WO2007021219A1

WO2007021219A1 - Stimulant of hair growth based on a pantothenic acid derivative

Info

Publication number: WO2007021219A1
Application number: PCT/RU2006/000421
Authority: WO
Inventors: Vasily Nikolaevich Osipov
Original assignee: Vasily Nikolaevich Osipov
Priority date: 2005-08-15
Filing date: 2006-08-10
Publication date: 2007-02-22
Also published as: RU2295949C1

Abstract

Stimulant of hair growth based on a pantothenic acid derivative The invention relates to cosmetic industry and relates to application of new derivatives of pantothenic acid, namely, ammonium salts with peptides of the following formula (I) where X i s GIy, Ala , Leu , lie , VaI , Pro , Tyr , Phe , Trp , D- IIe , Ϝ-aminobutyric acid ; Y is GIu , Z is OH, NH2, mono- or disubstituted amide (C1-C3) , alkyl ester (C1 - C3) , as well as conjugates of pantothenic acid with peptides of the following formula (II) where X is Gly, Ala , Leu, lie , VaI , Pro , Tyr , Phe , Trp ,D-Ile , Ϝ-aminobutyric acid; Y i s Glu, Z is OH, NH2, mono- or disubstituted amide (C1-C3) , alkyl ester (C1- C3) as a hair growth stimulant, and also a method of preparation of these derivatives.

Description

Stimulant of hair growth based on a pantothenic acid derivative

Field of the invention The invention relates to cosmetic agents, in particular, to hair growth stimulants and also relates to methods of producing biologically active derivatives of pantothenic acid.

Background of the invention Disturbances in normal hair growth down to their complete hair loss may have different reasons but irrespective of that such pathological developments have rather significant social effect.

The search of drugs stimulating hair growth began long time ago and still continues, however, an ideal and universal drugs equally effective in all cases apparently do not exist.

Wide use in hair care drugs in the form of different sorts of vegetative extracts is based either on the action of extracted substances as local irritants (extracts from onions, garlic, pepper) or on the action of some extracted products as active precursors for a biosynthesis of compounds important for a metabolism of the cells of follicles or a hair trunk (pantothenic acids in an extract of neetle, silica acid, in extracts of oat, equisetum) [Makhlyuk V. P., Medical Plants in Folk Medicine, Saratov, 1991] .

Pantothenic acid is vitamin of group B (Vitamin B₅) ; by its chemical nature it is a dipeptide consisting of residual amino acid of β-alanine and pantoic acid. It is well known that pantothenic acid and its various active derivatives are an internal factor important for passing metabolic processes in skin tissues, and it is specific with respective growth and pigmentation of hairs (US patent 3,376,334 A of June 17, 1965) . A lack of pantothenic acid in an organism of animals results in disturbances of the metabolism leading to dermatitise, depigmentation and loss of a hair, wool or feathers (V. M. Berezovsky, Chemistry of Vitamins, Moscow, 1973, page 69.). However attempts to obtain an effective medicine based on pantothenic acid stimulating hair growth have failed.

None the less, pantothenic acid was offered as a component of medicines for hair growth (JP 11246360, JP 2001288043, JP 2002316918, JP 2004091390). Alongside with pantothenic acid, these medicines include various biologically active substances including natural ones allowing one to inhibit hair loss and stimulate hair growth in complex.

Also known in the art is a medicine stimulating hair growth and containing low-molecular RNA of nucleuses of cells of follicles of physiologically young animals and capable to specifically stimulate hair growth (RU 2144366 Cl, Closed Joint-Stock Company "Astera" RU, January 20, 2000) . A disadvantage of the known medicine is a complex process of producing useful substances from animal raw material and, in this connection, such a composition is inconstant.

Known in the art are examples of application of peptides and protein derivatives as hair growth stimulants. Also known in the art are pharmaceuticals incorporating isolated oligopeptide having a length between 5 and 104 amino acid residues as an active ingredient (RU 2002130441 Al, Japan Sumitomo Electric Industries, Ltd. JP, 10.06.04; RU 2002135669 A, Japan Sumitomo Electric Industries, Ltd. JP, 20.07.04). The labor consumption for obtaining such oligopeptides is obvious.

As a close analog of our invention we may refer to the patent (RU 2043762 Cl, September 20, 1995, granted to the Research-and-Production Company "Protein Pav", RU) , in which for hair growth stimulation it is proposed to use N-acyl derivatives of protein hydrolysates obtained at interaction of acid chlorides and anhydrides of higher carboxylic acids with products of fragmentation of various protein substances. However, not precisely determined and changeable

(precisely non-reproducible) composition of the medicine can result in occurrence of undesirable side effects. Thymus peptides have pronounced immunomodulatory properties, they also carry out specific tissues action directed to recovery of normal functioning of immune cells

(V. G. Morozov, V. H. Khavinson, V.V. Malinin, Thymomimetic

Peptides. S. P, Science, 2000, page 33). Peptides, isolated from a thymus gland recover the immunological reaction of skin cells, control the cell immunity reactions, stabilize the metabolism in the skin cells, stimulate repair processes. These peptides also activate growth of hair follicles strengthening the hair roots and accelerating the hair growth. The peptides also enter compositions of some hair care means. These drugs are a set of polypeptides of various length acting on various parts of immune response, as well as on some other physiological functions of an organism, and this results in appearance of undesirable side effects. Besides, practical use of such drugs is difficult due to complexity of the methods of their extraction from natural raw materials, a low yield, appreciable variability of their physical and chemical properties. The latter circumstance was a stimulus for creation of synthetic peptides having immunoregulatory properties.

It is of great interest to create peptide immunoregulators based on short, selectively acting highly active peptides whose synthesis is economically expedient. Isolated from a thymus gland is series of short peptides having immunobiological activity (V. G. Morozov, V. H. Khavinson, V. V. Malinin, Thymomimetic Peptides. S. P, Science, 2000, pages 63-64) . Some chemically synthesized peptides are widely used in medical practice (for example Glu-Trp (thymogene, WO 8906134), Lys-Glu (vilone) ) . There are claimed some synthetic peptide thymomimetic drugs (γ-Glu-Trp (bestim, RU 2091389) , Ile-Glu-Trp (neogene, RU 2107691, WO 9626955, US 6,159,940) as immunoregulators .

Thymogene is also proposed as a basic component for a skin conditioner (patent RU 2008894 of March 15, 1994) helping to recover the protective properties of skin.

According to the data known to the inventors the patent and scientific literature includes no information on joint application of pantothenic acid and active peptides for cosmetic preparations, and there is also no data on methods of production of salts and conjugates of pantothenic acid and peptides.

Disclosure of the invention An object of the present invention is to obtain substances accelerating hair growth, strengthening the hair roots and recovering hair pigmentation.

This object is attained by obtaining derivatives of pantothenic acid and short peptides being thymomimetics and using them in cosmetic preparations for hair care.

We have obtained ammonium salts of pantothenic acid with peptides of the following formula I

where X is GIy, Ala, Leu, lie, VaI, Pro, Tyr, Phe, Trp,

D-IIe, γ-aminobutyric acid;

Y is GIu, GIy, Ala, Leu, lie, VaI, Pro, Tyr, Phe, Trp;

Z is OH, NH₂, mono- or disubstituted amide (C₁-C₃) , alkyl ester (Ci - C₃) .

Conjugates of pantothenic acid with peptides of the following formula II are also obtained.

where X is GIy, Ala, Leu, lie, VaI, Pro, Tyr, Phe, Trp, D-IIe, γ-aminobutyric acid;

Y is GIu, GIy, Ala, Leu, lie, VaI, Pro, Tyr, Phe, Trp; Z is OH, NH₂, mono- or disubstituted amide (C₁-C3) , alkyl ester (Ci - C₃) having properties of stimulation of hair growth, their strengthening and recovery of a hair pigmentation.

Brief description of the drawings

The claimed invention is described on concrete examples of its realization with reference to the appended drawing, in which:

Fig. 1 illustrates a control mouse (a)- to the left) and a mouse treated with compound 15 on the 7th day (b) - to the right) .

The best embodiment of the invention

The peptides described in the specification can be prepared through chemical synthesis, using the methods known in chemistry of peptides such as the solid-phase synthesis

(Merrifield, 1964, J. Am. Chem. Soc. 85:2149-2154) or the synthesis in a homogeneous solution (Houben-Weyl, 1987,

Methods of Organic Chemistry, ed. E. Wansch, Vol.15 I and

II, Thieme, Stuttgart) .

The peptide synthesis process provides use of a maximum amount of blocking function groups, starting from alkyl ethers of amino acids, using the activated esters method and the method of mixed anhydrides, preferably using BOC-group as a protection for the amino group which must not react during the synthesis. The suitable blocking agents are known in the peptide synthesis. After synthesis of peptides the protecting groups are removed.

The methods of obtaining some peptides are disclosed in a number of patents (Rϋ 2091389, RU 2107691, RU 2120446, US 6159940) .

Ammonium salts of pantothenic acid with peptides are obtained by successively treating calcium pantothenate with sulfuric acid, then by an appropriate peptide. The conjugates of pantothenic acid with peptides can be obtained by forming an amide bond by any known method used in the peptide chemistry. It is advisable to use the method of activated esters using perfluorophenyl ester of pantothenic acid. The hydroxyl groups of pantothenic acid are preferably preliminarily protected by an acetal group with subsequent removal of the protective group.

The claimed compounds may be used in various cosmetic preparations for hair treatment, such as creams, masks, balms, shampoos, tonics etc. The composition may include one or several claimed compounds.

Pantothenic acid is not toxic (toxicity on rabbits >2000 mg/kg intravenously) (V. M. Berezovsky, Chemistry of Vitamins, Moscow, 1973, page 72).

Peptides also cause no toxic reactions, no cumulatation in an organism and have no chronic toxicity. The results of researches have shown that at intra-abdominal introduction of 1000-fold dose the described peptides do not render acute toxic action and at these doses it has appeared impossible to achieve their LD₅₀ (RU 2091389, WO 9640740, US 6159940) . The essence of the invention is illustrated by the following examples.

Example 1

Obtaining a salt of pantothenic acid with Ile-Glu-Trp (compound 15)

Calcium pantothenate 86 mg (1.81*10⁴ mole) is dissolved in 2 ml of water, acidified with 0.09 ml of 10% solution H₂SO₄, the precipitate of calcium sulfate is filtered off. The precipitate is washed on a filter of 2 ml absolute alcohol .

The obtained solution is added with a suspension of 161 mg (3.62-10^"4 mole) of Ile-Glu-Trp in 10 ml of water. A reaction mixture is refluxed for one hour. The solvent is evaporated, the oil residue in ether. An amorphous powder of light-brown color is obtained. The yield is 237 mg (96%) .

The salts of pantothenic acid with the above peptides (compounds 1-18 in Table 1) are obtained in a similar way.

Example 2

Preparation of conjugate Ile-Glu-Trp with pantothenic acid (compound 15a) .

1. Preparation of pantothenic acid acetonide

To suspension of 5.00 g (10.5 mmole) of calcium pantothenate and 0.94 g (10.5 mole) of anhydrous oxalic acid in acetone was added 7.1 ml (0.058 mole) of 2,2- dimethoxypropane and refluxed in the presence of a molecular sieves 4A for 15 hours (TLC control) . The calcium oxalate deposit is filtered off, the acetone is evaporated, the residue is dissolved in ethyl acetate, washed with water and dried above magnesium sulfate. The solvent is evaporated, the residue is triturated in hexane to obtain 4.5 g of white crystals requiring no additional cleaning (yield 78%) .

Melting point = 87-89°C.

TLC: chloroform : methanol - 3:1 (Sorbfil) , R_f = 0.46.

2. Preparation of pentafluorophenyl ester of pantothenic acid acetonide.

1 g (3.30 mole) of pantothenic acid acetonide and 0.68 g (3.68 mmole) of pentafluorophenol are dissolved in 30 ml of anhydrous methylene chloride, the mixture is cooled down to -10⁰C, added with 0.76 g (3.68 mmole) dicyclohexylcarbodiimide and agitated for 3 hours at -10 to 0⁰C, then for 4 hours at 2O⁰C (TLC control) . The precipitate of dicyclohexylurea is filtered off, the solvent is evaporated and the residue is crystallized from hexane. 1.1 g (72%) of pentafluorophenyl ester of pantothenic acid acetonide is obtained. Melting point = 112-114°C.

TLC: chloroform (sorbfil) , R_f = 0.40.

3. Preparation of pantothenic acid conjugate with Ile-Glu- Trp. 4 ml of IM solution of complex of zinc chloride with pyridine is prepared in dimethylformamide. Dissolved therein are 0.15 g (0.34 mmole) of tripeptide and 0.19 g (0.4 mmole) of pentafluorophenyl ester obtained as described above, the mixture if added with 10% (wt) of imidazole, agitated for 10 hours at 40⁰C (TLC control) .

After the reaction has been completed, the dimethylformamide is evaporated and the product is diluted with 10 ml of 0.5 M solution of hydrochloric acid and agitated for 30 minutes. The reaction mass is extracted with ethyl acetate and dried above magnesium sulfate. The solvent is on evaporated under vacuum and the residue is triturated in ether to obtain 0.1 g (43%) of yellow crystals, which crystallized from ethyl acetate/methanol . Melting point = 145-147°C. TLC: chloroform: methanol : acetic acid - 8:1:1 (Sorbfil) , R_f = 0.15.

Conjugates of pantothenic acid with other peptides are obtained in a similar way (compound la-18a in Table 1) . Table 1 .

Characteristics of salts of pantothenic acid with peptides (compounds 1-18) and conjugates of pantothenic acid with peptides (compounds la-18a) .

Example 3

The pharmacological study has been conducted on 120 nondescript mice (3 males and 3 females on each compound) with a mass of 20-22 g. The animals were selected without defeat of a hair integument. The control and experimental groups contained under identical conditions of a vivarium and on a standard ration. The mice were gently shaved until no hair remained in test area (lower back) .

The definition of specific pharmacological action was carried out by applying onto the experimental group animals 1% of solutions of the tested compounds in 50% aqueous solution of dimethylsulfoxide in 100 mg on one depilated area of the skin, single-pass, at the same time of day, by way of mild massage. The treatment was applied on the 1st, 2nd, 4th and 5th days. A pure vehicle solution was applied onto the control group of animals on one depilated cite of the skin.

The hair growth (small grey fur well thus visible against pink skin) was observed for mice treated by the compound 15 already on the 2nd day.

On the 5th day of beginning the treatment the hair growth was observed on all mice treated by the compounds, i.e. the salts and conjugates of pantothenic acid with peptides. Noticeable growth was observed on the 7th day on the mice treated by the described compounds (Fig.l). A pronounced effect was observed for compounds 10, 12, 15, 16, 10a, 15a, 16a. In application of the solutions of tested substances no inflammation, desiccation of skin or another side effects on the skin were observed. In the control group of mice on the 7th day no hair growth effect was observed (Fig. 1) . Example 4

The clinical trials of lotion N-500 (developed on base of compound 15) for external use have been conducted on 30 patients. Report on the clinical trials of lotion N-500 for external use for patients with different form of alopecia areata are shown below.

REPORT ON THE CLINICAL TRIALS OF LOTION N-500 FOR EXTERNAL USE FOR

PATIENTS WITH DIFFERENT FORMS OF ALOPECIA AREATA. AIMS AND PURPOSES OF RESEARCH 1. To appreciate the therapeutic efficiency of Lotion N-500 for patients with alopecia areata.

2. To evaluate the effect of Lotion N-500 on hair growth.

3. To estimate the endurance of Lotion N-500, the frequency 5 of undesirable reactions and complications during the external use of this remedy.

THE DESIGN AND PERIOD OF TRIALS

The clinical trials have been carried out by the 10 physician trichologist, D. M., Member of European Society of Trichologists, practicing the hair cure for more than 20 years and having over 70 publications in this field.

The design of research is the multicentric visual trial of 30 patients with diagnosis of alopecia areata. 15 The beginning of clinical tests - 01.09.2005.

The ending of clinical tests - till present time.

DESCRIPTION OF N-500 LOTION FORMULA Lotion N-500 has been applied for research:

201. Lotion N-500 for external use only. Packing: 60 ml flask (manufacturer - "New Chemistry Horizons Laboratories" Ltd. , Russia) .

The Lotion N-500 contains:

Ammonium salt of pantothenic acid and tripeptide (Code 25 NCH Pep 0035) in water solution of glycerin. The concentration of basic substance is 500 ppm.

Pantothenic acid (Vitamin B₅) is dipeptide, influent on metabolic process in skin and hair growth.

Tripeptide (Code NCH Pep 0035) has strong 30 immunomodulatory properties and carries out the specific action for restoration of normal functioning of immune cells .

The Lotion doesn't contain aromatizers, conservants and has aqueous structure. 35 CRITERIA OF INCLUSION OF PATIENTS INTO CLINICAL TRIAL 1. Patients aged from 18 till 50 years suffering of various forms of alopecia areata.

2. Patients who have given their written approval for participation in clinical trial. CRITERIA OF EXCEPTION OF PATIENTS FROM TRIAL

Patients who have received the traditional system-defined therapy less than 2 weeks prior to the beginning of trial.

DESCRIPTION OF CLINICAL GROUP OF PATIENTS 30 patients (18 female and 12 male) with diagnosis of alopecia areata were under clinical supervision. 19 persons of them suffered of heavy total and subtotal forms of disease. 11 persons of them suffered of multifocal uptake and multicentric forms of alopecia. Main criteria of survey of defeated area: totally hairless space, state of skin in alopecia niduses, state of eyelashes and eyebrows, state of fine hair on trunk, thickness of retained hair on different parts of scalp' s skin and state of growing hair zone surrounding niduses in case of alopecia areata. Subjective data were also taken into consideration: itch, burning pain, infringement of sleep.

All patients have made an independent estimation of hair status before and after treatment, endurance of preparations. Results of treatment were estimated by 4-point scale: 1 point - growth of individual, colorless hair in the alopecia niduses,

2 points - active growth of fine hair in the niduses,

3 points - intensive growth of barbate hair in the niduses, 4 points - full restoration of hair at niduse forms and restoration of hair more than by 50% at the total form. Results of treatment were estimated in 1, 3 and 5 months from the beginning of clinical trial.

THE SCHEME OF TREATMENT Lotion was applied to all patients in form of monotherapy and was put on hairless sites plus 3 cm a surrounding zone 2 times a day.

RESALTS OF CLINICAL TRIALS

By the end of first month of treatment the following results have been received: at 74,3% of all patients has been marked a significant improvement of the status of skin cellular fatty tissue (its thickness has increased) , has been marked a reviving of follicle bulbs, was noticed a growth of fine hair at 42,6% of patients with nidus alopecia form and at 17,2% with total forms of alopecia.

After 3 months of treatment at 24,4% of patients with nidus alopecia form it was marked a total clinical remission, at 37,7% - a good growth of barbate hair in the niduses. Among the patients with total form of alopecia: 15,2% have shown a growth of barbate hair on scalp skin; 28,9% a growth of fine hair on head and trunk skin.

Examination of 5 months' treatment has shown a clinical remission at 68,1% of patients with nidus forms of disease, while at 37,2% of patients with total forms of disease it was marked a remission of barbate hair by more than 50%. At 9% of patients with total form of alopecia the efficiency of treatment was not significant and was revealed in approved status of skin cellular fatty tissue and moderate growth of fine hair. All patients with no exception have endured the treatment, did not make no subjective complaints, positively reported about preparation. Side effects were not described with no one patient. The lotion has absorbed well in the skin. It is to notice the lotion had no harmful effect on a scalp skin - did not provoke no dryness, peeling or irritation. The skin has remained soft, elastic and without coloring.

CONCLUSION The preliminary clinical trials of Lotion N-500 for hair cure has shown its high therapeutic efficiency for treatment of various forms of alopecia areata, and it can be recommended for a further random clinical research for obtaining the certificate and production application.

L. P. Mazitova, physician richologist, Ph. D.

Industrial applicability

The invention may be used in the cosmetic industry (in cosmetics) , in particular, in various cosmetic preparations for hair treatment such as creams, masks, balms, shampoos, tonics etc. The medicine composition may include one or more claimed compounds .

Claims

1. Application of derivatives of pantothenic acid, namely, ammonium salts with peptides of formula I:

where X is GIy, Ala, Leu, lie, VaI , Pro, Tyr, Phe , Trp, D-IIe , γ-aminobutyric acid;

Y is GIu, Z is OH, NH₂, mono- or disubstituted amide (Ci-C₃) , alkyl ester (Ci - C₃) as a hair growth stimulant.

2. Application of derivatives of pantothenic acid, namely, conjugates of pantothenic acid with peptides of formula II:

where X is GIy, Ala, Leu, lie, VaI, Pro, Tyr, Phe, Trp, D-IIe, γ-aminobutyric acid; Y is GIu,

Z is OH, NH2, mono- or disubstituted amide (Ci-C₃) , alkyl ester (Ci - C₃) as a hair growth stimulant.

3. A method of preparation of ammonium salts of pantothenic acid with peptides as claimed in claim 1, consisting in obtaining pantothenic acid from a calcium pantothenate by sulfuric acid treatment with subsequent formation of ammonium salts by means of boiling an aqueous- alcoholic solution of pantothenic acid and respective peptide .

4. A method of preparation of derivatives of pantothenic acid as claimed in claim 2 consisting in acetal protection of hydroxyl groups of pantothenic acid by 2,2- dimethoxypropane, preparation of pentafluorophenyl ether of protected pantothenic acid, its further interaction with appropriate peptide to form an amide bond and removal of protective acetal group.