MXPA96006553A

MXPA96006553A - Composition for the stimulation of hair growth through complexes of peptide-co

Info

Publication number: MXPA96006553A
Application number: MXPA/A/1996/006553A
Authority: MX
Inventors: J Pallenberg Alexander; M Patt Leonard; E Trachy Ronald
Original assignee: Procyte Corporation
Priority date: 1994-06-17
Filing date: 1996-12-17
Publication date: 1997-12-01

Abstract

The present invention relates to peptide-copper complexes that stimulate hair growth in warm-blooded animals. In one aspect of this invention, the peptide-copper complexes are dipeptides or tripeptides chelated with copper in a molar ratio of about 1: 1 to 3: 1, with the second position of the peptide being from the amino terminus, histidine, arginine, or a derivative thereof. Peptide-copper complexes can be formulated for administration by, for example, local application or injection. Any affliction associated with hair loss, including hair loss associated with both androgenetic and secondary alopecia, can be treated with the peptide-copper complexes of this invention.

Description

STIMULUS OF THE GROWTH OF THE HAIR THROUGH COMPLEXES OF PEPTIDO-COPPER Technical Field This invention relates generally to peptide-copper complexes, and more specifically, to compositions containing peptide-copper complexes to stimulate hair growth.

Background of the Invention Hair loss is a prevailing affliction of many humans, the most common being androgenetic alopecia (AGA), where men lose scalp hair as they age (ie, male pattern baldness). ). Other afflictions of hair loss include alopecia areata (AA), female pattern baldness, and hair loss secondary to chemotherapy and / or radiation treatment (ie, secondary alopecia). Hair is usually divided into two types: "terminal" and "hair". The terminal hair is thick pigmented hair that leaves the follicles, which develop deep inside the dermis. Hair hairs are typically thin, non-pigmented hairs that grow from hair follicles that are smaller and superficially located in the dermis. As the alopecia progresses, there is a change from the terminal hair to the hair. Other changes that contribute to alopecia are alterations in the hair growth cycle. Hair typically progresses through three cycles, anagen (active hair growth), catagen (transition phase), and telogen (resting phase during which the hair shaft is housed before re-growth). As baldness progresses, there is a change in the percentages of hair follicles in each phase, changing most from anagen. The size of the hair follicles is also known to decrease while the total number remains relatively constant. A variety of procedures and drugs have been used in an attempt to treat hair loss. A common technique involves hair transplantation. Briefly, skin plugs containing hair are transplanted from the areas of the scalp where the hair was growing to the bald or balding areas of the scalp. However, this procedure is slow and relatively painful. Other approaches include ultraviolet radiation and exercise therapy. More recently, the stimulation of hair growth has been achieved, although with "limited success, through drug therapy." One of the most recognized hair growth agents, it is sold under the trade name "Minoxidil", as described in U.S. Patent No. 4,596,812 assigned to Upjohn, however, although the results generated through the use of minoxidil have seemed promising, there is still a need in the art for better compositions capable of stimulating hair growth. in warm-blooded animals For this purpose, peptide-copper complexes which are effective hair growth agents have been discovered.For example, U.S. Patent Nos. 5,177,061; 5,120,831 and 5,214,032 describe certain peptide complexes. -bread that are effective to stimulate hair growth in warm-blooded animals. In the stimulation of hair growth by drug treatment, there is still a need in the art for compounds that have a greater stimulating effect on hair growth. The present invention satisfies this need, while also providing other related advantages.

SUMMARY OF THE INVENTION Briefly stated, the present invention relates to peptide-copper complexes, and to compositions containing them, to stimulate hair growth in warm-blooded animals. The compositions of this invention include one or more peptide-copper complexes in combination with an acceptable carrier or diluent. As used in this, the term "copper" is used to designate copper (II) (ie, Cu + 2). The peptide-copper complexes of this invention are administered to an animal in need, in a manner that results in the application of an effective amount of the peptide-copper complex. As used herein, the peptide "effective amount" means an amount of the peptide-copper complex that stimulates hair growth associated with a hair loss affliction (such as male pattern baldness), or caused by a aggression of hair loss (such as radiation or chemotherapy). Accordingly, the peptide-copper complexes can be used prophylactically, as well as therapeutically and cosmetically. Administration of the peptide-copper complexes is preferably by local application, although other means of administration may be employed, such as injection (eg, intramuscular, intravenous, subcutaneous, and intradermal). Typically, the peptide-copper complexes of this invention are formulated as a solution, cream, or gel for local application, or as a solution for injection, and include one or more acceptable carriers or diluents.

As used herein, the term "peptide-copper complex" means a peptide having at least two amino acids (or amino acid derivatives) chelated with copper, wherein the second amino acid from the amino terminus of the peptide is histidine. , arginine, or a derivative thereof. These peptide-copper complexes have the following general structure A: A: [R-L-P ^]: copper (II) wherein: R-L is an amino acid or an amino acid derivative; Y R2 is histidine, arginine, or a derivative thereof. The peptide-copper complexes of this invention have a ratio of the peptide to copper of about 1: 1 to about 3: 1, and more preferably from about 1: 1 to about 2: 1. In summary, a peptide component occupies at least one copper ion coordination site, and multiple peptides can be chelated with a single copper ion. In a preferred embodiment, the copper peptide complex comprises an additional chemical moiety linked to the fraction R2 of structure A via an amide or peptide linkage. (That is, -C (= 0) NH-). In this embodiment, the peptide-copper complex has the following structure B: B: [R-L-R2-R3]: copper (II) where: R? is an amino acid or an amino acid derivative; R2 is histidine, arginine, or a derivative thereof; and R3 is a chemical moiety linked to R2 by an amide bond. In a further preferred embodiment, R3 of structure B is at least one amino acid linked to R2 by a peptide bond. In this embodiment, the peptide-copper complex has the following structure C: C: [R1-R2-R3]: copper (II) wherein: R-L is an amino acid or an amino acid derivative; R2 is histidine, arginine, or a derivative thereof; and R3 is an amino acid or an amino acid derivative linked to R2 by a peptide bond, with the proviso that R- is not glycyl, alanyl, seryl, or vally when R2 is histidyl or (3-methyl) histidyl and R3 is lysine, lysyl-propyl-va lily-phenol lalan i lo-va 1 ina, 1 is i lo-val i lo-phenylalanyl-valine, lysyl-tryptophan, or lysyl- (glycyl) 1_2-tryptophan, and with the additional condition that RL is not lysyl when R is histidyl or (3-methyl) histidyl and R3 is glycine, glycyl-propyl-varyl-phenylalanyl-valine, glycyl-valyl-phenylalanyl-valine, glycyl-tryptophan, or glycyl- (glycyl) 1_2-tryptophan. In yet a further embodiment of the present invention, an additional chelating agent can be added to the peptide-copper complexes described above, to form a ternary complex of peptide-copper chelating agent. Other aspects of the present invention will become apparent upon a reference to the following detailed description.

Detailed Description of the Invention This invention relates to peptide-copper complexes that stimulate hair growth in warm-blooded animals. These complexes are typically administered as a composition containing acceptable diluents and / or vehicles. The administration of preference is by local application directly to the area where stimulation of hair growth is desired, such as the scalp, although other routes of administration may be employed.

The peptide-copper complexes of this invention can be used to stimulate hair growth in animals (including humans) afflicted with androgenetic alopecia (AGA). Animals afflicted with this condition are usually males, and the condition results in the loss of the scalp with age (also called "male pattern baldness"). Accordingly, the peptide-copper complexes can be administered in order to stimulate hair growth, thus eliminating or reducing the severity of hair loss and / or the rate at which androgenetic alopecia progresses. Other afflictions of hair loss include alopecia areata (AA), female pattern baldness, and hair loss secondary to treatment with chemotherapy and / or radiation (ie, secondary alopecia). In the case of secondary alopecia, the peptide-copper complexes can be used in advance of certain hair loss aggressions, such as chemotherapy or radiation regimens, to stimulate hair growth before aggression, and in this way reduce the amount of hair loss resulting from the same. As mentioned above, the peptide-copper complexes of the present invention have at least two amino acids (or amino acid derivatives), one of which is histidine, arginine, or a derivative thereof. In this context, the peptide-copper complexes have structure A as identified above. For example, when Rx is an amino acid and R is histidyl, or when R? is an amino acid and R2 is arginine, the peptide-copper complex has the following structures D and E, respectively: D: [(amino acid) -histidine]: copper (II) E: [(amino acid) -arginine]: copper (II) As used in structure A above, the terms "amino acid" and "amino acid derivative" are defined hereinafter. An amino acid of this invention includes any carboxylic acid having an amino moiety including (but not limited to) the naturally occurring a-amino acids (in the following listing, the single-letter amino acid designations are given in parentheses): alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I) ), leucine (L), plant (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine ( V). Other naturally occurring amino acids include (but are not limited to) hydroxyproline and β-carboxyglutamate. In a preferred embodiment, the amino acid is a naturally occurring a-amino acid, which has an amino moiety (ie, the -NH2 group, rather than a secondary amine, -NH-, such as is present in the proline. (attached to the α-carbon of the amino acid which, when quenched with copper, occupies a coordination site thereof) As used herein, "hydrophilic amino acids" include (but are not limited to) amino acids selected from K , R, H, D, E, N, Q, C, M, S, and T. An amino acid derivative of this invention includes any compound having the structure: wherein R is a derivative of an amino acid side chain that occurs naturally. In one embodiment, R1 and R "in the above structure, a saturated or unsaturated, branched or cyclic, straight-chain substituted or unsubstituted alkyl fraction containing from 1 to 20 carbon atoms may be selected from hydrogen. , and a substituted or unsubstituted aryl fraction containing from 6 to 20 carbon atoms (including heteroarytic fractions) In a preferred embodiment, R1 and R "ee may be selected from the chemical fractions identified in the following Table 1 .

