minoxidil, nicotinic acid esters, 5α-reductase inhibitors and other peptides with dermatological activity such as those found in PCT application WO97 / 18239 or WO2005 / 009456).

[0040] Also advantageously, the present invention relates to a peptide conjugate of formula (I) as defined herein, for its dermatological use, as the sole active ingredient.

[0041] The present invention further relates to a peptide conjugate of formula (I) as defined herein, for its use in the treatment or prevention of alopecia.

[0042] In addition, the object of the present invention therefore relates to a pharmaceutical, dermatological or cosmetic composition comprising at least one peptide conjugate of formula (I) as defined herein. Definitions " Peptide conjugate » / « Peptide strand

[0043] The term "peptide" (equivalent to "oligopeptide") should be understood as a polymer of amino acids, these amino acids being linked together by a peptide bond. A peptide bond is an amide bond linking two amino acids. A peptide generally contains between 2 and 80 to 100 amino acids; the upper limit is not clearly defined, but generally falls within the domain of proteins. Thus, a "dipeptide" is a polymeric fragment of two amino acids linked together by a peptide bond. A tripeptide is a polymeric fragment of three amino acids linked together by a peptide bond, and so on.

[0044] In the context of this application, peptides or peptide conjugates are tetrapeptides (i.e., composed of 4 amino acids) or pentapeptides (composed of 5 amino acids).

[0045] When reference is made to the expression " "peptide conjugate", "peptide strand" or even " peptide fragment"These expressions simply refer to the sequence of amino acids considered."

[0046] In the traditional manner, peptides (or proteins) can be extracted from a biological medium or synthesized. Preferably, the peptides according to the present invention are synthesized. Peptide synthesis techniques are described in Paul Lloyd-Williams, Fernando Albericio, and Ernest Giralt, "Chemical Approaches to the Synthesis of Peptides and Proteins," CRC Press, 1997, or Houben-Weyl, "Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics," Vol. E 22a, Vol. E 22b, Vol. E 22c, Vol. E 22d, M. Goodmann Ed., Georg Thieme Verlag, 2002. Peptide synthesis can be carried out in a liquid medium or on a solid support. Both techniques are applicable to the present invention. However, for practical reasons, synthesis on a solid support is preferred according to the present invention.

[0047] The designations typically used in the field of peptide synthesis are applicable in this case. For example, the terms "Ala" or "A" correspond to alanine, "Pro" or "P" to proline, "Arg" or "R" to arginine, "Lys" or "K" to lysine, "Phe" or "F" to phenylalanine, etc.

[0048] The amino acids involved in the peptide conjugate of formula (I) according to the present invention may be of L or D configuration, depending on the stereochemistry of their asymmetric carbon. The "L" form exclusively is preferred, as natural amino acids are of this form. Furthermore, mixtures of L and D forms of the same amino acid may be used for the synthesis of a peptide conjugate of formula (I) according to the present invention. The 50 / 50% (±10%) racemic mixture by mass is designated "L / D". In the case of amino acid mixtures, the L / D mixture is preferred because it is readily obtained. However, the object of the present invention also relates to a peptide of formula (I) above obtained by using any type of mixture of the relevant amino acid in either "L" or "D" form.

[0049] Furthermore, according to the present invention, the term "side chain of an amino acid" represents the fragment attached to the α carbon of an amino acid. For example, the side chains of natural amino acids such as glycine, valine, alanine, and aspartic acid correspond to the hydrogen atom, the isopropyl group, the methyl group, and the CH2COOH group, respectively.

[0050] The side chains of amino acids can be protected by protecting groups (P) and more particularly N-protectors or O-protectors when these chains contain the corresponding heteroatoms.

