WO1991014437A2

WO1991014437A2 - Use of copper(ii) containing compounds to accelerate wound healing

Info

Publication number: WO1991014437A2
Application number: PCT/US1991/002028
Authority: WO
Inventors: Loren R. Pickart
Original assignee: Procyte Corporation
Priority date: 1990-03-26
Filing date: 1991-03-26
Publication date: 1991-10-03
Also published as: US5164367A; EP0522004A1; WO1991014437A3; AU7565091A; EP0522004B1; JPH05505808A; ATE187646T1; CA2078347C; DE69131847D1; CA2078347A1; DE69131847T2

Abstract

Methods for the use of compositions of copper(II) containing compounds to accelerate healing of wounds in warm-blooded animals. The methods include systemic loading of copper(II) to accelerate the rate of wound healing following injury or surgery. The copper(II) containing compounds include copper(II) complexes with amino acids and peptides, and copper(II) salts.

Description

USE OF COPPER(II) CONTAINING COMPOUNDS TO ACCELERATE WOUND HEALING

Technical Field

The present invention relates in general to the use of compositions of copper(II) containing compounds as active therapeutic substances, including their use to accelerate healing of wounds in warm-blooded animals, and more specifically to systemic loading of copper(II) compounds to accelerate the rate of wound healing following injury or surgery to warm-blooded animals.

Background of the Invention

Wound healing and tissue repair in humans and other warm¬ blooded animals are often inadequate or unacceptably slow. This is especially true in certain chronic diseases such as diabetes in the elderly, and in cancer patients. Chronic non-healing wounds create serious medical problems with infections, pain, extended hospitalization, and costly treatment.

Wound healing is a very complex process which involves the following phases or events. The first phase involves the recruitment of the proper types of white blood cells to cleanse the wound and initiate the healing response. The body seals off blood flow into the area and a white cell type called "neutrophils" secrete toxic molecules (such as superoxide anion) into the wound to kill bacteria and induce a general inflammation.

The second phase involves the formation of granulation tissue (a mixture of fibroblasts, macrophages and new blood vessels in a loose matrix of collagen and other material) and the suppression of the inflammatory response. This occurs while while macrophages, monocytes, and other white blood cells invade to clean up tissue debris and open a path for mast cells and fibroblasts to follow and secrete angiogenic factors to attract capillary endothelial cells. There is a great proliferation of fibroblasts that secrete the structural protein collagen and glycosaminoglycans into the wound area. Reepithelialization of skin surface wounds will also commence early in the second phase.

The third and final stage involves the remodeling and formation of new connective tissue components, and wound closure. A variety of approaches have been pursued to develop materials and methods which will aid in the healing process in warm blooded animals. One approach for the treatment of chronic wounds and skin ulcers has been the topical application of protein growth factors to the wound area (G.L. Brown et al., N_, Eng. J. Med.. 321:76-79. 1989). Other methods include the topical application of vasoactive peptides PΛ. Janssen, J. Am. Acad. Dermatol.. 21:85-90, 1989) and the systemic administration of macrophage activating agents such as tetrachlorodecaoxygen (R.A. Hatz et al., Plast. and Reconst. Surg.. £4:953-959, 1989). In addition, certain peptide and peptide-derivative copper(II) complexes have been shown to accelerate wound healing in animals when applied topically or by injection at the locality of the wound (U.S. Patent Nos. 4,665,054, 4,760,051, 4,810,693 and 4,877,770).

However, even though these compounds have shown positive results in wound healing, there still exists a need in the art for compositions and methods which accelerate the healing of wounds in warm-blooded animals.

Summary of the Invention

Briefly stated, the present invention discloses compositions for use as active therapeutic substances. In addition, the instant compositions find use in accelerating wound healing in warm-blooded animals, and may be used in the manufacture of medicaments for such use. The compositions include copper(H) (i.e., Cu²⁺) containing compounds in combination with a pharmaceutically acceptable carrier or diluent. The present invention also discloses methods for the systemic administration of a therapeutically effective amount of a composition containing copper(ϋ) compounds. The methods are particularly useful to accelerate the rate of wound healing following injury or surgery in warm-blooded animals.