Table 1 Amino Acid Derivatives NH-CH-COOH 5 I | R 'R " where R "= H or the following fractions: 10 - (CH2) nCH3 where n = 1-20 - (CH2) nCH (CH3) (CH2) mCH3 where nm = 0-20 (where n = 0, m? O or l, and when n = l, m? 0) - (CH2) nNH2 where n = 1-20 (n? 4) - (CH2) nCONH2 where n = 3-20 15 - (CH2) nCOOH where n = 3-20 ~ (CH2) ñ? Q) where n = 2-20 - (CH2)? / 0H at don = 2-20 where n = 2-20 - (CH2) nSH where n = 2-20 - (CH2) nS (CH2) mCH3 where n, m = 1-20 (where n = 2, m? 0) - (CH2) nCH2OH where n = 1-20 25 - (CH2) nCH (CH3) OH where = 1-20 Y where Rj = H or the following fractions: - (CH2) nCH3 where n = 0-20 - (CH2) nCH (CH3) (CH2) mCH3 where n, = 0-20 The histidine derivatives of this invention include compounds having the structure: wherein n = 1-20, and X and Y are independently selected from the alkyl fractions containing from 1 to 12 carbon atoms, or an aryl fraction containing from 6 to 12 carbon atoms. In the preferred embodiments, n is 1, X is methyl, and Y is H (ie, 3-methylhistidyl), or X is H and Y is methyl (ie, 5-methylhistidine). In a similar manner, the arginine derivatives of this invention include the compounds having the structure: where n = 1-20 (excluding n = 3).

In another embodiment of this invention, the peptide-copper complexes of structure A, further comprise a chemical moiety linked to the R2 moiety by an amide or peptide bond (ie, -C (= 0) NH-). The peptide-copper complexes of this embodiment are illustrated above as structure B. As used herein, a chemical moiety (ie, R3) linked to the R2 moiety by an amide bond, includes any chemical moiety that has an amino group capable of forming an amide bond with the carboxyl terminus of R2 (i.e., the carboxyl terminus of histidine, arginine, or derivatives thereof). Suitable R3 fractions include (but are not limited to) -NH2, alkylamino fractions having from 1 to 20 carbon atoms, and arylamino fractions having from 6 to 20 carbon atoms, as well as amino acids and their derivatives. As used herein, "alkylamino moieties" include alkyl moieties that contain an amino moiety, wherein the alkyl moiety is as defined above, and includes (but is not limited to) octyl amine and amine propyl In a similar manner, "arylamin fractions" include the aryl moieties that contain an amino moiety, wherein the aryl moiety is as defined above, and includes (but is not limited to) benzyl amine and (CH2) ? _i4 Benzyl amine. Other examples of suitable chemical moieties having amino groups capable of forming an amide bond with the carboxyl terminus of R2 include the polyamines, such as spermine and sperimidine. For example, in structure B, when R- is an amino acid, R2 is histidine or arginine, and R3 is an amino fraction, the peptide-copper complex has the following structures F and G, respectively: F: [(amino acid) -histidine-NH2]: copper (II) G: [(amino acid) -arginine-NH2]: copper (II) In a similar manner, when R1 is an amino acid, R2 is histidine or arginine, and R3 is an alkylamino moiety, the peptide-copper complex has the following structures H and I, respectively: H: [(amino acid) -histidine- NH-alkyl] -copper (II) I: [(amino acid) -arginine-NH-alkyl]: copper (II) In still a further embodiment (as represented by structure C above), the fraction R3 of structure B is at least one amino acid or an amino acid derivative as defined above. In a preferred embodiment, R3 is an α-amino acid that occurs naturally bound to R2 by a peptide bond. For example, when R? and R2 of structure C are amino acids, and R2 is histidine or arginine, the peptide-copper complexes of this invention have the following structures J and K, respectively: I: [(amino acid) -histidine- (amino acid)]: copper (II) K: [(amino acid) -arginine- (amino acid)]: copper (II) It should be understood that, although only one amino acid is illustrated in the R3 position of structures H and I, other chemical fractions, including additional amino acids and / or amino acid derivatives, may also be present. For example, R3 in structures H and I may be a peptide such as phenylalanine-phenylalanine (glycyl) n-tryptophan where n = 1-4, prolyl-XL-phenylalanyl-X2, or X? -phenylalanyl-X2, where X? and X2 are selected from valine, alanine, and glycine. Peptides of the peptide-copper complexes of this invention can be generally classified as dipeptides (ie, structure A), dipeptides with a chemical moiety linked to the carboxyl terminus via an amide bond (ie, structure B), or as tripeptides (ie, 'structure C above). In the case of the peptide complexes ido-c? Bib e of structures D and C, additional chemical fractions, including amino acids, can be bound with the dipeptide or tripeptide, to produce peptides containing four or more amino acids. For purposes of illustration, Table 2 presents different representative examples of peptide-copper complexes of this invention.

Table 2 Representative Peptide-Copper Complexes Structure A: glycyl-histidine: copper alanyl-his idine: copper glieyl- (3-methyl) histidine: copper alanyl- (3-methy1) histidine: copper glycyl- (5-methyl) histidine : copper alanyl- (5-methyl) histidine: copper glycyl-arginine: copper alanyl-arginine: copper (N-methyl) glycine-histidine: copper (N-methyl) glycine-arginine: copper Structure B; glycyl-histidyl-NH2: copper glycol-arginyl-NH2: copper glycyl- (3-methyl) histidyl-NH2: copper alanyl- (3-methylJhistidyl-NH ,: cobr glycyl-arginyl-NH- ,: copper alanyl-arginyl- NH- ,: copper (N-methyl) glycine-histidyl-NH-: copper (N-methyl) glycine-arginyl-NH2: cobr glycyl-histidyl-NHoctyl: copper glycyl-NHoctyl: copper Structure C; glycyl-histidyl-lysine : glycolic-arginyl-lysine copper: glycyl- (3-methyl) histidyl-lysine copper: glycyl- (5-methyl) histidyl-lysine: cob alanyl-histidyl-lysine: copper alanyl-arginyl-lysine: copper alanyl- ( 3-methyl) histidyl-lysine: copper alanyl- (5-methyl) histidyl-lysine: cob glycyl-histidyl-phenylalanine: copper glycyl-arginyl-phenylalanine: copper glycyl- (3-methyl) histidyl-glycyl- (5-methyl) ) his idilo-phenylalanine: phenylalanine copper: alañilo-histidyl-phenylalanine copper: alanyl-arginyl-phenylalanine copper: glycyl- (3-methyl) histidyl-glycyl- (5-methyl) histidyl-phenylalanine: copper phenylalanine: copper glyc ilo-histidyl-lysyl-phenylalanyl-glj.cilo-arginyl-lysyl-phenylalanyl phenylalanyl: copper phenylalanyl: copper glycyl- (3-methyl) histidyl-lysyl-glycyl- (5-methyl) histidyl-lysyl-phenylalanyl-phenylalanyl: copper phenyl lanyl-phenylalanyl: copper (N-methyl) glycyl-histidyl-lysine: copper (N-methyl) glycyl-arginyl-lysine: co Other examples of the peptide-copper complexes of this invention are described in U.S. Patent Nos. 5,118,665 and 5,164,367, as well as in U.S. Patent Nos. 4,760,051; 4,665,054; 4,877,770; 5,177,061; 4,810,693; 4,767,753; 5,135,913; 5,023,237; 5,059,588, and 5,120,831, all of which are incorporated herein by reference in their entirety. Accordingly, the peptide-copper complexes described in the patents of the prior United States of America, can be used to stimulate hair growth in animals (including humans) afflicted with androgenetic alopecia (AGA) or male pattern baldness, eliminating in this way or reducing the severity of hair loss and / or the rate at which androgenetic alopecia progresses. These peptide-copper complexes can also be used to treat other hair loss afflictions, including alopecia areata, female pattern baldness, and hair loss secondary to treatment by chemotherapy and / or radiation (ie, secondary alopecia). In the case of secondary alopecia, the peptide-copper complexes can be used to stimulate hair growth before an assault that normally results in hair loss, such as chemotherapy or radiation regimens. Accordingly, the peptide-copper complexes of this invention can be used to prevent hair loss. In the practice of this invention, the molar ratio of the peptide to copper is greater than 0 to 1 (eg, 0.1: 1, 0.2: 1, etc.). The molar ratio of the peptide to copper will depend, in part, on the number of copper coordination sites that are occupied by the peptide. In a preferred embodiment, the molar ratio of the peptide to copper is from about 1: 1 to 3: 1, and more preferably from about 1: 1 to 2: 1. For example, in the case of a tripeptide (such as GHF: copper), the preferred ratio of the peptide to copper is from 1: 1 to 2: 1, with each tripeptide occupying three copper coordination sites. In a similar manner, with a dipeptide (such as GH: copper), the preferred ratio of the peptide to copper is from 1: 1 to 3: 1, with each dipeptide occupying two coordination sites of the copper ion. In another embodiment of this invention, a chelating agent can be added to the peptide-copper complex to form a ternary complex of peptide-copper chelating agent. Suitable chelating agents include imidazole or imidazole-containing compounds, such as histidine, and sulfur-containing amino acids, such as cysteine or methionine. Accordingly, if the peptide-copper complex is GHF: copper, histidine may be added to produce the ternary complex of GHF: copper: histidine. However, to form this ternary complex, the molar ratio of the copper to the peptide to the chelating agent must be considered. For example, if the ratio of the peptide to copper is 2: 1, the addition of a chelating agent to the peptide-copper complex, although possible, is difficult, due to the site's occupation by the peptide. However, by maintaining the ratio of the peptide to copper about 1: 1, a chelator group can easily be added to form the ternary group. Accordingly, the preferred ratio of the peptide to copper to the chelating agent is about 1: 1: 1. Although the chiral amino acids of the present invention (particularly amino acids) have not been specifically designated, the present invention encompasses both the naturally occurring L form and the D form. For example, any of the naturally occurring L-amino acids (or amino acid derivatives) described herein, can be replaced by a corresponding D-amino acid (or amino acid derivative). In the practice of this invention, it is critical that the second position of the peptide (ie, R of structures A, B, and C) be histidine, arginine, or a derivative thereof. It is believed that the superior effect of the peptide-copper complexes of the present invention is achieved, at least in part, by the binding of copper by an amino fraction of the amino acid side chain of histidine., arginine, or derivative thereof. For example, in the case of histidine, an amine group of the imidazole ring of histidine occupies a copper coordination site (ie, residual valences or non-shared electrons of the amine group are shared with copper). In the case of arginine, an amine group of the amino acid side chain similarly occupies a copper coordination site. The binding of R2 to the copper atom is preferably combined with the coordination of an amine group from the fraction R? of structures A, B >; And C, to produce the peptide-copper complex. Accordingly, a peptide of this invention chelates copper by donating the amine group R2, and preferably both amine groups R1 and R2, to the peptide-copper complex. The peptide-copper complexes of structures B and C can also occupy additional coordination sites on copper. Specifically, the amine group of the amide bond of structure B, and the peptide bond of structure C, can still occupy additional coordination sites. As mentioned above, the peptide-copper complexes of this invention have utility as hair growth agents. More particularly, the peptide-copper complexes stimulate hair growth in warm-blooded animals. Accordingly, peptide-copper complexes can be used to treat a variety of disease states associated with hair loss, including (but not limited to) androgenetic alopecia (also known as male pattern baldness), alopecia areata, and Female pattern baldness. In these cases, the peptide-copper complexes stimulate hair growth after the establishment of hair loss application. Alternatively, the peptide-copper complexes can be administered prophylactically for conditions such as secondary alopecia. For example, the complexes can be administered before an assault that normally results in hair loss, such as a treatment with chemotherapy and / or radiation. Accordingly, the peptide-copper complexes of this invention can be used to prevent hair loss. The administration of the peptide-copper complexes of the present invention can be carried out in any manner that results in the application of an effective amount or dose of the peptide-copper complex to the animal, including application to the hair follicles. For example, administration can be by local application directly to the scalp, or to another area where hair stimulation is desired (subsequently in the present "treatment area"). Alternatively, administration can also be performed by injection (such as intradermal injection) into the treatment area, including the scalp. Typically, the peptide-copper complexes are formulated as a composition containing the peptide-copper complex in combination with one or more acceptable carriers or diluents, including formulations that provide sustained release of the peptide-copper complexes over time. . In one embodiment, the peptide-copper complexes are formulated for intradermal injection to the treatment area. In those cases, the formulations preferably contain one or more peptide-copper complexes of this invention in combination with a vehicle suitable for intradermal injection, the peptide-copper complex being present in the composition in a concentration of 10 micrograms to 2000 micrograms per 0.1 milliliter of vehicle (ie 1.0 milligram / milliliter to 20 milligram / ililiter). Vehicles suitable for intradermal injection include (but are not limited to) serum and sterile water. In another embodiment, the peptide-copper complexes are formulated for local administration. Suitable local formulations include one or more peptide-copper complexes in the form of a liquid, lotion, cream, and / or gel. Local administration can be done by applying directly to the treatment area. For example, this application can be performed by rubbing the formulation (such as a lotion or gel) on the skin of the treatment area, or by spray application of a liquid formulation on the treatment area. Any amount of local formulation sufficient to accelerate the rate of hair growth, or to prevent the subsequent loss of hair, is effective, and the treatment can be repeated as frequently as indicated by the progress of hair growth. Preferably, the local compositions of this invention contain one or more peptide-copper complexes in an amount of 0.1 percent to 20 percent by weight of the composition, and more preferably from 0.1 percent to 5 percent by weight of the composition. In addition to the carriers and diluents, the peptide-copper complexes may also be formulated to contain additional ingredients, such as penetration enhancing agents and / or surface active agents. For example, local formulations may contain 0.5 percent to 10 percent of one or more agents of superlicial activity (also caller; aqonto. emulsifiers). The non-ionic surface active agents and the ionic surface active agents can be used for the purposes of the present invention. Examples of suitable non-ionic surface active agents are nonylphenoxypolyethoxyethanol (Nonoxynol-9), polyoxyethylene oleyl ether (Brij-97), different polyoxyethylene ethers (Tritons), and block copolymers of ethylene oxide and propylene oxide. different molecular weights (such as Pluronics 68). Examples of suitable ionic surface active agents include sodium lauryl sulfate and similar compounds. Penetration enhancing agents may also be present in the local formulations. Suitable penetration enhancing agents include dimethyl sulfoxide (DMSO), urea, and substituted urea compounds. In the case of a liquid formulation for local administration, the concentration of the penetration enhancing agent (such as DMSO) may be from 30 percent to 80 percent of the liquid formulation. The rest of the local formulations may include inert, physiologically acceptable carriers or diluents. Suitable carriers or diluents include, but are not limited to, water, saline, bacteriostatic serum (serum containing 0.9 milligrams / milliliter 'of benzyl alcohol), petrolatum-based creams (eg, hydrophilic ointment USP and similar creams, Unibase , Par e-Davis, for example), different types of pharmaceutically acceptable gels, and short chain alcohols and glycols (for example, ethyl alcohol and propylene glycol). In another embodiment of the invention, local formulations may also contain the peptide-copper complex encapsulated in liposomes to aid in the application of the peptide-copper complex to the hair follicle. Alternatively, the peptide-copper complex can be formulated in an instrument to apply the compound by means of iontophoresis. The peptide-copper complexes of this invention exhibit superior skin permeability when applied locally. This results in a higher effective dose to the treatment area, and consequently, a correspondingly greater stimulus of hair growth. In the practice of this invention, amino acids or hydrophobic amino acid derivatives are preferably used for administration by injection (such as intradermal injection), while amino acids and hydrophilic amino acid derivatives are used for local administration, although the use of amino acids or Hydrophobic amino acid derivatives generally improve the activity of the copper-peptide complexes of this invention, the use of amino acids or hydrophilic amino acid derivatives is preferred for local administration, due to the improved permeability of the skin associated with them. For purposes of illustration, Table 3 presents examples of suitable local formulations within the context of the present invention. As used below, "% (w / w)" represents the weight percentage of a component, based on the total weight of the formulation: Table 3 Representative Local Formulations Preparation A; Peptide-copper complex 1.0% (weight / weight) Hydroxyethyl cellulose 3.0% (weight / weight) Propylene glycol 20.0% (w / w) Nonoxynol-9 3.0% (weight / weight) Benzyl alcohol 2.0% (weight / weight) Aqueous phosphate regulator (0.2N) 71.0% (weight / weight) Preparation B; Peptide-copper complex 1.0% (weight / pee) Nonoxynol-9 3.0% (pee / weight) Ethyl alcohol 96.0% (w / w) Preparation C; Peptide-copper complex 5.0% (weight / weight) Ethyl alcohol 47.5% (weight / weight) Isopropyl alcohol 4.0% (w / w) Propylene glycol 20.0% (w / w) Lanoeth-4 1.0% (weight / peeo) Water 27.5% (peeo / peeo) Preparation D; Peptide-copper complex 5.0% (w / w) Eetéril water 95.0% (w / w) Preparation E: Peptide-copper complex 2.5% (peeo / peeo Hydroxypropyl cellulose 2.0% (w / w) Glycerin 20.0% (weight / weight Nonoxynol-9 3.0% (w / w) Sterile water 72.5% (weight / weight Preparation F) Peptide-copper complex 0.5% (weight / weight Sterile water 16.5% (weight / weight) Propylene glycol 50.0% (w / w) Ethanol 30.0% (weight / weight Nonoxynol-9 3.0% (weight / weight) Preparation G: Peptide-copper complex 5.0% (w / w) Sterile water 10.0% (w / w) Hydroxypropyl cellulose 2.0% (weight / pee Propylene glycol 30.0% (weight / peeo Ethanol 50.0% (weight / weight Nonoxynol-9 3.0% (pee / weight) The peptides of the present invention can be synthesized by solid phase or solution phase techniques known to those skilled in the art of peptide synthesis. The general procedure involves the stepwise addition of protected amino acids to construct the desired peptide sequence. The resulting peptide can then be complexed with copper (in the desired molar ratio of the peptide to copper) by dissolving the peptide in water, followed by the addition of copper chloride, and adjusting the pH. A more detailed description directed to the synthesis of the peptide-copper complexes of this invention, as well as the activity of certain representative peptide-copper complexes, is presented below.

EXAMPLES The following examples are offered by way of illustration, and not by way of limitation. To summarize the examples that follow, Example 1 describes the general preparation of peptide-copper complexes of the present invention, by chelating a peptide with copper in an aqueous solution. Examples 2 to 10 describe the synthesis of peptides that can be chelated with copper to produce peptide-copper complexes. Examples 11 to 16 describe the ability of the representative peptide-copper complexes of this invention to stimulate hair growth.

Source of Chemical Products The peptide intermediate and peptide products used in the following examples can be purchased from a number of suppliers, including: Sigma Chemical 'Co., St. Louie Missouri; Peninsula Laboratories, San Carlos, California; Aldrich Chemical Company, Milwaukee, Wisconsin, Vega Biochemicals, Tucson, Arizona; Pierce Chemical Co., Rockford, Illinois; Research Biochemical, Cleveland, Ohio; Van Waters and Rogers, South San Francisco, California; and Bachen, Inc., Torrance, California.

Example 1 Preparation of Eptide-Copper Complex The peptide-copper complexes of the present invention can be synthesized by dissolving the peptide in distilled water, followed by the addition of copper chloride (eg, 99.999 percent, available from Chemical Dynamics, NJ), and then the pH of the solution is adjusted to about 7.0. For example, glycyl-L-hietidyl-L-phenylalanine copper complexes (GHF) with a molar ratio of the peptide to copper of 1: 1, 2: 1, or greater (eg, 3: 1), ee may prepare by dissolving a given weight of GHF in distilled water (e.g., 50 milligrams / milliliter), and adding the desired molar amount of purified copper chloride. The pH of the resulting peptide solution is then adjusted to about 7.0 by the addition of, for example, a sodium hydroxide solution. Alternatively, copper salts other than copper chloride may be used, for example, copper acetate, copper sulfate, or copper nitrate.

Example 2 Synthesis of Glycyl-L-Histidyl-L-Caprolactam L (-) - 3-amino-e-caprolactam was dissolved in tetrahydrofuran (THF), and then coupled with Na-t-butyloxycarbonyl-N? M-benzyloxycarbonyl- L-histidine (Na-BOC-N? M-CBZ-L-histidine) using isobutyl chloroformate and N-methyl morpholine in tetrahydrofuran. After 2 hours at -20 ° C, and an additional 1 hour at room temperature, the reaction was quenched with 2N aqueous potassium bicarbonate. This product was extracted into ethyl acetate, washed with 1M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave Na-BOC-N? M-CBZ-L-histidyl-L-caprolactam. The above compound was diered in 30 percent trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated to form N? M-benzyloxycarbonyl-L-histidyl-L-caprolactam. This was then dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methyl morpholine, and benzyloxycarbonyl-glycine were added, to form benzyloxycarbonyl-glycyl-N? M-benzyloxycarbonyl-L-histidyl-L-caprolactam. This product was recrystallized once from ethyl acetate, and then dissolved in acetic acid, and hydrogenated overnight in the presence of a 10-percent Pd catalyst. The resulting glycidyl-L-histidyl-L-caprolactam was lyophilized from water several times, then purified by liquid chromatography on a C-18 reverse phase column, to give the peptide as a diacetate salt.

Example 3 Synthesis of L-Alanyl-L-Hlstidyl-L-Phenylalanine To a stirred solution of Na-BOC-N? M-CBZ-L-histidine (9.74 grams, 25.0 mmol) and N-methyl morpholine (5.8 milliliters, 5.3 grams, 52.5 mmol) in tetrahydrofuran (50 milliliters) at -15 ° C, was added isobutyl chloroformate (3.4 milliliters, 3-6 grams, 26.3 millimoles). After 2 minutes, phenylalanine benzyl ester tosylate (10.7 grams, 25.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters) water, and brine. The resulting solution was dried over sodium eulfate, filtered, and evaporated to yield 13.7 grams (87 percent) of the blocked dipeptide as a white semisolid. { Rj = 0.75, 10 percent methanol / dichloromethane), which was used in the next transformation without further purification. A solution of the tertiary-butyl-carbonyl protected dipeptide (12.9 grams, 20.6 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potaeium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 13.3 grams (approximately 100 percent highest solvent) of the free amino compound as a white solid: Rf = 0.49 (10 percent methanol / dichloromethane) . To a stirred solution of N-CBZ-L-alanine (6.03 grams, 27.0 mmol) and N-methyl morpholine (3.3 milliliters, 3.0 grams, 29.7 mmol) in tetrahydrofuran (50 milliliters) at -15 ° C, chloroformate was added. of isobutyl (3.7 milliliters, 3.9 grams, 28.4 'millimoles). After 2 minutes, a solution of the suitably protected dipeptide (11.4 grams, 21.8 millimoles) in tetrahydrofuran (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 100 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, KHC03 2M (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give the blocked tripeptide as a white solid. { Rf = 0.55, 10 percent methanol / dichloromethane), which was recrystallized from 95 percent ethanol to give 12.6 grams (79 percent) of a free flowing white powder: p.f. 147-147.5 ° C; Analysis calculated for C41H40N508: C, 67.39; H, 5.52; N, 9.58. Found: C, 66.78; H, 5.64; N, 9.24. At a suspension of the blocked tripeptide (12.6 grams, 17.6 millimoles) in ethanol (150 milliliters), water was added, until the mixture became very cloudy (approximately 150 milliliters). The resulting mixture was stirred with palladium chloride (1.56 grams, 8.8 mmol) under an atmosphere of hydrogen (5 atm) for 16 hours. The catalyst was removed by filtration through a plug of Celite® and the filtrate was concentrated to remove volatile organic matter. The rest was lyophilized to give 8.30 grams of a white powder. This material was dissolved in water, filtered through a 0.2M nylon membrane, and lyophilized to give 6.27 grams (87 percent) of the desired tripeptide dihydrochloride as a free-flowing white powder: [a] D 5.1 ° (c 2.0, water); XH NMR (500 MHz, DMS0-D6) d 8.71 (1H, d, J = 7.9), 8.49 (1H, d, J = 7.8), 8.21 (1H, e), 7.30-7.22 (4H, m), 7.20. -7.15 (1H, m), 7.12 (1H, s), 4.54 (1H, br q, J = 7.1), 4.37 (1H, m), 3.86 (1H, q, J = 6.8), 3.12 (1H, dd , J = 4.3, 13.8), 3.05-2.90 (2H, m), 2.88 (1H, dd, J = 9.5, 13.8), 1.27 (3H, d, J = 6.8); 13 C NMR (125 MHz, DMSO-d 6) d 173.5, 169.9, 169.5, 138.1, 134.2, 130.5, 129.2, 128.2, 126.4, 117.8, 54.4, 52.5, 48.0, 36.8, 28.5, 17.2.

EXAMPLE 4 Synthesis of Glycyl-L-Histidyl-L-Glutamic Acid To a stirred solution of Na-BOC-N? M-CBZ-L-histidine (9.74 grams, 25.0 milliliters) and N-methyl morpholine (5.8 milliliters, 5.3 grams, 52.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, was added isobutyl chloroformate (3.4 milliliters, 3.6 grams, 26.3 millimoles). After 2 minutes, dibenzyl ester tosylate of glutamic acid (12.5 grams, 15.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.2 grams (87 percent) of the blocked dipeptide as a white semisolid (Rf = 0.74, 10 percent methanol / dichloromethane), which was used in the next transformation without further purification. A solution of the tertiary-butyl-carbonyl protected dipeptide (15.1 grams, 21.6 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over eodium eulfate, filtered, and evaporated to give 14.8 grams (about 100 percent plus solvent pooled) of the free amino compound as a white solid: Rf = 0.48 (10 percent methanol / dichloromethane) . To a stirred solution of N-CBZ-glycine (5.23 grams, 25.0 mmol) and N-methyl morpholine (3.0 milliliters, 2.8 grams, 27.5 mmol) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate was added. (3.4 milliliters, 3.6 grams, 26.3 millimoles). After 2 minutes, an adequately protected dipeptide (12.9 gram, 21.6 mmol) in tetrahydrofuran (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 100 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and concentrated to a syrup, which was diluted with absolute ethanol, and kept overnight at -20 ° C. The resulting precipitate was collected on a filter to provide 9.93 grams (58 percent) of the blocked tripeptide as a white solid. { Rf = 0.58, 10 percent methanol / dichloromethane): m.p. 114-116 ° C. Analisis calculated for C43H43N5010: C, 65.39; H, 5.49; N, 8.87. Found: C, 64.93; H, 5.56; N, 8.41. At a suspension of the blocked tripeptide (9.6 grams, 12.2 mmol) in ethanol (150 milliliters), water was added, until the mixture became very cloudy (approximately 150 milliliters). The resulting mixture was stirred with palladium chloride (2.22 grams, 12.5 milliliters) under a hydrogen atmosphere (5 atmospheres) for 16 hours. The catalyst was removed by filtration through a plug of Celite®, and the filtrate was concentrated to remove volatile organic matter. The rest was lyophilized to give 4.72 grams of a white powder. This material was dissolved in water, filtered through a 0.2M nylon membrane, and lyophilized to give 4.64 grams (93 percent) of the desired tripeptide dihydrochloride as a free flowing white powder: [a] D -16.6 ° (c 2.0, water); H NMR (500 MHz, D20) d 8.65 (1H, s), 7.35 (1H, s), 4.77 (1H, s), 4.46 (1H, m), 3.88 (2H, s), 3.28 (1H, dd) , J = 15.3, 6.1), 3.21 (1H, dd, J = 15.3, 8.0), 2.47 (2H, m), 2.21 (2H, m), 2.00 (2H, m); 13 C NMR (125 MHz, D 20) d 179.9, 177.3, 174.3, 169.8, 136.5, 130.8, 120.4, 55.6, 54.9, 43.3, 32.8, 29.3, 28.5; Analysis calculated for C13H21C12N506: C, 37.69; H, 5.11; N, 16.91; Cl, 17.12. Found: C, 37.23; H, 5.07; N, 16.01; Cl, 17.95.