[0051] Protecting groups (P) are groups known to those skilled in the art. These protecting groups and their use are described in works such as, for example, Greene, "Protective Groups in Organic Synthesis", Wiley, New York, 2007 4th edition; Harrison et al. "Compendium of Synthetic Organic Methods", Vol. 1 to 8 (J. Wiley & sons, 1971 to 1996); Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt, "Chemical Approaches to the Synthesis of Peptides and Proteins", CRC Press, 1997 or Houben-Weyl, "Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics", Vol E 22a, Vol E 22b, Vol E 22c, Vol E 22d., M. Goodmann Ed., Georg Thieme Verlag, 2002. Depending on whether these protecting groups are attached to a nitrogen atom, they will be designated as N-protecting groups. The same applies to O-protecting groups, etc.For example, a hydroxyl group can be protected by a trityl group, or a carboxylic acid can be protected as a tert-butyl ester. If the synthesis is performed on a solid support, the resin acts as the protecting group for the C-terminal carboxyl function.

[0052] The protection of the amino group of the amino acid can be achieved, for example, by a tert-butyloxycarbonyl group (hereinafter referred to as Boc-) or a -9-fluorenylmethyloxycarbonyl group (hereinafter referred to as Fmoc) represented by the formula:

[0053] In particular, the so-called "Fmoc / tBu" strategy is preferred due to its ease of implementation. "tBu" refers to the side chains being protected by acid-labile groups such as tBu, which is most commonly used in this strategy. Other protecting groups for functional groups on the side chains include the "Boc" group (for lysine, for example) or "Pbf" (for arginine, for example). Protection is thus achieved using methods known in the prior art. For example, protection by the Boc group can be obtained by reacting the amino acid with di-tert-butylpyrocarbonate (Boc2O).

[0054] As indicated above, It may have a hydrogen atom, an acyl group at C6 to C20, or a cholesterol residue.

[0055] When A is an acyl group, it is a linear C6 to C20 acyl group, particularly a linear C10 to C16 acyl group, and even more particularly a linear C16 acyl group, that is to say a palmitoyl group.

[0056] In the context of this invention, the term acyl refers to a radical or functional group obtained by removing the hydroxyl group from a carboxylic acid. The acyl group corresponding to a carboxylic acid with the formula RCOOH will have the formula RCO-, where the carbon and oxygen atoms are linked by a double bond (carbonyl group). A C6 to C20 acyl group therefore corresponds to a saturated hydrocarbon chain, linear or branched, preferably linear, comprising 6 to 20 carbon atoms, including a "-C=O-" group, linking this hydrocarbon chain to the molecule that bears it.

[0057] A could also be a cholesterol residue.

[0058] Cholesterol is a lipid in the sterol family that is involved in many biochemical processes.

[0059] The formula for cholesterol is:

[0060] However, several chiral centers are present in this molecule, and preferably, the cholesterol used for the present invention is:

[0061] Thus, according to the present invention, by "cholesterol residue" it is understood that the OH group of the cholesterol molecule is engaged in an ester bond with the X1 group.

[0062] The term "hydroxyl" according to the present invention refers to the fragment -OH, and possibly its salts, for example sodium or potassium. Detailed description

[0063] The object of the present invention thus relates to a peptide conjugate of formula (I) as defined herein, characterized in that group A represents an acyl group at C10 to C20, preferably a C16 palmitoyl acyl group or a cholesterol residue according to formula (II): in which: Y represents the carbon atom of the ester bond between the cholesterol residue and X1.

[0064] 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 palmitoyl group or a cholesterol residue.

[0065] When A is a cholesterol residue, it is understood that the Y group in formula (II) above represents the carbon atom of the ester bond formed between cholesterol and X1.

[0066] 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 acidic addition salt, wherein the acid is, for example, hydrochloric acid, trifluoroacetic acid, and / or acetic acid. Preferably, the acid is chosen to increase the lipophilicity of the peptide conjugate of formula (I) and thus aid its penetration into the skin.

[0067] The object of the present invention further relates to a peptide conjugate of formula (I) as defined herein, characterized in that X 1 is an alanine or a covalent bond, and X 2 is an arginine or an alanine.

[0068] 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: (2) Palm-Ala-Arg-Pro-Ala-Lys-OH, (3) Palm-Ala-Arg-Pro-Ala-Lys-NH 2, (4) Palm-Ala-Arg-Pro-Ala-Ala-NH 2, (5) Palm-Ala-Arg-Ala-Ala-Lys-NH 2, (6) Palm-Ala-Ala-Pro-Ala-Lys-NH 2, (7) Chol-Ala-Arg-Pro-Ala-Lys-NH 2, (9) Palm-Arg-Pro-Ala-Lys-OH, in which Palm represents a palmitoyl group and Chol represents the cholesterol residue.

[0069] Preferably, the object of the present invention relates to a peptide conjugate of formula (I) for its use in the treatment or prevention of alopecia, characterized in that the alopecia is chosen from androgenetic, congenital, acquired, localized, diffuse, acute or chronic alopecia.

[0070] 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 as a composition for topical use.

[0071] 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 diastereomers, preferably the amino acids of the peptide conjugate being of form L or D exclusively (i.e. greater than a ratio of 95 / 5%) or L / D (i.e. 50 / 50% ± 10%).

[0072] The object of the present invention further relates to a composition as defined above characterized in that it is a composition for the scalp.

[0073] The object of the present invention thus relates to a composition as defined above characterized in that said composition is chosen from a lotion, a serum, a shampoo, such as a treatment shampoo, a spray, a gel or a cream such as a treatment cream.

[0074] 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 between 10⁻⁹ and 10⁻⁴ mole / litre of composition, preferably between 10⁻⁷ and 10⁻⁵ mole / litre of composition.

[0075] The object of the present invention thus relates to the cosmetic use of a composition as defined above to reduce hair loss and / or stimulate hair growth.

[0076] The object of the present invention also relates to a composition as defined above, as a medicinal product.

[0077] The object of the present invention relates preferably to a composition as defined above for its dermatological use comprising optionally in association with the peptide conjugate of formula (I) according to the invention, another preferably dermatological active principle such as those defined above.

[0078] The object of the present invention further relates to a composition as defined above for its use in the treatment or prevention of alopecia.

[0079] The object of the present invention relates to a composition as defined above for its use as above, characterized in that the alopecia is chosen from androgenetic, congenital, acquired, localized, diffuse, acute or chronic alopecia. Examples

[0080] The following examples illustrate the present invention but do not in any way limit its scope. I. Summary

[0081] The synthesis of solid-phase peptides, according to the following examples, was carried out using an Fmoc / tBu strategy.

[0082] A known quantity of resin is introduced into a reactor, then cycles of coupling and deprotection follow one another until the desired peptide is obtained.

[0083] The peptide is then cleaved from the resin, isolated by precipitation, and then purified.

[0084] The peptides were synthesized on Rink-amide resin. Couplings were performed with HBTU as the coupling agent and DIEA as the base.

[0085] At the end of the synthesis, the cleavage and deprotection of the side chains were carried out according to the following protocol for the Rink-amide resin. A. Resin cleavage:

[0086] Rink-amide resin is cleaved with TFA at a rate of approximately 10mL / g of resin for 1 hour.

[0087] After one hour, the resin is filtered and then rinsed with DCM. The filtrate is then concentrated under vacuum and the peptide is isolated by precipitation in ether and then centrifuged.

[0088] The supernatant is then removed, and the pellet is then dried under vacuum.

[0089] The final compounds were characterized by analytical HPLC and by LC / MS mass spectrometry.

[0090] Yields were calculated relative to the initial resin charge. B. Peptide acylation : P1: Palmitoylation of peptides

[0091] Add DCM to the reactor containing the supported peptide (the peptide must be unprotected at the N-terminal end) and let the resin swell for 15 min.

[0092] Dissolve 3 equivalents of Palm-Cl (equivalents relative to the resin charge) in DCM. Then empty the reactor and add the dissolved Palm-Cl to the DCM, then stir for 3 hours.

[0093] Wash the resin and dry under vacuum. P2: Grafting cholesterol onto peptides

[0094] Add DCM to the reactor containing the supported peptide (the peptide must be deprotected at the N-terminus) and allow the resin to swell for 15 min. Solubilize 3 equivalents of cholesterol (equivalents relative to the resin charge) as cholesterol chloroformate in DCM.

[0095] Add the Cholesteryl-Cl (synonym for cholesterol chloroformate) dissolved in the DCM and then shake for 3 hours.

[0096] Wash the resin and dry under vacuum. • Synthesis of the peptide (3) Palm-Ala-Arg-Pro-Ala-Lys-NH2

[0097] The peptide was palmitoylated according to protocol P1, then cleaved from the resin and purified by preparative HPLC. Yield: 40%; Purity: 93%; LCMS: m / z 779,7 [M+H] +< • Synthesis of peptide (7) Chol-Ala-Arg-Pro-Ala-Lys-NH2

[0098] Cholesterol was grafted according to the P2 protocol, then the peptide was cleaved from the resin and purified by preparative HPLC. Yield: 50%; Purity: 97%; LCMS: m / z 953,8 [M+H] +< • Synthesis of the peptide (5) Palm-Ala-Arg-Ala-Ala-Lys-NH2

[0099] The peptide was palmitoylated according to protocol P1, then cleaved from the resin and purified by preparative HPLC. Yield: 60%; Purity: 93%; LCMS: m / z 753,6 [M+H] +< • Synthesis of peptide (6) Palm-Ala-Ala-Pro-Ala-Lys-NH2

[0100] The peptide was palmitoylated according to protocol P1, then cleaved from the resin and purified by preparative HPLC. Yield: 75%; Purity: 99%; LCMS: m / z 694,6 [M+H] +< • Synthesis of the peptide (9) Palm-Arg-Pro-Ala-Lys-OH

[0101] The peptide was palmitoylated according to protocol P1, then cleaved from the resin and purified by preparative HPLC. Yield: 60%; Purity: 99%; LCMS: m / z 709,6 [M+H] +<

[0102] The peptides whose use is the subject of the invention have been subjected to pharmacological tests which have shown their anti-hair loss and hair regrowth activity. II. Evaluation of the biological activity of 9 peptides 1. Study of the effect of peptides on the proliferation and viability of cells forming part of the human hair follicle

[0103] Cell proliferation and viability were evaluated. in vitro with a Promega kit, CellTiter-Blue ®< Cell Viability Assay which allows estimation of the number of living cells present in the culture. This kit is based on the detection, by fluorescence measurement, of the conversion of the dye resazurin into a fluorescent product (resorufin) by living and thus metabolically active cells.

[0104] The effect of the tested peptides (1) And (9) at 4 different concentrations (10⁻⁵, 10⁷, 10⁻⁹ and 10⁻¹¹ M) was examined on five cell types: immortalized human cutaneous keratinocytes, HaCaT; human follicular keratinocytes, HHFK ( Human Hair Follicular Keratinocytes) ; human dermal fibrobroblasts, NHDF ( Normal Human Dermal Fibroblasts ) ; human hair follicle dermal papilla cells, HFDPC ( Hair Follicle Dermal Papilla Cell ) and human epidermal melanocytes, NHEM (Normal Human Epidermal Melanocytes) exposed to the molecule under study for 72 hours.

[0105] The results obtained show that all the peptides studied at the 4 concentrations tested do not exhibit any toxicity towards the four types of cells used in this experiment.

[0106] On the other hand, a slight stimulation of NHDF proliferation by (6) at 10 -5< M (+11%) and of HaCaT by (1) at 10 -5< M (+15%) was observed. 2° Quantitative PCR analysis (TLDA) of gene expression encoding protein factors potentially involved in male pattern baldness

[0107] To study the effect of 9 peptides on these genes, HFDPC follicular cells were treated for 24 hours with tested molecules at concentrations of 10⁻⁷< M and 10⁻⁹< M.

[0108] RNA was extracted and purified from cell pellets using the RNAXS Kit (Macherey-Nagel). RNA quantification was performed using Nanodrop, and sample quality was verified on a Bioanalyzer with RNA6000nano chips (Agilent). cDNA was synthesized from 300 ng of RNA using the High Capacity cDNA Reverse Transcription Kit (LifeTech). Quality control qPCR of the cDNA was performed in microplates on the reference gene GAPDH and the SIRT1 gene.

[0109] The final QPCR experiments were performed in a 1µl volume on microfluidic boards (TaqMan Low Density Arrays, LifeTech) and the ABI 7900HT QPCR instrument. Amplification reactions were carried out using a duplicate technique, with 1 to 2 ng of cDNA per QPCR reaction and the TaqMan Universal Master Mix II (LifeTech).

[0110] Ct values ​​were obtained using RQ Manager software. For qPCR results analyses, Ct values ​​were limited to 35 cycles, and technical duplicates were averaged. GenexPro software was used for stability studies and selection of the best reference genes. Expression changes were calculated using the relative quantification method and expressed as a percentage increase in the expression of the gene of interest compared to untreated controls.

[0111] The results, presented in Table 1, show that (1), (2), (3), (5), (6) And (9) They stimulate several genes whose expression is reduced in subjects with alopecia. The intensity of the effect varies depending on the structure of the peptide tested and its concentration. Table 1: qPCR analysis of the in vitro effect of peptides (10⁻⁵ < M) on the modification of gene expression encoding growth factors involved in hair growth % stimulation Formulas KGF HGF CEO F VEGF IGF1 BMP2 1 H-Ala-Arg-Pro-Ala-Lys-OH (example not included in this invention) 14% 32% (10 -7< ) 26% (10 -9< ) 2 Palm-Ala-Arg-Pro-Ala-His-OH 71% 29% 11% 136% (10 -9< ) 3 Palm-Ala-Arg-Pro-Ala-Lys-NH 2 83% 4 Palm-Ala-Arg-Pro-Ala-Ala-NH 2 5 Palm-Ala-Arg-Ala-Ala-Lys-NH 2 10% 29% (10 -7< ) 16% (10 -9< ) 6 Palm-Ala-Ala-Pro-Ala-Lys-NH 2 24% 94% (10 -9< ) 116% (10 -9< ) 7 Chol-Ala-Arg-Pro-Ala-Lys-NH 2 49% 8 H-Arg-Pro-Lys-OH (example outside the invention) 14% 9 Palm-Arg-Pro-Ala-Lys-OH 156% 304% 93% Study conducted on HFDPc cells 3° Study of the effect of peptides on the secretion of growth factors involved in the regulation of the hair cycle

[0112] Given that hair growth remains under the control of numerous growth factors whose roles are well established, the ability of peptides to induce the secretion of these factors by hair cells was evaluated. Furthermore, it was of interest to determine whether these peptides have an effect on the secretion of protein factors encoded by genes repressed in alopecia, the expression of which was increased following treatment with the tested compounds.

[0113] NHDF, HFDPC, HHFK, and NHEM cells were treated with the studied peptides for 48 hours. At the end of the treatment, the cell supernatants were collected and stored at -80°C until analysis. The factors of interest secreted into the supernatant were quantified by ELISA® (R&D) or BioPlex® (BioRad®).

[0114] The results obtained (Table 2) demonstrated a stimulatory effect of peptides at concentrations of (10⁻⁷ < M and 10⁻⁵ < M) on the secretion of several growth factors involved in hair growth, such as PDGF, KGF, VEGF, HGF, SCF, and NGF. Furthermore, treatment of cells within the hair follicle led to an increase in the secretion of protein factors encoded by genes whose expression is reduced in subjects developing alopecia (BMP2). (6) as well as (1) exhibit the most significant activity.

[0115] The results are expressed as a percentage increase in the concentration of the factor present in the supernatant compared to the untreated control. The magnitude of the effect varies depending on the concentration of the peptide studied and the type of cells treated. in vitro Table 2. Effect of peptides on the secretion of factors involved in hair growth by hair follicle cells Peptide [M] HFPDC NHDF HHKF NHEM 10 -7< PDGF 38% NGF 12% HGF 52% VEGF 15% KGF 13% VGF 407% SCF 9% NGF 10% (6) 10 -5< PDGF 15% VEGF 40%, 275% KGF 7% NT NT VEGF 18% NGF 51% BMP2 44% SCF 8% (1) (example not an invention) 10 -7< PDGF 31% VEGF 4% NT not tested KGF 8% VEGF 9% 10 -9< PDGF 23% VEGF5% KFG 25% HGF 18% VEGF 4% NGF 13% BMP2 22% Peptide [M] HFPDC NHDF HHKF NHEM (5) 10 -7< PDGF 55% HGF 21% HGF 104% VEGF 5% HGF 8% 10 -5< PDGF 39% VEGF 12% NT NT NGF 15% (3) 10 -7< KGF 53% NT VEGF 9% 10 -9< PDGF 39% VEGF 48% KFG 45% HGF 5% HGF 33% NGF 89% NGF 6% (7) 10 -7< NT HGF 104% SCF 10% NGF 11% 10 -9< NT NT (4) 10 -7< PDGF 8% HGF 188% VEGF 9% SCF 6% NGF 12% 10 -9< NT HGF 95% SCF 8% NGF 18% Peptide [M] HFPDC NHDF HHKF (9) 10 -7< PDGF 117% SCF 8% HGF 94% SCF 11% 10 -5< PDGF 62% VEGF 24% VEGF 10% (2) 10 -7< HGF 565% HGF 13% HGF 28% SCF 37% 10 -5< HGF 403% SCF 24% Peptide [M] HFPDC NHDF (8) (example not based on invention) 10 -7< HGF 129% 10 -5< HGF 26% HGF 8% III. Study of the stimulatory activity of (1), (6) and (9) on the growth of isolated hairs ex vivo, and on the associated morphological changes

[0116] This study was performed on microdissected hairs from scalp grafts taken from women aged 52 to 71 years. Isolated hairs were incubated for 11 to 12 days in Williams medium under standard cell culture conditions (37°C, 5% CO2). The peptide was incorporated into the culture medium at two concentrations: 10⁻⁷ M and 10⁻⁹ M, every 2 days. Minoxidil® (2,4-diamino 6-piperidinopyrimidine 3-oxide) sulfate was used as a positive control at a concentration of 10⁻⁵ M.

[0117] To assess the effect of (1), (6) Or (9) Regarding hair growth, the hairs were photographed and their lengths measured using image analysis software. Hair growth between day 0 and day 11 / 12 was then calculated and compared to that of the untreated control. At the end of the treatments (day 11 or day 12), the hairs were collected, frozen, or fixed for immunostaining and histological staining.

[0118] The results obtained, summarized in Table 3, show that the peptides (1) And (6), and more weakly (9), stimulate hair growth after 11 to 12 days of treatment ex vivo.

[0119] Increased growth with (1) at 10⁻⁹ < M is close to that obtained after treatment with minoxidil sulfate (+48% at day 11). The increase in growth with (6) at 10 -9< M is higher than that obtained after treatment with minoxidil sulfate (+28% at J12).

[0120] On the other hand, these three peptides stimulate the expression of follicular markers involved in the regulation of stem cells (CK15, CK19), in follicular growth (IGF1), and in the anchoring of the hair to the extracellular matrix (collagen IV, laminin-5). ex vivo Table 3. Effect of 9 peptides on hair growth vs Witness (1) (example not an invention) (6) (9) 10 -7< M 10 -9< M 10 -7< M 10 -9< M 10 -7< M 10 -9< M Growth at day 11 / day 12 No effect +40% +28% +38% #< No effect +5% Follicular markers ↗ IGF1 Laminin-5 ↗ CK19 ↗ CK19 ↗ CK19 CD34 CK15 CK15 Coll. IV Coll. IV ↗ increase; #< Student t-test significant with p<0.1.

[0121] This invention relates to hair growth stimulants containing as an active ingredient synthetic peptides of the general formula AX1-X2-Pro-Ala-XB. All observations obtained under the operating conditions of this study identify these peptides as activators of repressed gene expression in the case of alopecia, as well as stimulators of the production of growth factors essential for hair growth.

[0122] The evaluation ex vivoTheir biological activity highlights their hair growth-inducing effect. The peptides studied induce all the described changes at concentrations ranging from 10⁻⁹ M to 10⁻⁵ M. Thus, the preparation according to the invention can be effectively used as a topical preparation, such as a pharmaceutical or cosmetic product, to promote hair growth and / or increase hair density and / or reduce hair loss.

[0123] The following formulation examples further illustrate the present invention: Example 1 : lotion comprising peptide (1) (peptide not included in the invention) In g - Peptide (1) 5.10 -6< - 95% Ethanol 20 - Propylene glycol 10 - Water preservatives qsp 100 Example 2 : lotion containing peptide (9) In g - Peptide 10 -5< - Water 81 - Keltrol T 0,5 - Techpolymer MB - 4°C 1 - Sepigel 305 0,5 - Silicone Oil 140 2 - Butylene Glycol 5

Claims

1. A peptide conjugate of formula (I):         A-X1-X2-Pro-Ala-X-B     (I) wherein: - A represents a C6 to C20 acyl group or a cholesterol residue; - X1 represents a covalent bond, an alanine or a proline; - X2 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 salts, preferentially pharmaceutically, dermatologically or cosmetically acceptable salts.

2. Peptide conjugate of formula (I) according to claim 1, wherein group A represents a C10 to C20 acyl group, preferentially a palmitoyl group, or a cholesterol residue according to formula (II): wherein: - Y represents the carbon atom of the ester bond between the cholesterol residue and X1.

3. Peptide conjugate of formula (I) according to claim 1 or 2, wherein the salt is an acid addition salt, wherein the acid is for example hydrochloric acid, trifluoroacetic acid and / or acetic acid.

4. Peptide conjugate of formula (I) according to any one of claims 1 to 3, wherein X1 is an alanine or a covalent bond, and X2 is an arginine or an alanine.

5. Peptide conjugate of formula (I) according to any one of claims 1 to 4, wherein the peptide conjugate of formula (I) is selected from: - (2) Palm-Ala-Arg-Pro-Ala-Lys-OH, - (3) Palm-Ala-Arg-Pro-Ala-Lys-NH2, - (4) Palm-Ala-Arg-Pro-Ala-Ala-NH2, - (6) Palm-Ala-Ala-Pro-Ala-Lys-NH2, - (7) Chol-Ala-Arg-Pro-Ala-Lys-NH2, - (9) Palm-Arg-Pro-Ala-Lys-OH, and wherein - Palm represents a palmitoyl group and - Chol represents a cholesterol residue.

6. A process for manufacturing the peptide conjugate of formula (I) according to claim 1, wherein said process comprises the following successive steps: a. synthesis, at least partially on solid support, of the peptide strand of formula H-X1-X2-Pro-Ala-X-B, wherein - X1, X2, X are as defined in any one of claims 1 to 5 and are suitably protected; - B is the solid support or a suitably protected OH or NH2 group; b. grafting of group A onto the peptide strand of step (a) by acylation reaction, preferentially by the use of an activated acyl in the presence of a base; c. deprotection of the protected amino acids optionally together with cleavage of the peptide from the resin, when B is a solid support; d. optional purification of the peptide conjugate of formula (I) obtained, for example by HPLC; and e. collection of the product of formula (I).

7. Peptide conjugate of formula (I) according to any one of claims 1 to 5, as drug.

8. Peptide conjugate of formula (I) according to any one of claims 1 to 5, for dermatological use preferably in combination with another dermatological active ingredient.

9. Peptide conjugate of formula (I) according to any one of claims 1 to 5, for use in the treatment or the prevention of alopecia.

10. Peptide conjugate of formula (I) according to any one of claims 1 to 5 for use according to claim 9, wherein alopecia is selected from androgenetic, congenital, acquired, localized, diffuse, acute or chronic alopecia.

11. Pharmaceutical, dermatological or cosmetic composition comprising at least one peptide conjugate of formula (I) according to any one of claims 1 to 5 and 8 to 10.

12. Composition according to claim 11, further characterized in that it is a composition for topical use.

13. Composition according to claims 11 or 12, wherein said composition comprises the 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 or of L / D form.

14. Composition according to any one of claims 11 to 13, characterized in that the composition is for the scalp.

15. Composition according to any one of claims 11 to 14, characterized in that said composition is chosen from the group consisting of a lotion, a serum, a shampoo, such as a medicated shampoo, a spray, a gel, or a cream such as a treating cream.

16. Composition according to any one of claims 11 to 15 characterized in that said composition comprises at least one peptide conjugate of formula (I) according to any one of claims 1 to 5 at a concentration ranging from 10-9 to 10-4 mole / litre of composition, preferably from 10-7 to 10-5 mole / litre of composition.