Administration of compositions of the present invention may be accomplished in any manner which will result in a systemic dose of copper(II) to the animal. For example, such administration may be by injection (intramuscular, intravenous, subcutaneous or intradeπnal), oral, nasal, or suppository applications. Typically, compositions of the present invention include copper(II) containing compounds in solution for various forms for injection, or in pharmaceutical preparations which are formulated for the sustained release of the copper(H) compounds for oral, nasal, or suppository dosage application. The balance of the composition or pharmaceutical preparation comprises an inert, physiological acceptable carrier. Preferably, this carrier does not interact with the effectiveness of the copper(II) compounds.

Compounds of the present invention include copper(II) compounds that are capable of delivering copper(H) by systemic application. Such compounds include copper(II) complexed with naturally occuring substances such as amino acids, including glycine, alanine, valine, lysine, histidine, arginine, trytophan, phenylalanine, serine, leucine, isoleucine, proline, or any other naturally occurring amino acids. In addition to naturally occurring amino acids, a number of other substances may similarly be complexed to copper(H), including amino acid derivatives such as 3-methyl-histidine, and organic compounds such as citrate. Preferably, copper(II) complexes of the present invention bind copper(II) in such a manner that, after systemic administration, the copper(II) may be exchanged with binding sites in the body of the warm-blooded animal. For example, the binding site for copper of bovine serum albumin (BSA). Compounds of the present invention also include combinations of two or more amino acids or amino acid derivatives complexed with copρer(H), such as the naturally occuring dipeptide /9-alanyl-histidine (i.e., caraosine). Compounds of the present invention also include coρper(II) complexed with, for example, glycyl-glycine, glycyl-glycyl-glycine, alanyl-(3-methyl)-histidine, histidyl- valine, valyl-histidine, glycyl-serine, leucyl-glycine, phenylalanine-glycine, arginine- lysyl-glycine, histidyl-serine, lysyl-alariine and tyrosyl-lysine.

In yet another embodiment, compounds of the present invention include copper(II) salts, such as copper(II) sulfate, copper(H) acetate, and copper(II) chloride. In addition, compounds of the present invention also include coppeπpeptide and copper peptide derivative complexes of the following general formulas designated A through I:

A: [glycyl-I^histidyl-L-lysine-R] :copper(II) wherein

R is an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is I^tryptophan, (glycyl)y-___rtryptophan, where y = 1-4, L-prolyl-Xi-L- phenylalanyl-X2 or Xι-L-phenylalanyl-X2, where Xj and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where X and X2 are not both I^valine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n. where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5; B: [glycyl-Rι-R2-R3]:copper(II) wherein

Rl is selected from the group consisting of L-histidyl, I_^(3-W)- histidyl and L-(5-W)-histidyl, where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R₂ is -NHCH((CH2)_nNH3⁺)CO- where n = 5-10; and

R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is .Utryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-Lr phenylalanyl-X2 or X -L-phenylalanyl-X2, where X and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n. where X4 is a naturally occurring carbohydrate such as glucose, maπnose, galactose, glucosamine or galactosamine and n = 1-5;

C: [glycyl-Rι-R₂-R3]:copper(π) wherein

Rl is selected from the group consisting of L-(3-W)-histidyl and Lr (5-W)-histidyl where W is an alkyi moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl; and

R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)yLrtryptophan, where y = 1-4, L-prolyl-Xχ-1_^ phenylalanyl-X2 or X -L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where X and X2 are not both L-valine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_> where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5;

D: [glycyl-Rι-R2-R3]:copper(II) wherein

Rl is selected from the group consisting of I_^histidyl, L-(3-W)- histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; R2 is a basic amino a d such as arginine, a basic amino moiety such as cadaverine, spermine or spermidine, or a modified basic amino acid such as caprolactone; and R3 is hydrogen, -NH2, ^an -~~y^ moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6- 12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tιyptophan, where y = 1-4, L-prolyl-Xi-L- phenylalanyl-X2 or X -L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5;

E: [R -R2-glycine-R3]:copper(II) wherein

Rl is L-lysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; R2 is selected from the group consisting of I iistidyl, L-(3-W)- histidyl and I_^(5-W)-histidyl where W is an alkyl moiety contaimng from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-

12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is l-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-Lr phenylalanyl-X2 or X -L-phenylalanyl-X2, where X and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5;

F: [Rι-R2-R3-R4]:copper(π) wherein

Rl is L-lysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10;

R2 is selected from the group consisting of L-histidyl, L-(3-W)- histidyl and I_^(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; R3 is a basic amino acid such as arginine, an amino moiety such as cadaverine, spermine or spermidine, or a modified basic amino acid such as caprolactone; and R4 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6- 12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L- phenylalanyl-X2 or X -L-phenyIalanyl-X2, where X and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n» where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5;

G: [Rι-glycyl-R2-R3]:copper(II) wherein

Rl is selected from the group consisting of L-histidyl, ____-(3-W)- histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl, -NHCH((CH₂)_nNH3⁺)CO-, where n = 5-10, or a basic amino acid such as arginine, an amino moiety such as cadaverine, spermine or spermidine, or a modified basic amino acid such as caprolactone; and R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6- 12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)yL-tryptophan, where y = 1-4, L-prolyl-Xi-Lr phenylalanyl-X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, l-alanine and glycine, (X3)_n-I_^tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5;

H: [L-alanyl-Rι-R2-R3]:copper(II) wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)- histidyl and ___r(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L- phenylalanyl-X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where X and X2 are not both Lrvaline, (X3)_n-I^tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_. where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5; and

I: [glycyl-Ri-R₂-R3]:copper(n) wherein

Rl is L-lysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10 R2 is selected from the group consisting of L-histidyl, L-(3-W)- histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and R3 is hydrogen, -NH2» ^an a^ l moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6- 12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is l-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L- phenylalanyl-X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-I_^tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate such as glucose, mannose, galactose, glucosamine or galactosamine and n = 1-5. In another embodiment of the present invention, an additional chelating agent may be added to the coppeπpeptide complex to form a ternary metal:peptide:chelating agent complex. Suitable chelating agents include imidazole and imidazole containing compounds, such as histidine, and sulfur- containing amino acids, such as cysteine and methionine. Methods of the present invention include the systemic administration to the warm-blooded animal of a therapeutically effective amount of a composition which includes a copper(II) containing compound of the present invention. Any pharmaceutically acceptable form of copper(II) compound may be used in the method of this invention. The method results in a loading of a therapeutic amount of copper(II) in the animal resulting in the acceleration of wound healing. Other aspects of the present invention will become evident upon reference to the following detailed description.

Detailed Discription of the Invention The present invention discloses compositions which accelerate the healing of wounds, lesions, surgical incisions, and other hard or soft tissue defects in warm-blooded animals. The invention also discloses methods for the general enhancement or acceleration of wound healing by the systemic administration of compositions or pharmeceutical preparations containing copper(II) compounds of the present invention.

Compositions containing copper(H) compounds can be liquids with inert ingredients or pharmaceutical preparations comprising a slow release form of copper(H) compounds and suitable inert ingredients or pharmaceutical preparations for either oral, nasal, suppository, or parenteral applications. These pharmaceutical preparations can be prepared according to methods well known in the art

Administration of compositions of the present invention may be accomplished in any manner which will result in a systemic dose of copper H) to the animal. For example, such administration may be by injection (intramuscular, intravenous, subcutaneous or intradermal), oral, nasal, or suppository applications. Typically, compositions of the present invention include copper(H) containing compounds in solution for various forms for injection, or in pharmaceutical preparations which are formulated for the sustained release of the copper(II) compounds for oral, nasal, or suppository dosage application. The balance of the composition or pharmaceutical preparation comprises an inert, physiological acceptable carrier. Preferably, this carrier does not interact with the effectiveness of the copper(II) compounds.

Copper(II) containing compounds of the present invention accelerate healing throughout the animal and need not be administered directly to the damaged or afflicted tissue. Rather, acceleration of wound healing in internal wounds is achieved by systemic administration of the copper(ϋ) containing compound. Many wounds are too deep or are too irregular to treat adequately by local administration of healing agents. It is also difficult to administer healing agents to wounds remaining after internal surgery of bone repair. Systemic administration permits the copper(II) containing compounds of the present invention to be transported to the wound site via the blood vessel network, thus permitting a natural delivery of the copper(H) compounds to the cell and tissues. Compounds of the present invention include copper(II) compounds that are capable of delivering copper(II) by systemic application. Such compounds include copper(II) complexed with naturally occuring substances such as amino acids, including glycine, alanine, valine, lysine, histidine, arginine, trytophan, phenylalanine, serine, leucine, isoleucine, proline, or any other naturally occurring amino acids. In addition to naturally occurring amino acids, a number of other substances may similarly be complexed to copper(II), including amino acid derivatives such as 3-methyl-histidine, and organic compounds such as citrate. Preferably, copper(II) complexes of the present invention are capable of binding copper(II) in such a manner that after systemic administration, the copper(II) may be exchanged with binding sites in the body of the warm-blooded animal, such as, for example, the binding site for copper of BSA.

Compounds of the present invention also include combinations of two or more amino acids or amino acid derivatives complexed with copper(II), such as the naturally occuring dipeptide S-alanyl-histidine (i.e., carnosine). Compounds of the present invention also include copper(H) complexed with, for example, glycyl-glycine, glycyl-glycyl-glycine, alanyl-(3-methyl)-histidine, histidyl- valine, valyl-histidine, glycyl-serine, leucyl-glycine, phenylalanine-glycine, arginine- lysyl-glycine, histidyl-serine, lysyl-alanine and tyrosyl-lysine. In yet another embodiment, compounds of the present invention include copper(II) salts, such as copper(II) sulfate, copper(II) acetate, and copper(II) chloride.

In still another embodiment, compounds of the present invention include the complexes of the general formula designated A through I above. For example, in Formula F if Ri is L-lysyl, R2 is L-histidyl, R3 is arginine, R4 is an n- octyl alkoxy moiety, and the metal is copper(II), the structure of the copper(II):peptide complex would be as follows: O(CH₂)₇CH₃

:copper(ll)

Similarly, if R4 were an aminoalkyl moiety containing three carbon atoms of the formula -NH(CH2)2CH3, the metahpeptide complex would have the following structure:

NH(CH₂)₂CH₃

:copper(ll)

The chiral amino acids of the present invention have been designated as the L form. However, one skilled in the art would readily appreciate that the D forms of the amino acids may be utilized as a substitute for the L forms.

It will be recognized to one skilled in the art that compounds of the present invention also include penicillamine, ethylenediaminetetraacetic acid, desserroxamine, and other copper(II) chelating agents complexed with copper(ϋ).

The ability of a chelating agent to bind copper(II) is typically expressed as a "log K" binding or stability constant (Martell et. al., The Determination and Use of Stability Constants. V.C.H. Publishers Inc, New York, N.Y., 1988, incorporated herein by reference). Preferably, the copper(II) containing compounds of the present invention possess a log K binding constant for copper(II), at physiological pH, less than the binding constant of EDTA for copper(II). Chelating agents with binding constants equal to or greater than EDTA bind copper(II) too tightly, and thus will not exchange the copper with the binding sites in the body of the warm-blooded animal.

The peptides of the present invention may be synthesized either by solution chemical techniques or by solid phase techniques. The general procedure involves the stepwise addition of protected amino acids to build up the desired peptide sequence. Such methodology is well known to those skilled in the art. Illustrative syntheses of complexes of the present invention are presented in the examples hereinbelow.

Within the present invention, one may utilize a molar ratio of peptide or amino acid to copper(II) of, for example, 1:1, 2:1 or greater (e.g., 3:1). Preferably, the peptide or amino acid to copper(II) molar ratio is 2:1.

In another embodiment of the present invention, a chelating agent may be added to the copper(II):peptide complex to form a ternary copper(π):peptide:chelating agent complex. Suitable chelating agents include imidazole or imidazole-containing compounds, such as histidine, and sulfur containing amino acids, such as cysteine or methionine. Thus, if the copper(II):peptide complex is glycyl-L-histidyl-L-lysine:copper(II), histidine may be added to yield the ternary complex glycyl-L-histidyl-L- lysine:copper(π):histidine. However, to form such a ternary complex, the molar ratio of copper(II) to peptide to chelating agent must be considered. For example, if the ratio of peptide to copper(H) is 2:1, the addition of a chelating agent to the copper(II):peptide complex, although possible, is difficult due to site occupancy by the peptide. However, by maintaining the ratio of peptide to copper(II) near 1:1, a chelating group may readily be added to form the ternary complex. Preferably, the peptide to copper(II) to chelating agent ratio is 1:1:1.

Compositions or pharmaceutical preparations of the present invention may contain suitable inert ingredients for either oral or parenteral applications (i.e., pharmaceutically acceptable carriers). The diluent or carrier should not interact with the copper(II) containing compound to significantly reduce the effectiveness thereof.

Methods for encapsulating compositions (such as in a coating of hard gelatin) for oral or suppository administration are well known in the art (Baker et al., Controlled Release of Biological Active Agents. John Wiley and Sons, 1986, incorporated herein by reference). