WO2008067833A1

WO2008067833A1 - Use of ghrelin to reverse hair loss

Info

Publication number: WO2008067833A1
Application number: PCT/EP2006/011703
Authority: WO
Inventors: Nathalie Mondoly; Rakesh Datta; Zheng Xin Dong; Stefan Anker; Jochen Springer; Sandra Palus
Original assignee: Charité Universitätsmedizin - Berlin; Societe De Conseils De Recherches Et D'applications Scientifiques Sas
Priority date: 2006-12-05
Filing date: 2006-12-06
Publication date: 2008-06-12
Also published as: WO2008067831A2

Abstract

Use of an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof in the preparation of a medicament for administering to a mammal so as to stimulate its hair growth.

Description

Use of Ghrelin to Reverse Hair Loss

This invention relates to ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof in the preparation of a medicament for administering to a mammal so as to stimulate its hair growth. Ghrelin is a recently discovered gastric hormone of 28 amino acids showing a unique structure with an n-octanoyl ester at its third serine residue (Kojima M et al. Nature 1999; 402(6762):656660). Though many synthetic peptidyl and nonpeptidyl growth hormone (GH) secretagogues (GHS) were identified as ligands of GHS-R, ghrelin is shown to be a physiological ligand for the GHS-R. Ghrelin powerfully stimulates GH secretion through its interaction with GHS-R both in animals and in humans (Ukkola, O et al., 2002 Ann. Med. 34:102-108). The GH-releasing activity of ghrelin is mediated by activation of GHS-R at the pituitary and, mainly, at the hypothalamic level (Kojima M et al. Nature 1999; 402(6762):656660) likely by enhancing the activity of growth hormone releasing hormone (GHRH)-secreting neurons and, concomitantly, acting as a functional somatostatin (SS) antagonist (Ghigo E et al. Eur J Endocrinol 1997; 136(5):445460). Other mechanisms have been postulated recently as well (Ahnfelt-Ronne I et al. Endocrine 2001; 14(1): 133- 135).

The GHS-R and its subtypes are not restricted to the hypothalamus-pituitary unit but are present also in other central and peripheral tissues (Papotti M et al. J CHn Endocrinol Metab 2000; 85(10):3803-3807) and the physiological actions of ghrelin, as well as those of synthetic GHS are not restricted to GH secretion, hi fact, ghrelin stimulates lactotroph and corticotroph hormone secretion, has orexigenic and cardiovascular actions, shows anti proliferative effects on thyroid and breast tumors and regulates gastric motility and acid secretion through vagal mediation (Ukkola, O et al., 2002, Anti. Med. 34:102-108). In humans, fasting leads to elevated serum GH concentrations. Traditionally, changes in hypothalamic GHRH and somatostatin have been considered as the main mechanisms, which induce elevations in GH secretion during fasting. As ghrelin administration in man also stimulates GH release, and serum ghrelin concentrations are elevated during fasting, increased ghrelin actions might be another mechanism whereby fasting results in the stimulation of GH release.

Although ghrelin is likely to regulate pituitary GH secretion in interplay with GHRH and SS; GHS receptors have also been identified on hypothalamic neurons and in the brainstem (Nakazato M et al. Nature 2001; 409(6817):194-198). Apart from potential paracrine effects, ghrelin may thus offer an endocrine link between the stomach, hypothalamus and pituitary, suggesting an involvement in the regulation of energy balance. Tschop et al. have shown that daily peripheral administration of ghrelin in mice and rats caused weight gain by reducing fat utilization (Tschop M et al. Nature 2000; 19; 407(6806):908-913). Intracere broventricular administration of ghrelin generated a dosedependent increase in food intake and body weight. Rat serum ghrelin concentrations increased by fasting and decreased by re-feeding or oral glucose administration, but not by water ingestion. Apparently ghrelin, in addition to its role in regulating GH secretion, signals the hypothalamus when an increase in metabolic efficiency is necessary (Tschop M et al. Nature 2000; 19; 407(6806):908-913; Muller A F et al.. Clin Endocrnol (Oxf) 2001; 55(4):461-467).

Studies by Kojima and others have shown that unacylated ghrelin (UAG) has no affinity to the known GHS-R ((GHS-RIa receptor), which is responsible for GH release from the pituitary gland (Kojima M et al. Nature 1999; 402(6762):656-660). This was confirmed later by Bednarek M A et al (Bednarek M A et al, J. Med Chem. 2000, 43:4370-4376), who showed that unacylated ghrelin could not be a physiological ligand of the GHS-RJa receptor (IC50> 10,000 nM), since it poorly activated GHS-RIa at micromolar concentrations; large hydrophobic acyl group is obligatory at position 3 of ghrelin for its biological response on GH secretion.

Ghrelin was discovered as the peptide hormone that stimulates release of growth hormone from the anterior pituitary. It was subsequently determined that ghrelin, along with several other hormones, has significant effects on appetite and energy balance. Further, ghrelin analogues have been proposed for glycemic control in certain metabolic diseases (US 2005/0080007).

Subject of the present invention is the surprising effect of ghrelin, analogues thereof and pharmaceutically acceptable salts thereof in the preparation of a medicament for administering to a mammal so as to stimulate its hair growth. Especially preferred is human ghrelin.

Therefore, subject of the present invention is the use of an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof in the preparation of a medicament for administering to a mammal so as to stimulate its hair growth.

Ghrelin is a amino acid peptide of SEQ ID No. 1. Analogue of ghrelin according to this invention means a) Peptide-analogs, full length or short peptides that mimic ghrelin action on the GHS- receptor and other putative pathways. b) Non-peptide-analogs that mimic ghrelin action on the GHS-receptor and other putative pathways.

Unless otherwise stated, those amino acids with a chiral center are provided in the L- enantiomer.

The present invention also encompasses derivatives of the inventive amino acid peptides. Reference to "a derivative thereof refers to a modified amino acid such as the corresponding D-amino acid, a N-alkyl-amino acid, a labeled amino acid.

Preferred derivatives of analogs of the invention comprise D-amino adds, N-alkyl-amino acids, β-amino acids, and/or one or more labeled amino adds (including a labeled version of a D-amino acid, a N-alkyl-amino acids, or a β-amino add). A labeled derivative indicates the alteration of an amino acid or amino add derivative with a detectable label. Examples of detectable labels include luminescent, enzymatic, and radioactive label. Both the type of label and the position of the label can effect analog activity. Labels should be selected and positioned so as not to substantially after the activity of the ghrelin analog at the GHS receptor. The effect of a particular label and position on ghrelin activity can be determined using assays measuring ghrelin activity and/or binding.

A protecting group covalently joined to the C-terminal carboxy group reduces the reactivity of the carboxy terminus under in vivo conditions. The carboxy terminus protecting group is preferably attached to the a-carbonyl group of the last amino acid. Preferred carboxy terminus protecting groups include amide, methylamide, and ethyla- mide protecting group is preferably attached to the a-carbonyl group of the last amino acid. Preferred carboxy terminus protecting groups include amide, methylamide, and ethylamide.

Suitable peptidyl analogues include those which are disclosed in US 2005/0148515 and which are incorporated herein by reference.

The present invention features ghrelin peptidyl and non-peptidyl analogs active at the GHS receptor.

Certain amino adds present in compounds of the invention are represented herein as follows:

A3 c 1 -amino- lcyclopropanecarboxylic acid

A4c 1 -amino- 1- cyclobutanecarboxylic acid

A5c 1 -amino- 1- cyclopentanecarboxylic acid

A6c 1 -amino- 1- cyclohexanecarboxylic acid

Abu α- aminobutyric acid

Ace 1 -amino- l-cyclo(C₃ C₉) carboxylic acid

Act 4-amino-4-carboxytetrahydropyran, i.e.,:

Aib α-aminoisobutyric acid

Ala or A alanine

β-Ala beta-alanine

Ape amino piperidinylcarboxylic aid, i.e.

Arg or R arginine hArg homoarginine

Asn or N asparagine Asp or D aspartic acid

BaI 3-Benzothienylalanine, i.e.

D-BaI D-3-Benzothienylalanine, i.e.

Bip 4,4'-Biphenylalanine, i.e.

4-Benzoylphenylalanine, i.e.

Cha β-cyclohexyalanine;

Cys or C cysteine;

Dab 2,4-diaminobutyric acid, (α,γ-Diaminobutyric acid);

Dap 2,3-diaminopropionic acid, (α,β-Diaminopropionic add) Dip β,β-Diphenylalanine, i.e.:

Dhp 3,4-dehydroproline

Dmt 5,5-dimethylthiazolidine-4-carboxylic acid

2Fua β-(2-furyl)-alanine, i.e.:

Gin or Q glutamine GIu or E glutamic acid Glu(NH-hexyl), i.e.:

GIy or G glycine

His or H histidine 3Hyp trans-3-hydroxy-L-proline, i.e., (2S, 3S)-3-hydroxypyrrolidine-2-carboxylic acid;

4Hyp 4-hydroxyproline, i.e., (2S, 4R)-4-hydroxypyrrolidine-2-carboxylic acid;

He or I isoleucine

Inc indoline -2-carboxylic acid Inp isonipecotic acid, i.e.:

Ktp 4-ketoproline Leu or L leucine hLeu homoleucine Lys or K lysine Met or M methionine INaI β-(l-Naphthyl)alanine; 2NaI β-(2-Naphthyl)alanine; NIe norleucine Nva norvaline

Oic octahydτoindole-2-carboxylic acid Orn ornithine 2PaI β-(2-Pyridyl)-alanine, i.e.,

3PaI β-(3-Pyridyl)-alanine, i.e.

4PaI β-(4-Pyridyl)-alanine, i.e.

Pff pentafluorophenylalanine, i.e.

Phe or F phenylalanine hPhe homophenylalanine

Pirn 2'-(4-Phenyl)imidazolyl, i.e.

Pip pipecolic acid

Pro or P proline

Ser or S serine

Taz β-(4-thiazolyl)alanine, i.e.,

2Thi β-(2-thienyl)alanine, i.e.:

3Thi β-(3-thienyl)alanine, i.e.

Thr or T threonine

Thz thiazolidine-4-carboxylic acid

Tic 1,2,3,4-tetrahydroisoquinoline-carboxylic acid Tie tert-leucine

Tφ or W tryptophan

Tyr or Y tyrosine

VaI or V valine

Certain other abbreviations used herein are defined as follows: Boc: tert-butyloxycarbonyl

BzI: benzyl

DCM: dichloromethane

DIC: N,N-diisopropylcarbodiimide DIEA: diisopropylethyl amine

Dmab:4{N-(l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl)-amino}benzyl

DMAP :4-(dimethylamino)pyridine

DMF dimethylformamide DNP:2,4-dinitrophenyl

Fmoc: Fluorenylmethyloxycarbonyl

HBTU:2-( 1 H-benzotriazole- 1 -yl)- 1 ,1,3,3 -tetramethyluronium hexafluorophosphate cHex cyclohexyl

HO AT:O-(7-azabenzotriazol-l-yl)l,l,3,3-tetramethyluronium hexafluorophosphate HOBt 1-hydroxy-benzotriazole

HOSu: N-hydroxysuccinimide

Mmt 4-methoxytrityl

NMP: N-methylpyrrolidone

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl tBu: tert-butyl

TIS: triisopropylsilane

TOS: tosyl trt trityl

TFA: trifluoro acetic acid TFFH: tetramethylftuoroforamidinium hexafluorophosphate

Z: benzyloxycarbonyl Unless otherwise apparent, abbreviations (e.g. Ala) of amino acids in this disclosure stand for the structure of -NH-C(R)(R')-CO-, wherein R and R' each is, independently, hydrogen or the side chain of an amino acid (e.g., R=CH₃ and R'=H for Ala), or R and R' may be joined to form a ring system.

"Alkyl" refers to a hydrocarbon group containing one or more carbon atoms, where multiple carbon atoms if present are joined by single bonds. The alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups.

"Substituted alkyl" refers to an alkyl wherein one or more hydrogen atoms of the hydrocarbon group are replaced with one or more substituents selected from the group consisting of halogen, (i.e., fluorine, chlorine, bromine, and iodine), -OH, -CN, -SH, -NH₂, -NHCH₃, -NO₂, -C_1-2 alkyl substituted with 1 to 6 halogens, CF₃, -OCH₃, -OCF₃, and - CH₂)_0-4-COOH. In different embodiments 1, 2, 3 or 4 substituents are present. The presence of -(CH₂)_o-4-COOH results in the production of an alkyl acid. Examples of alkyl acids containing, or consisting of, -CH₂)_o-4 COOH include 2-norbornane acetic acid, tert-butyric acid and 3-cyclopentyl propionic acid.

"Heteroalkyl" refers to an alkyl wherein one of more of the carbon atoms in the hydrocarbon group are replaced with one or more of the following groups: amino, amido, -0-, or carbonyl. In different embodiments 1 or Z heteroatoms are present.

"Substituted heteroalkyl" refers to a heteroalkyl wherein one or more hydrogen atoms of the hydrocarbon group are replaced with one or more substituents selected from the group consisting of halogen, (i.e., fluorine, chlorine, bromine, and iodine), -H, -CN, -SH, -NH₂, - NHCH₃, -NO₂, -C_1-2 alkyl substituted with 1 to 6 halogens, -CF₃, -OCH₃, -OCF₃, and - CH₂)_o-4 COOH. In different embodiments 1, 2, 3 or 4 substituents are present.

"Alkenyl" refers to a hydrocarbon group made up of two or more carbons where one or more carbon-carbon double bonds are present. The alkenyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups.

"Substituted alkenyl" refers to an alkenyl wherein one or more hydrogens are replaced with one or more substituents selected from the group consisting of halogen (i.e., fluorine, chlorine, bromine, and iodine), -H, -CN, -SH, NH₂, -NHCH₃, -NO₂, -C_1-2 alkyl substituted with 1 to 6 halogens, -CF₃, -OCH₃, OCF₃, and -(CH₂)_O-4COOH. In different embodiments 1, 2, 3 or 4 substituents are present. "Aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups. Preferably, the aryl Is a 5 or 6 membered ring. Preferred atoms for a heterocyclic aryl are one or more sulfur, oxygen, and/or nitrogen. Examples of aryl include phenyl, 1 -naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and 9-anthracene. Aryl substituents are selected from the group consisting of -C_1-4 alkyl, -C_1-4 alkoxy, halogen (i.e., fluorine, chlorine, bromine, and iodine), -OH, -CN, OH, NH₂, -NO₂, -C_1-2 alkyl substituted with 1 to 5 halogens, -CF₃, - OCF₃, and - (CH₂)_O-4 COOH. In different embodiments the aryl contains O, 1, 2, 3, or 4 substituents.

"Alkylaryl" refers to an "alkyl" joined to an "aryl".

When a non-amino acid imidazole moiety, (e.g., Pirn, defined above), is present at the C- terminus of a compound of the invention It Is understood that the imidazole moiety Is attached to the adjacent amino acid via a pseudo-peptide bond, wherein a bond is formed between the position 2 carbon of the imidazole ring and the alpha carbon of the amino acid. For example, in the case where the adjacent amino acid is D-tryptophan (D-Trp) and the imidazole moiety is Pirn, the C-terminus of the peptide would appear as follows:

For clarity, in the written formula for such a compound the presence of this bond is indicated by the Greek letter "ψ" alone in parentheses. For example, the written formula H-Inp-D-Trp-D-2Nal(ψ)-Pim denotes the structure:

A peptide of this invention is also denoted herein by another format, e.g., Glu3(NH- hexyl))hGhrelin(1-28)-NH2; with the substituted amino acids from the natural sequence placed between the first set of parentheses (e.g., A5c⁸ for Ala⁸ in hGhrelin). The abbreviation hGhrelin means human Ghrelin. The numbers between the parentheses refer to the number of amino acids present in the peptide (e.g., hGhrelin(l-28) is amino acids 1 through 28 of the peptide sequence for human Ghrelin). The sequence for hGhrelin is found is SEQ ED NO:1.

The compounds of the invention can be formulated and administered to a subject using the guidance provided herein along with techniques well known in the art. The preferred route of administration ensures that an effective amount of compound reaches the target

Guidelines for pharmaceutical administration in general are provided in, for example,

Remington's Pharmaceutical Sciences 18^th Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics 2^nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby incorporated by reference herein.

The compounds of the invention can be prepared as acidic or basic salts. Pharmaceutically acceptable salts (in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases. Examples of such salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, tosylate, and undecanoate; and base salts such as ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine. Ghrelin and its analogues may be further modified e.g. may be acylated or unacylated. Such peptides are described e.g. in US 2005/0080007 and are incorporated herein by reference.

Homologues of ghrelin are also included into this invention. The term "Homology" refers to sequence similarity between two peptides while retaining an equivalent biological activity. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences so that an homologues sequence refers to a sequence sharing homology and an equivalent function or biological activity. The activity which should be retained by all peptides according to the present invention is to mimic ghrelin action on the GHS-receptor and other putative pathways. They further stimulate hair growth according to the persent invention. Due to the high sequence homology between ghrelins of different species (only 2 aa exchanged between rat and human ghrelin (Kojima et al., Nature 402:656-660 (1999)), all natural variants of ghrelin with more than 80 % identity, preferably more than 90 %, more preferably more than 95 %, and even more preferably more than 99 % with human ghrelin are encompassed as homologues of ghrelin by the present invention.

General methods and synthetic strategies used in providing functional and structural analogues of peptides is described in publications such as "Solid phase peptide sythesis" by Stewart and Young, W.H. Freeman & Co., San Francisco, 1969 and Erickson and Merrifield, "The Proteins", Vol. 2, p 255 et seq. (Ed. Neurath and Hill) Academic Press, New York, 1976.

The agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof may be administered through a route selected from the group consisting of intravenous, subcutaneous, transdermal, or oral (only non-peptide analoga).

Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.

The compounds of the invention may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. When administered by injection, the injectable solution or suspension may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as

Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

hi a preferred embodiment the agent is selected from a group consisting of amino acid sequences according SEQ ID No. 1 -5.

1) AAU93610 (human) SEQ ID No.1 : GSSFLSPEHQRVQQRKESKKPPAKLQPR the third serine residue S is a n-Octanoyl modified serine

2) AAU93611 (rat)

SEQ ID No.2: GSSFLSPEHQKAQRKESKKPP AKLQPR the third serine residue S is a n-Octanoyl modified serine 3) NP 067463 (mouse)

SEQ ID No.3: GSSFLSPEHQKAQQRKESKKPP AKLQPR

4) BAC75929 (dog)

SEQ ID No.4: GSSFLSPEHQKLQQRKESKKPP AKLQPR

5) BAD34670

SEQ ID No.5: GSSFLSPEHQKVQQRKESKKPP AKLQPR

More preferably the agent is native full length human gherlin, having the formula human Ghrelin(1-28)-OH or hGhrelin(1-28)-OH.

hi another preferred embodiment, the agent is a ghrelin analogue having the formula H-Inp- D-Bal-D-Trp-Phe-Apc-NH2.

hi another preferred embodiment, the agent is a ghrelin analogue having the formula (Aib2, Glu3(NH-hexyl))hGhrelin(1-28)-NH2.

hi another preferred embodiment, the agent is a ghrelin analogue having the formula (Lys5)hGhrelin(1-28)-NH2. In another preferred embodiment the agent is administered by an implanted osmotic pump in a therapeutically effective amount.

In another preferred embodiment the agent is topically administered in a therapeutically effective amount.

Suitable dosing regimens are preferably determined taking into account factors well known in the art including type of subject being dosed; age, weight, sex and medical condition of the subject the route of administration; the renal and hepatic function of the subject; desired effect; and the particular compound employed.

Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium , and elimination of a drug. Further embodiment of this invention is a method for stimulating hair growth of a mammal comprising a therapeutically effective amount of an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof.

In a preferred embodiment of this method the agent is selected from the group consisting of amino acid sequences according to SEQ ED No. 1 -5.

Ghrelin, ghrelin analogues and pharmaceutically acceptable salts can be incorporated into pharmaceutical compositions. Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one ghrelin or ghrelin analogue in association with a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption enhancers, isotonic and absorption delaying agents, skin penetration enhancers and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. The agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof may be administered through a route selected from the group consisting of intravenous, subcutaneous, transdermal, or oral (only non-peptide analoga).

The compounds of the invention may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. When administered by injection, the injectable solution or suspension may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. In another preferred embodiment the agent is administered by an implanted osmotic pump in a therapeutically effective amount.

The therapeutically effective amount of the agent is between 10 nM/kg/d and 1000 nM/kg/d, preferred between 50 nM/kg/d and 700 nM/kg/d, more preferred between 50 nM/kg/d and 500 nM/kg/d, more preferred between 200 nM/kg/d and 500 nM/kg/d, with 500 nM/kg/d being the more effective dose.

In case of the administration by an implanted osmotic pump the pharmaceutical effective composition may be 2% inactivated serum and 5% Tween 80 in 0.9% saline. This might be understood as one example but is not meant as limitation. In a preferred embodiment, the pharmaceutical composition is intended for transdermal (topical) administration. Where the pharmaceutical composition is intended for transdermal (topical) administration skin penetration enhancers appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, anionic surfactants, zwitterionic surfactants, non-ionic surfactants, fatty acids, fatty esters, fatty amines, or azone-like molecules. Preferably the skin penetration enhancer is N- methyl-2-pyrrolidone (NMP) chemical penetration enhancer. For transdermal administration, the active compounds are formulated into lotions, ointments, salves, gels, or creams as generally known in the art.

In a preferred embodiment the penetrant is an aqueous vehicle, for example aqueous NaCl, more preferably NaCl, 0.9% wt/vol.

In a preferred embodiment the penetrant is propylene glycol.

In a preferred embodiment, the composition is a lotion. More preferably the lotion comprises ghrelin or a ghrelin analogue dissolved at concentration of 0.5% wt/vol in ethanol with 10% wt/vol NMP as an enhancer. Preferably the composition is made by adding ghrelin or ghrelin analogue to the ethanol and mixed until it is dissolved; then, NMP is added and mixed to uniformity. In a prefered embodiment the ghrelin or ghrelin analogue is H-Inp-D-Bal-D-Trp- Phe-Apc-NH2.

In a preferred embodiment, the composition is a gel. More preferably, the gel comprises ghrelin or a ghrelin analogue dissolved at concentration of 0.5% wt/vol in ethanol with 10% wt/vol NMP as an enhancer and 25% wt/vol PF 127. Preferably the composition is made by adding ghrelin or ghrelin analogue to the ethanol and mixed until it is dissolved; 10% wt/vol NMP is added and 25% wt/vol PF127 is added to the mixture and stirred well. The mixture is then diluted with water (to get a 1 :1 ethanol:water ratio) and stirred well. The resulting solution is then stored at 4°C overnight to ensure complete polymer dissolution. In a prefered embodiment the ghrelin or ghrelin analogue is H-Inp-D-Bal-D-Trp-Phe-Apc-NH2.

The agents, compositions and methods may be used to support and stimulate the natural hair growth. Another application of the present invention is the regeneration of hair after operation or after hair loss due to chemotherapy or stress-related hair loss or hormone related hair loss. For example the agents, compositions and methods maybe used to treat alopecia.

In an embodiment, the agents, compositions and methods may be used in cosmetic methods to stimulate hair growth. For example a cosmetic process for stimulating hair growth comprising topically administering an effective amount of the an agent selected from the group comprising ghrelin, an analogue thereof or a pharmaceutically acceptable salt thereof to a mammal, more preferably an agent selected from the group consisting of amino acid sequences according to SEQ ID NO: 1-5. An effective amount of agent is an amount sufficient to stimulate hair growth.

A preferred embodiment is use of an agent selected from the group comprising ghrelin, an analogue thereof or a pharmaceutically acceptable salt thereof for manufacturing a product for stimulating hair growth, preferably a product for topical administration.

The patent and scientific literature referred to herein represents knowledge that is available to those with skill in the art. All patents, patent publications and other publications cited herein are hereby incorporated by reference in their entirety.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the claims.

Figure Description

Figure 1 shows hair growth of rats in shaved areas, where the osmotic pumps were inserted, 14 days post surgery. Depending on the dose given the differences in hair growth were seen over a period of 14 days.

Examples

Preparation of Gherlin Analogues

The analogues of the invention can be produced using the techniques disclosed in the examples herein as well as techniques that are well known in the art. For example, a polypeptide region of a ghrelin analog can be chemically or biochemically synthesized and modified. Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook et al, in Molecular Cloning, A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, 1989. Techniques for chemical synthesis of polypeptides are also well known in the art (See e.g., Vincent in Peptide and Protein Drug Delivery, New York, N. Y., Dekker, 1990). For example, the peptides of this invention can be prepared by standard solid phase peptide synthesis (See, e.g., Stewart, J.M., et al, Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984)).

The substituents R² and R³ of the above generic formula may be attached to the free amine of the N-terminal amino acid by standard methods known in the art. For example, alkyl groups, e.g., (C₁-C₃₀)alkyl, may be attached using reductive alkylation. Hydroxyalkyl groups, e.g., (C₁-C₃₀)hydroxyalkyl, may also be attached using reductive alkylation wherein the free hydroxy group is protected with a t-butyl ester. Acyl groups, e.g., COE¹, may be attached by coupling the free acid, e.g., E¹COOH, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for one hour. If the free acid contains a free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the coupling should be performed with an additional 3 molar equivalents of HOBt.

When R¹ is NH-X²-CH₂-CONH₂, (i.e., Z⁰=CONH₂), the synthesis of the peptide starts with BocHN-X²-CH₂-COOH, which is coupled to the MBHA resin. If R¹ is NH-X²-CH₂- COOH (i.e., Z°=COOH), the synthesis of the peptide starts with Boc-HN-X²-CH₂-COOH, which is coupled to PAM resin. For this particular step, 4 molar equivalents of Boc-HN-X²- COOH, HBTU and HOBt and 10 molar equivalents of DIEA are used. The coupling time is about 8 hours. The protected amino acid l-(N-tert-butoxycarbonyl-amino)-l-cyclohexane-carboxylic acid (Boc-A6c-OH) was synthesized as follows: 19.1 g (0.133 mol) of 1-amino-l- cyclohexanecarboxylic acid (Acros Organics, Fisher Scientific, Pittsburgh, PA) was dissolved in 200 ml of dioxane and 100 ml of water. To it was added 67 ml of 2N NaOH. The solution was cooled in an ice-water bath and 32.0 g (0.147 mol) of di-tert-butyl-dicarbonate was added to this solution. The reaction mixture was stirred overnight at room temperature. Dioxane was then removed under reduced pressure and 200 ml of ethyl acetate was added to the remaining aqueous solution. The mixture was cooled in an ice- water bath. The pH of the aqueous layer was adjusted to about 3 by adding 4N HCl. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (1 x 100 ml). The two organic layers were combined and washed with water (2 x 150 ml), dried over anhydrous MgSO₄, filtered, and concentrated to dryness under reduced pressure. The residue was recrystallized in ethyl acetate/hexanes and 9.2 g of the pure product was obtained, 29% yield.

Boc-A5c-OH was synthesized in an analogous manner to that of Boc-A6c-OH. Other protected Ace amino acids can be prepared in an analogous manner by a person of ordinary skill in the art as enabled by the teachings herein.

In the synthesis of a ghrelin analogue of this invention containing A5c, A6c and/or Aib, the coupling time is 2 hours for these residues and the residue immediately following them.

Methods

Left coronary artery ligation was performed on Sprague Dawley (SD) rats to induce a myocardial infarction (body weight at the time of surgery approx. 220 g). Alternatively, rats received sham-surgery. Twenty-six days after surgery, the animals were randomized to 8 infarct and 2 sham groups (sham: 12 animals per group; infarct: 18 animals per group at the start of therapy) and received one of three ghrelin-analoga or placebo via osmotic pumps implanted under the skin of the back.

Results Macroscopical Pathology

In the shaved areas, where the osmotic pumps were inserted, differences in hair growth were seen over a period of 14 days. Images of the areas were acquired and scored in a blinded fashion.

ghrelin, 5OnM n=9 ghrelin, 50OnM n=10 ghrelin analogue 1, 5OnM n=8 ghrelin analogue 1, 50OnM n=10 ghrelin analogue 2, 5OnM n=8 ghrelin analogue 2, 50OnM n=10

Sham, Placebo n=4 Sham, Plac, diuretic n=10 Table 1

The scores were analysed by the Chi-square test, resulting in X ² = 76.63 und p < 0.001.

Transdermal Permeation

The following experimental results demonstrate the effectiveness of transdermal administration of ghrelin and ghrelin analogues. The applicant has discovered that hair over the incision of rats treated with ghrelin analogue appeared to grow back much faster than control animals, confirming an effect upon hair growth. The effect of ghrelin and ghrelin analogues with or without absorption enhancer was evaluated. Two adsorption enhancer vehicles were tested: an aqueous vehicle, NaCl, 0.9% and an excipient, propylene glycol.

Preparation of transdermal permeation experiments Skin membrane

Disks of pig skin having a diameter of 3 cm and a thickness of 600μm were placed between a donor compartment and a receptor compartment of a Franz cell (PermeGear). Tightness was ensures around the exposed skin area using a clamp and seal. The receptor compartment of the Franz cell was filled with the composition to be tested. The skin was calibrated for 15 minutes with its underside in contact with the receptor medium continuously stirred and maintained at 32°C. Receptor compartment volume was adjusted at 15 ml via the sampling port until the gauge line.

Test Solutions

Compositions of the ghrelin analogue H-Inp-D-Bal-D-Trp-Phe-Apc-NH2 were prepared as follows:

Solution 1 : 6.06mg of H-Inp-D-Bal-D-Trp-Phe-Apc-NH2 (equivalent to 5 mg of pure

API) + ImI NaCl 0.9%, to provide a solution of 5mg/ml.

Solution 2: 245.14mg of H-Inp-D-Bal-D-Trp-Phe-Apc-NH2 (equivalent to 203 mg of pure API) + ImI propylene glycol, to provide a solution of 5mg/ml.

Both preparations were mixed until clear solutions were obtained. The solutions were analysed by HPLC in order to determine the exact concntration applied to the skin.

Transdermal Permeation Study 177μl of a sample was applied topically to each skin membrane, equivalent to 100μl/cm². For solution 1 0.5mg/cm² is equivalent to 0.885 mg. For solution 2 20.3mg/cm² is equivalent to 35.93 mg.

500 μl samples were withdrawn from the receptor compartment at TO, 2h, 4h, 6h, 8h, 24h, 28h, 32h, and 48h and immediately replaced with fresh receptor medium. Samples were analysed using HPLC.

Results

HPLC analyses of donor solution laid on skins gave the following results: - solution 1 : 4.69±0.56 mg/ml

- solution 2: 160.36±7.08 mg/ml.

Cumulative permeated amounts of H-Inp-D-Bal-D-Trp-Phe-Apc-NH2 in the receptor fluid were calculated and the results shown in table 2: Table 2:

The quantity of the ghrelin analogue that has crossed the skin after 24h is important. Propylene glycol allows to apply a higher ghrelin analogue quantity under soluble form on the skin and consequently the permeated amount after 24h is much higher (28 fold higher) than NaCl as a vehicle.

Results

The results demonstrate that compositions of ghrelin and ghrelin analogues for topical administration are effective for transdermal permeation.

Claims

1. Use of an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof in the preparation of a medicament for administering to a mammal so as to stimulate its hair growth.

2. Use of an agent according to claim 1 whereas that agent is selected from a group consisting of amino acid sequences according SEQ ID No. 1 -5.

3. Use of an agent according to any of claims 1 or 2 whereas the agent is administered by an implanted osmotic pump in a therapeutically effective amount.

4. Use of an agent according to any of claims 1 or 2 whereas the agent is administered topically in a therapeutically effective amount.

5. Method for stimulating hair growth of a mammal comprising administering a therapeutically effective amount of an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof.

6. Method for stimulating hair growth of a mammal according to claim 5 comprising administering a therapeutically effective amount of an agent selected from the group consisting of amino acid sequences according SEQ ID No. 1-5.

7. Method for stimulating hair growth of a mammal according to claim 5 or 6 whereas the agent is administered by an implanted osmotic pump in a therapeutically effective amount.

8. Method for stimulating hair growth of a mammal according to claim 5 or 6 whereas the agent is topically administered in a therapeutically effective amount.

9. Method for stimulating hair growth of a mammal according to claim 8, wherein the method is a cosmetic method.

10. A pharmaceutical composition for stimulating hair growth comprising an agent selected from the group comprising ghrelin, an analogue thereof and a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, characterised in that the composition is suitable for topical administration.

11. A pharmaceutical composition according to claim 10, wherein said agent is selected from a group consisting of amino acid sequences according SEQ ID No. 1 -5.

12. A pharmaceutical composition according to claim 10, wherein said agent is H-Inp-D-

Bal-D-Trp-Phe-Apc-NH2.

13. A pharmaceutical composition according to claim 10, wherein said agent is (Aib2, Glu3(NH-hexyl))hGhrelin(1-28)-NH2.

14. A pharmaceutical composition according to claim 10, wherein said agent is (Lys5)hGhrelin(1-28)-NH2.

15. A pharmaceutical composition according to any one of claims 10 to 14, wherein said pharmaceutically acceptable carrier comprises a skin penetration enhancer.

16. A pharmaceutical composition according to claim 15, wherein said skin penetration enhancer is NMP.

17. A pharmaceutical composition according to claim 15, wherein said skin penetration enhancer is Propylene glycol.

18. Use of an agent selected from the group comprising ghrelin, an analogue thereof, or a pharmaceutically acceptable salt thereof for manufacturing a product for stimulating hair growth.

19. A cosmetic process for stimulating hair growth comprising topically administering an effective amount of an agent selected from the group consisting of ghrelin, an analogue thereof, or a pharmaceutically acceptable salt thereof to a mammal.