Example 5 Synthesis of Glycyl-L-Histidyl-L-Phenylalanine To a stirred solution of Na-BOC-N? M-CBZ-L-histidine (9.74 grams, 25.0 mmol / L) and N-methyl morpholine (5.8 milliliters, 5.3 grams, 52.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate (3.4 milliliters, 3.6 grams, 26.3 millimoles) was added. After 2 minutes, phenylalanine benzyl ester tosylate (10.7 grams, 25.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 13.0 grams (83 percent of the blocked dipeptide as a white semisolid { Rf = 0.79, 10 percent methanol / dichloromethane), which used in the next transformation without further purification. A solution of the tertiary-butyl-carbonyl protected dipeptide (12.9 grams, 20.6 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo, and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 12.3 grams (about 100 percent plus solvent pooled) of the free amino compound as a white solid: Rf = 0.50 (10 percent methanol / dichloromethane) . To a stirred solution of N-CBZ-glycine (5.23 grams, 25.0 mmol) and N-methyl morpholine (3.0 milliliters, 2.8 grams, 27.5 mmol) in tetrahydrofuran (50 milliliters) at -15 ° C, was added chloroformate. isobutyl (3.4 milliliters, 3.6 grams, 26.3 millimoles). After 2 minutes, a solution of the suitably protected dipeptide (10.8 grams, 20.6 millimoles) in tetrahydrofuran (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 100 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 14.0 grams (95 percent) of the blocked tripeptide as a white solid. { Rf = 0.64, 10 percent methanol / dichloromethane), which was recrystallized from absolute ethanol to give a free flowing white powder. To a suspension of the blocked tripeptide (6.0 grams, 8.3 mmol) in ethanol (150 milliliters), water was added, until the mixture became very cloudy (approximately 150 milliliters). The resulting mixture was stirred with palladium chloride (1.47 grams, 8.3 mmol) under an atmosphere of hydrogen (5 atm) for 16 hours. The catalyst was removed by filtration through a plug of Celite®, and the filtrate was concentrated to remove the volatile organic materials. The rest was lyophilized to give 1.46 grams of a white powder. This material was dissolved in water, filtered through a 0.2M nylon membrane, and lyophilized to give 1.38 grams (38 percent) of desired tripeptide dihydrochloride as a free flowing white powder: [a) D -7.5 ° (c 1.0, water); H NMR (500 MHz, D20) d 8.59 (1H, s), 7.39-7.25 (5H, m), 7.21 (1H, s), 4.70 (1H, t, J = 7), 3.80 (2H, s) , 3.24 (1H, dd, J = 14.0, 5.5), 3.16 (1H, dd, J = 15.4, 6.9), 3.10 (1H, dd, J = 15.4, 7.4), 3.03 (1H, dd, J = 14.0, 9.1); 13C NMR (125 MHz, DMS0-d6) d 172.7, 169.5, 166.0, 137.6, 133.3, 129.2, 128.9, 128.3, 126.5, 116.8, 53.9, 51.8, 40.1, 36.4, 27.3.

Example 6 Synthesis of Glycyl-L-Histidyl-L-Lysyl-L-Phenylalanine To a stirred solution of Na-BOC-N? M-CBZ-L-lysine (9.5 grams, 25.0 mmol) and N-methyl morpholine (5.8 milliliters) , 5.3 grams, 52.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate (3.4 milliliters, 3.6 grams, 26.7 millimoles) was added. After 2 minutes, phenylalanine benzyl ester tosylate (10.7 grams, 25.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.76 grams (approximately 100 percent highest solvent) of the blocked dipeptide as a white solid. { Rf = 0.84, 10% methanol / dichloromethane), which was used in the next transformation without further purification. A solution of the tertiary-butyl-carbonyl protected dipeptide (15.4 grams, 25.0 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 100 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 15.8 grams (about 100 percent more solvent pooled) of the free amino compound as a white semisolid: Rf = 0.55 (10 percent methanol / dichloromethane) . To a stirred solution of Na-BOC-N? M-CBZ-L-histidine (9.74 grams, 25.0 mmol) and N-methyl morpholine (3.0 milliliters, 2.8 grams, 27.5 mmol) in tetrahydrofuran (50 milliliters) at -15 °. C, isobutyl chloroformate (3.4 milliliters, 3.6 grams, 26.7 millimoles) was added. After 2 minutes, a solution of the suitably protected dipeptide (12.9 grams, 25.0 mmol) in tetrahydrofuran (30 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 20.58 grams (93 percent) of the blocked tripeptide as a white semi-solid. { Rf = 0.67, 10 percent methanol / dichloromethane), which was used in the next transformation without further purification. A solution of the tripeptide protected with tertiary-butyl carbonyl (20.5 grams, 23.1 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 20.5 grams (approximately 100 percent plus solvent pooled) of the free amino compound as a white solid: Rf = 0.51 (10 percent methanol / dichloromethane) . To a stirred solution of N-CBZ-glycine (7.24 grams, 34.6 millimoles) and N-methyl morpholine (4.2 milliliters, 3.9 grams, 38.1 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate was added. (4.7 milliliters, 5.0 grams, 36.3 millimoles). After 2 minutes, an adequately protected tripeptide (18.2 grams, 23.1 mmol) solution in 1: 1 tetrahydrofuran / dimethyl formamide (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 21.6 grams (95 percent) of the blocked tetrapeptide as a white solid. { Rf = 0.85, 10% methanol / dichloromethane), which was used in the next transformation without further purification. To a suspension of the blocked tetrapeptide (21.5 grams, 21.9 millimoles) in ethanol (150 milliliters), water was added, which the mixture became very turbid (approximately 125 milliliters). The resulting mixture was stirred with palladium chloride (3.89 grams, 21.9 millimoles) under a hydrogen atmosphere (5 atmospheres) for 16 hours. The reaction mixture became clear within about half an hour, which may indicate completion of the reaction. The catalyst was removed by filtration, and the filtrate was evaporated to give 13.7 grams of a colorless semi-solid. This material was dissolved in water and lyophilized to give 11.5 grams (94 percent) of the desired tetrapeptide dihydrochloride as a free flowing white powder: [a] D -12.4 ° (c 2.0, H20); 1 H NMR (500 MHz, D 20) d 8.72 (1H, d, J = 7.7), 8.40 (1H, d, J = 7.8), 8.00 (1H, s), 7.30-7.19 (5H, m), 7.01 (1H , s), 4.62 (1H, br q, J = 4.7), 4.44 (1H, m), 4.22 (1H, br q, J = 4.9), 3.58 (2H, s), 3.10-2.90 (4H, m) , 2.72 (2H, t, J = 7.3), 1.65-1.20 (6H, m).

Example 7 Synthesis of Glycyl-L-Histidyl-L-Lysyl-L-Phenylalanyl-L-Phenylalanine To a stirred solution of Na-BOC-L-phenylalanine (10.6 grams, 40.0 mmol) and N-methyl morpholine (4.8 milliliters, 4.5 grams, 44.0 mmol) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate (5.5 milliliters, 5.7 grams, 42.0 millimoles) was added. ). After 2 minutes, a solution prepared by the mixture of phenylalanine benzylester tosylate (7.1 grams, 40.0 mmol), tetrahydrofuran (50 milliliters), and N-methyl morpholine (4.4 milliliters, 4.0 grams, 40.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 19.8 grams (98 percent) of the blocked dipeptide as a white solid (Rf = 0.98, 10 percent methanol / dichloromethane). A solution of the tertiary-butyl-carbonyl protected dipeptide (19.7 grams, 39.2 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo, and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 100 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 19.3 grams (about 100 percent plus solvent pooled) of the free amino compound: Rf = 0.65, (10 percent methanol / dichloromethane). To a stirred solution of Na-BOC-N? M-CBZ-L-lysine (15.2 grams, 40.0 mmol) and N-methyl morpholine (4.8 milliliters, 44.0 mmol) in tetrahydrofuran (100 milliliters) at -15 ° C, He added isobutyl chloroformate (5.5 milliliters, 5.7 grams, 42.0 millimoles). After 2 minutes, the protected dipeptide was added (15.8 grams, 39.2 mmol). The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29.9 grams (98 percent) of the blocked tripeptide as a white solid. { Rf = 0.84, 10 percent methanol / dichloromethane). A solution of the tripeptide protected with tertiary-butyl carbonyl (15.4 grams, 25.0 mmol) in 35 percent trifluoroacetic acid / dichloromethane (300 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 100 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.7 grams (about 100 percent plus solvent pooled) of the free amido compound, as a fluffy white solid: Rf = 0.72 (10 percent methanol / dichloromethane).

To a stirred solution of N ^ BOC-N ^ -CBZ-L-hietidine (15.6 grams, 40.0 mmol) and N-methyl morpholine (4.8 milliliters, 4.5 grams, 44.0 mmol) in tetrahydrofuran (80 milliliters) at -15 ° C , isobutyl chloroformate (5.5 milliliters, 5.7 grams, 42.0 millimoles) was added. After 2 minutes, a solution of the appropriately protected tripeptide (12.9 grams, 25.0 mmol) in dimethyl formamide (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29.1 grams (72 percent) of the blocked tetrapeptide as a white solid. { Rf = 0.97, 10 percent methanol / dichloromethane). A tertiary-carbonyl butyloxy-protected tetrapeptide solution (29.1 grams, 28.0 mmol) in 35 percent trifluoroacetic acid / dichloromethane (300 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.4 grams (about 100 percent more pooled solvent) of the free amino compound as a white solid. To a stirred solution of N-CBZ-glycine (7.32 grams, 35.0 mmol) and N-methyl morpholine (4.2 milliliters, 3.9 grams, 38.1 mmol) in tetrahydrofuran (100 milliliters) at -15 ° C, isobutyl chloroformate was added. (4.8 milliliters, 5.0 grams, 36.7 millimoles). After 2 minutes, a suitably protected tetrapeptide solution (26.3 grams, 28.0 mmol) in 1: 1 tetrahydrofuran / dimethyl formamide (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 27.3 grams (87 percent) of the blocked pentapeptide as a white solid (Rf = 0.95, 10 percent methanol / dichloromethane). To a suepeneion of the blocked pentapeptide (27.3 grams, 24.2 mmol) in ethanol (200 milliliters), water was added, until the mixture became very cloudy (approximately 100 milliliters). The resulting mixture was stirred with palladium chloride (4.3 grams, 24.4 millimoles) under an atmosphere of hydrogen (5 atmospheres) for 16 hours. The reaction mixture became clear within about half an hour, which may indicate completion of the reaction. The catalyst was removed by filtration, and the filtrate was evaporated to give 14.6 grams (82 percent) of the desired pentapeptide dihydrochloride as a free flowing white powder: [α] D -12.1 ° (c 2.0, methanol).

Example 8 Synthesis of Glycyl-L-Arginyl-L-Lysine To a stirred solution of Na-BOC-Ng-nitro-L-arginine (8.0 grams, 25.0, millimoles) and N-methyl morpholine (3.0 milliliters, 2.8 grams, 27.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, was added isobutyl chloroformate (3.4 milliliters, 3.6 grams, 26.3 millimoles).

After 2 minutes, a solution of benzyl ester hydrochloride of L- (N? M-CBZ) lysine (10.2 grams, 25.0 mmol) and N-methyl morpholine (2.8 milliliters, 2.5 grams, . 0 mmol) in tetrahydrofuran (30 milliliters). The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.3 grams, 97 percent) of the blocked dipeptide as a white solid. { Rf = 0.57, 10 percent methanol / dichloromethane). A solution of the tertiary-butyl-carbonyl protected dipeptide (16.3 grams, 24.3 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo, and neutralized with 2M aqueous potaeium bicarbonate. The product was extracted into ethyl acetate (3 x 100 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 17.0 grams (about 100 percent more solvent pooled) of the free amino compound as a white semi-solid:. { Rf = 0.12, 10 percent methanol / dichloromethane). To a stirred solution of CBZ-glycine (7.3 grams, . 0 millimoles) and N-methyl morpholine (4.2 milliliters, 4.0 grams, 38.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate (4.8 milliliters, 5.0 grams, 36.8 millimoles) was added. After 2 minutes, a solution of the protected dipeptide (13.9 grams, 24.3 mmol) in tetrahydrofuran (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.7 grams (95 percent) of the blocked tripeptide as a white solid. { Rf = 0.517, 10 percent methanol / dichloromethane). To a suspension of the blocked tripeptide (17.7 grams, 23.2 mmol) in ethanol (250 milliliters) was added water, until the mixture became very cloudy (approximately 100 milliliters). The resulting mixture was stirred with palladium chloride (4.25 grams, 24.0 mmol) under an atmosphere of hydrogen (5 atmospheres) for 18 hours. The catalyst was removed by filtration, and the filtrate was evaporated to a white semi-solid. This material was dissolved in water, filtered through 0.45m nylon syringe filters, and lyophilized to give 10.2 grams (approximately 100 percent) of the desired tripeptide dihydrochloride as a white powder: [a] D -14.6 ° (c 2, water); 1 H NMR (500 MHz, D 20) d 8.81 (1 H, br s), 8.30 (1 H, br s), 7.92 (1 H, br s), 4.37 (1 H, br s), 3.96 (1 H, d, J = 4.8 ), 3.58 (2H, d, J = 8.8), 3.13 (2H, br s), 2.74 (2H, br s), 1.90-1.20 (10H, m); 13C NMR (125MHz, D20) d 175.2, 170.5, 166.9, 157.5, 115.0, 53.7, 52.6, 31.4, 29.2, 27.8, 26.8, 25.0, 22.5, 19.1.

Example 9 L-Alanyl-L-Histidyl-L-Lysine AHK can be obtained as an acetate salt in Bachem Bioscience Inc., Philadelphia, Pennsylvania (Catalog No. 1555). In an alternative manner, AHK can be synthesized as the dihydrochloride salt by the following procedure. To a stirred solution of Na-BOC-N? M-CBZ-L-histidine (9.74 grams, 25.0 millimoles) and N-methyl morpholine (5.8 milliliters, 5.3 grams, 52.5 millimoles) in tetrahydrofuran (50 milliliters) at -15 ° C, isobutyl chloroformate (3.4 milliliters, 3.6 grams, 256.3 millimoles) was added.

After 2 minutes, benzyl ester hydrochloride of (N-e-CBZ) -L-lysine (10.2 grams, 25.0 mmol) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.2 grams (93 percent) of the blocked dipeptide as a white semisolid (Rf = 0.61, 10 percent methanol / dichloromethane), which was used in the next transformation without further purification. A solution of the tertiary-butyl-carbonyl protected dipeptide (17.2 grams, 23.2 mmol) in 35 percent trifluoroacetic acid / dichloromethane (150 milliliters) was stirred for half an hour at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3 x 150 milliliters). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 16.8 grams (about 100 percent plus solvent pooled) of the free amino compound as a white solid: Rf = 0.26 (10 percent methanol / dichloromethane) . To a stirred solution of N-CBZ-L-alanine (6.28 grams, 25.0 ilimoles) and N-methyl morpholine (3.0 milliliter, 2.8 grams, 27.5 millimole) in tetrahydrofuran (50 milliliter) at -15 ° C, chloroformate was added. of isobutyl (3.4 milliliters, 3.6 grams, 26.3 millimoles). After 2 minutes, a solution of the above protected dipeptide (14.9 grams, 23.2 mmol) in tetrahydrofuran (50 milliliters) was added. The reaction mixture was stirred at -15 ° C for 1.5 hours, and then allowed to warm to 0 ° C. At this time, the reaction was quenched by the addition of 2M aqueous potassium bicarbonate. The products were extracted with ethyl acetate (3 x 150 milliliters). The combined extracts were washed with 1M citric acid (3 x 100 milliliters), water, 2M KHC03 (3 x 100 milliliters), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to a syrup, from which the blocked tripeptide was precipitated by dilution with 95 percent ethanol (300 milliliters). The resulting material was collected on a filter, washed with 95 percent ethanol, and dried to give a white solid:. { Rf = 0.49, 10 percent methanol / dichloromethane); p.f. 151-153 ° C. To a suspension of the blocked tripeptide (21.5 grams, 21.9 millimoles) in ethanol (200 milliliters) was added water (approximately 200 milliliters). The resulting mixture was stirred with palladium chloride (4.25 grams, 24.0 mmol) under an atmosphere of hydrogen (5 atm) for 1 hour. The resulting mixture, where the volume of the material (other than the catalyst) was dissolved, was filtered, and the filtrate was concentrated in vacuo to remove the volatile organics. The remaining aqueous solution was lyophilized to give 10.88 grams of a white solid. This material was dissolved in water, filtered through a 0.2M nylon membrane, and again lyophilized to give 10.50 grams (99 percent) of the desired tripeptide dihydrochloride as a white powder: [a] D -4.43 ° ( c, 3 H20); 1 H NMR (500 MHz, DMS0-d 6) d 8.73 (1 H, d, J = 7.8), 8.45 (1 H, d, J = 7.8), 8.45 (1 H, d, J = 7.5), 8.09 (1 H, s). , 7.08 (1H, s), 4.59 (1H, s), 4.59 (1H, dd, J = 5.4, 7.5), 4.12 (1H, m), 3.88 (1H, q, J = 6.9), 3.03 (1H, dd, J = 15.0, 4.8), 2.96 (1H, dd, J = 15.0, 7.7), 2.74 (2H, t, J = 7.5), 1.76-1.68 (1H, m), 1.66-1.51 (3H,), 1.41-1.21 (2H,), 1.32 (3H, d, J = 7.0); 13C NMR (125 MHz, DMS0-d6) d 174.0, 169.9, 169.5, 134.2, 130.5, 117.8, 52.6, 52.5, 48.0, 38.4, 30.3, 28.2, 26.5, 22.4, 17.2.

Example 10 Synthesis of Peptide-Copper Complexes in Different Molar Proportions of Peptide to Copper A. Peptide-Copper Complex in a Molar Ratio of 2; 1 An AHK solution was prepared by dissolving 2.6954 grams (0.0065 moles) of AHK acetate (Bachem Inc.) in approximately 10 milliliters of distilled water. The initial pH of this AHK solution was 6.71. Separately, a copper (II) chloride solution was prepared by dissolving 0.4479 grams (0.0033 moles) of anhydrous copper (II) chloride in approximately 2.0 milliliters of distilled water.

The copper (II) chloride solution was slowly added to the AHK solution in rapid stirring, and the pH was constantly monitored with a pH meter. After all the copper (II) chloride solution was added, the pH of the combined solution was 3.83. Then the pH was adjusted to 7.16 by the slow addition of a 0.5M NaOH solution, and the final volume was adjusted to 20.0 milliliters by the addition of distilled water. This procedure produced an aqueous solution containing AHK: Cu in a molar ratio of the peptide to copper of 2: 1, and in a concentration of 10 milligrams / milliliter. The solution was a dark blue-purple color, and had a characteristic absorption peak at 563 to 580 nanometers.

B. Peptide-Copper Complex in a Molar Ratio of 2: 1 AHK was prepared as the dihydrochloride salt as described in Example 9. A solution of AHK was prepared by dissolving 0.6388 grams (0.00146 moles) of L-alanyl hydrochloride -L-histidine in approximately 5 milliliters of distilled water. The initial pH of this AHK solution was 2.45. Separately, a copper (II) chloride solution was prepared by dissolving 0.0967 grams (0.0007 moles) of anhydrous copper (II) chloride in approximately 1.0 milliliters of distilled water. The copper (II) chloride solution was slowly added to the AHK solution in rapid stirring, and the pH was constantly monitored with a pH meter. After all the copper (II) chloride solution was added, the pH of the combined solution was 2.36. The pH was then adjusted to 7.05 by the slow addition of a 0.5 M NaOH solution, and the final volume was adjusted to 20.0 milliliters by the addition of distilled water. This procedure produced an aqueous solution containing AHK: Cu in a molar ratio of the peptide to copper of 2: 1, and in a concentration of 10 milligrams / milliliter. The solution was a dark blue-purple color, and had a characteristic absorption peak at 563 to 580 nanometers.

C Peptide-Copper Complex in a Molar Ratio of 1.1: 1 AHK was prepared as the dihydrochloride salt as described in Example 9. A solution of AHK was prepared by dissolving 1.6144 grams (0.0037 moles) of L-alanyl hydrochloride. L-histidyl-lysine in approximately 10 milliliters of distilled water. The initial pH of this AHK solution was 2.70. Separately, a copper (II) chloride solution was prepared by dissolving 0.4267 grams (0.0032 moles) of anhydrous copper (II) chloride, in approximately 2.0 milliliters of distilled water. The copper (II) chloride solution was slowly added to the AHK solution in rapid stirring, and the pH was constantly monitored with a pH meter. After all the copper (II) chloride solution was added, the pH of the combined solution was 2.14. The pH was then adjusted to 6.89 by the slow addition of a 0.5M NaOH solution, and the final volume was adjusted to 20.0 milliliters by the addition of distilled water. This procedure produced an aqueous solution containing AHK: Cu in a molar ratio of the peptide to copper of 1.1: 1, and in a concentration of 7.5 milligrams / milliliter. The solution was a dark blue-purple color, and had a characteristic absorption peak at 593 nanometers, and a broad peak at 586 to 607 nanometers.

D. Peptide-Copper Complex at a Molar Ratio of 1; 1 An AHK solution was prepared by dissolving 1.3007 grams (0.0007 mole) of AHK acetate (Bachem Bioscience Inc.) in approximately 5 milliliters of distilled water. The initial pH of this AHK solution was 6.95. By electionA solution of copper (II) chloride was prepared by dissolving 0.0966 grams (0.0007 mole) of anhydrous copper (II) chloride in approximately 2.0 milliliter of deethylated water. The copper (II) chloride eeolution was slowly added to the AHK solution in rapid stirring, and the pH was constantly monitored with a pH meter. After all the copper (II) chloride solution was added, the pH of the combined solution was 2.91. The pH was then adjusted to 7.08 by the slow addition of a 0.5M NaOH solution, and the final volume was adjusted to 0.15 milliliters by the addition of distilled water. This procedure produced an aqueous solution containing AHK: Cu in a molar ratio of the peptide to copper of 1: 1, and in a concentration of 10 milligrams / milliliter. The solution was a dark blue-purple color, and had a characteristic absorption peak at 595 nanometers, and a broad peak at 584 to 612 nanometers.

Example 11: Hair Growth Stimulus by Representative Peptide-Copper Complexes The following example illustrates the stimulation of hair growth in warm-blooded animals after an intradermal injection of representative peptide-copper complexes of this invention. In this experiment, the loins of C3H mice (60-day-old, telogen hair growth phase) were etched safely on day 1 using an electric e-transporter. A sterile saline solution containing the indicated peptide-copper complex (i.e., infiltrated under the skin) was then intradermally injected into two places within the shorn areas of the mice. The injection in two places provided two test places within the shorn area of each mouse. Each injection (0.1 milliliter) contained between 0.36 and 0.55 milligrams of the peptide-copper complex within the sterile saline solution. A group of mice injected with serum (0.1 milliliters) served as controls. Following the injection of the peptide-copper complexes, indications of hair growth were seen within 10 days. The first visual signs were a darkening of the skin in a circular region around the site of the injection. The size of this region generally depends on the dose, increasing with an increase in the dose. The 0.1 milliliter injections used in this experiment produced a circle of hair growth that measured approximately 0.5 square centimeters to 5.0 square centimeters in diameter. The growth of active hair occurred between 14 and 20 days after the injection, with a maximum effect seen on day 29. Both the number of mice with growing hair at the injection site and the diameter in the growth region of hair were determined on day 21. A positive response was expressed as the number of mice that exhibited hair growth at the injection sites, comparing to the total number of mice injected in the study. The results of this experiment are presented in the following Table 4 (the day of establishment is the day in which the hair follicle pigmentation was observed for the first time): Table 4 Hair Growth Stimulus by Peptide-Copper Complexes EXAMPLE 12 Hair Growth Stimulus by Peptide-Copper Representative Complexes The following example illustrates the growth stimulation of hair in warm-blooded animals after the intradermal injection of representative peptide-copper complexes of this invention. As in Example 11 above, the loins of C3H mice (60-day-old, telogen hair growth phase) were sheared tightly on day 1 using an electric clipper. A sterile saline solution containing the indicated peptide-copper complex (i.e., infiltrated under the skin) was then intradermally injected into two places within the shorn areas of the mice. The injection in two places provided two test sites within the area of each mouse. Each injection (0.1 milliliter) contained between 0.75 and 1.5 milligrams of the peptide-copper complex within the sterile saline solution. A group of mice injected with serum (0.1 milliliters) served as controls. Following the injection of the peptide-copper complexes, hair growth indications were seen within 10 days. The first visual signs were a darkening of the skin in a circular region around the site of the injection. The size of that region generally depends on the dose, increasing with an increase in the dose. The 0.1 milliliter injections used in this experiment produced a circle of hair growth that measured approximately 0.5 square centimeters to 5 square centimeters in diameter. Active hair growth occurred between 14 and 20 days after the injection, seeing a maximum effect on day 29. On day 21, both the number of mice that grew their hair at the site of the injection, and the diameter of the hair growth region were determined. A positive response was expressed as the number of mice that exhibited hair growth at the sites of the injection, compared to the total number of mice injected in the study. The results of this experiment are presented in Table 5.

Table 5 Hair Growth Stimulus by Peptide-Copper Complexes EXAMPLE 13 Hair Growth Stimulus by Peptide-Copper Complexes Containing D-Amino Acids This example illustrates the stimulation of hair growth in warm-blooded animals by intradermal injection of AHK: Cu (1.1: 1), using a D-amino acid instead of the D-amino acid that occurs naturally. In this experiment, the loins of C3H mice (60-day-old, telogen hair growth phase) were sheared closely on day 1, using an electric clipper. A sterile saline solution containing AHK: Cu (1.1: 1), or AHK: Cu (1.1: 1) containing D-amino acid (i.e., infiltrated under the skin (in two places within the cells) was injected intradermally. Sheared areas of the mice Injection at two sites gave two places within the shorn area of each mouse Each injection (0.1 milliliter) contained either 1.2 or 1.8 micromoles per injection of peptide-copper complex in the sterile saline solution. group of mice injected with serum (0.1 milliliters) served as controls.After the injection of the peptide-copper complex, indications of hair growth were seen within 10 days.The first visual signs were a darkening of the skin in a circular region surrounding the injection site The size of this region generally depends on the dose, increasing with an increase in the dosage.The injections of 0.1 milliliters used in this experiment or they produced a circle of hair growth that measured approximately 0.5 square centimeters to 5 square centimeters in diameter. Active hair growth was presented between * 14 and 20 days after the injection, with a maximum effect on day 29. The degree of hair growth was determined by measuring the total area of hair growth at the two sites of hair growth. injection. The data of this experiment are presented in Table 6.

Table 6 Hair Growth Stimulus by Peptide-Copper Complexes containing D-amino acids The above table illustrates that the substitution with D-a inoids by corresponding L-amino acids does not affect the hair growth activity of the peptide-copper complexes.

Example 14 Stimulus of Hair Growth by Local Application of a Peptide-Copper Complex This example illustrates the stimulation of hair growth in warm-blooded animals by the local application of a peptide-copper complex. In this experiment, female C3H mice were prepared in telogen cycle (from 60 to 65 days of age), shearing in the posterior dorsal region (ie, day 1). Local application of peptide-copper complexes was performed twice a day (Monday-Friday) using a cotton-tipped applicator that applied approximately 0.1 milliliters per treatment. The local formulation used in this experiment contained the following components: Peptide-copper complex 0.1-0.5% (w / w) Sterile water 16.9-16.5% (w / w) Propylene glycol 50.0% (pee / wt) Ethanol 30.0% (wt / wt) Nonoxynol-9 3.0% (wt. / weight) The local application of the previous formulation continued until the establishment of the follicle pigmentation, which proceeds to the emergence of the hair shaft. The measurement of the degree of response was performed using digital image analysis at weekly intervals, starting on day 14. The data were expressed as the response percentage of the treatment area using the following equation: % treatment area = (growth area / treatment area) x 100 For comparison purposes, to illustrate the effect of hydrophobic amino acid residues on hair growth after local application, AHK: Cu with AHF: Cu was compared. In this experiment, local formulations containing AHK: Cu (1.1: 1) and AHF: Cu (1.1: 1) were prepared in a concentration of 0.5 percent and 0.1 percent (w / w) as indicated above. The hair growth response (ie, "Treatment Area Percentage") was determined on day 20, on day 27, and on day 34. The results of this experiment are presented in Table 7.

Table 7 The data presented in Table 7 illustrate that peptide-copper complexes containing hydrophilic residues (ie, AHK: Cu amino acid) are more active in stimulating hair growth than similar peptides containing hydrophobic amino acid residues. (ie, the phenylalanine amino acid of AHF: Cu) following local administration. This contrasts with administration by injection, where peptide-copper complexes containing hydrophilic residues are less active than similar peptides containing hydrophobic amino acid residues.

EXAMPLE 15 Hair Growth Stimulus by Intraperitoneal Injection of Peptide-Copper Complexes The following experiment illustrates the maintenance of viability of the hair follicle (i.e., growth) by intraperitoneal (sietemic) injection of the peptide-copper complex GHKVFV: Cu during the treatment with the cytosine arabinoside chemotherapeutic agent (Ara-C). In this experiment, Sprague-Dawley rat pups (8 days old) were kept in four litters (n = 10 / litter) for the duration of this study. On day 0, the baits received intraperitoneal (IP) injections of GHKVFV: Cu (2: 1) in a sterile saline solution, or a serum control (one injection per animal, 0.1 milliliter per injection). On day 1, all animals initiated a series of 7 consecutive daily intraperitoneal injections with Ara-C (50 milligrams / kilogram). On day 8, all animals were evaluated for the extent of hair loss (alopecia) using the following evaluation scale: Rating Rating of Alopecia 0 Normal (no hair loss) 1 Light weight loss 2 Moderate weight loss 3 Scarce hair cover 4 Total hair loss Ara-C injections caused significant hair loss on days 5-6 in most animals. In order to evaluate the effect of GHKVFV: Cu, the degree of hair loss was evaluated daily. The injection of GHKVFV: Cu at a dosage of 50 milligrams / kilogram, caused a slight retention of hair on the body of the test animals. This was seen primarily on the head, with scarce remaining hair on the body. This contrasted with the serum control group (+ Ara-C) that showed total hair loss. Table 8 presents the results of this experiment evaluated on day 8, using the rating scale previously described, expressing the "Alopecia Degree" as the average response for all animals.

Table 8 The observation of the retained hair was confirmed histologically on day 8. Of the animals that received 50 milligrams / kilogram of GHKVFV: Cu, it was found that approximately 30 to 40 percent of the dorsal hair was in anagen, compared with 5 to 10 percent for animals that received serum plus Ara-C only. Control animals with serum that did not receive Ara-C had 100 percent anagen follicles.

EXAMPLE 16 Hair Growth Stimulus by Intradermal Invention of Peptide-Copper Complexes The following experiment illustrates the localized maintenance of the viability of the hair follicle (i.e. growth) by intradermal (local) injection of the peptide-copper complex AHK: Cu by treatment with the cytosine arabinoside chemotherapeutic agent (Ara-C). In this experiment, Sprague-Dawley rat puppies (8 days old) were kept in five litters (n = 10-11 / litter) for the duration of this study. On day-0, the baits received intradermal injections (ID) of AHK: Cu (1: 1) in a sterile ealine solution, or a serum control (one injection per animal, 0.05 milliliters per injection.) Each bait contained two normal control animals where AHK: Cu or Ara-C (ie, serum only) was not administered. On day 1, the designated animals initiated a series of 7 consecutive daily intraperitoneal (IP) injections with Ara-C (25). milligrams / kilogram) On day 10, all animals were evaluated for the extent of hair loss (alopecia) at the injection sites, using the evaluation identified in Example 15. The injections of Ara-C caused a significant loss of hair on days 5-6 in the majority of animals. In order to evaluate the stimulating effect of AHK: Cu, the degree of hair loss at the site of the injection was evaluated daily. The injection of AHK: Cu generally caused hair retention in a radius of 0.25 centimeters around the injection site, most notably in the groups of 0.1 to 0.5 milligrams of dose. Table 9 presents the results evaluated on day 10, using the rating scale described above, expressing the "degree of alopecia" as the average response seen at the site of the injection.

Table 9 Observation of the hair retained within the area of the AHK injection: Cu was examined histologically. Although hair follicles of anagen were seen to appear functioning normally at the site of the AHK: Cu injection, the follicles located far from the injection were dystrophic and non-functional (alteration of the integrity of the internal and external root sheaths, and rods of displaced hair). These data confirm the gross observations of the normal hair follicle function within the site of the AHK: Cu injection, and illustrate the stimulatory effect of AHK: Cu on the hair follicle that maintains the active growth cycle during treatment with chemotherapy. From the foregoing, it will be appreciated that, while the specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. In accordance with the above, the invention should not be limited, except by the appended claims.

Claims

CLAIMS 1. A composition for use as an active therapeutic substance, comprising a pharmaceutically acceptable carrier or diluent, and a peptide-copper complex having the structure: [R1-R2-R3t: copper (II) wherein R-L is an amino acid or an amino acid derivative; R2 is histidine, arginine, or a derivative thereof, and R3 is at least one amino acid or an amino acid derivative linked to R2 by a peptide bond, with the proviso that when R2 is histidyl or (3-methyl) histidyl , and R3 is lysine, lysyl-propyl-valyl-phenylalanyl-valine, lysyl-valyl-phenylalanyl-valine, lysyl-tryptophan, or lysyl- (glycyl) -l-2-tryptophan, then R is not glycyl, alanyl, seryl, or valyl , and with the additional proviso that when R2 is histidyl or (3-methyl) histidyl and R3 is glycine, glycyl-prolyl-valyl-phenylalanyl-valine, glycyl-valyl-phenylalanyl-valine, glycyl-tryptophan, or glycyl- ( glycyl) 1_ 2-tryptophan, then R1 is not lysyl. The composition according to claim 1, wherein the histidine derivative has the structure: wherein n = 20, and X and Y are independently selected from alkyl fractions containing from 1 to 12 carbon atoms, or an aryl fraction containing from 6 to 12 carbon atoms; and the arginine derivative has the structure: where n = 1-20 (excluding n = 3). 3. The composition according to claim 2, wherein the amino acid derivative has the structure: Me-CH-OOH I I 'R " wherein R 'and R "are independently selected from hydrogen, a saturated or unsaturated, substituted or unsubstituted, straight-chain, branched, or cyclic alkyl moiety, containing from 1 to 20 carbon atoms, and a moiety of substituted or unsubstituted aryl containing from 6 to 20 carbon atoms 4. The composition according to claim 1, wherein R? ee an amino acid, and R3 is at least one amino acid 5. The composition according to claim 1, wherein R is an amino acid 6. The composition according to claim 1, wherein R2 is histidine 7. The composition according to claim 1, wherein R3 is at least one amino acid. composition according to the claim 1, wherein R3 is an amino acid. 9. The composition according to claim 1, wherein R -? _ Is a hydrophilic amino acid. 10. The composition according to claim 1, wherein R3 is a hydrophilic amino acid. 11. The use of the composition according to any of claims 1 to 8, for the manufacture of a medicament for stimulating hair growth in an animal in need thereof. 12. The use according to claim 11, wherein the animal has a hair loss affliction selected from the group consisting of androgenetic alopecia, alopecia areata, female pattern baldness, and secondary alopecia. 13. The use according to claim 12, wherein the affliction of hair loss is androgenetic alopecia. 14. The use according to claim 12, wherein the affliction of hair loss is secondary alopecia. 15. A method for stimulating hair growth in an animal in need, which comprises administering to the animal an effective amount of the composition according to any of claims 1 to 10. The method of claim 15, in where the administration of the peptide-copper complex is by local administration. 17. The use of a peptide-copper complex according to any of claims 1 to 16, for the manufacture of a medicament for stimulating hair growth in an animal in need thereof. 18. The use according to claim 17, wherein the animal has a hair loss affliction selected from the group consisting of androgenetic alopecia, alopecia areata, female pattern baldness, and secondary alopecia. 19. The use according to claim 18, wherein the affliction of hair loss is androgenetic alopecia. 20. The use according to claim 18, wherein the affliction of hair loss is secondary alopecia. 21. A method for stimulating hair growth in an animal in need thereof, which comprises administering to the animal an effective amount of a peptide-copper complex according to any of claims 1 to 16. 22. The method of claim 18, wherein the administration of the peptide-copper complex is by local administration.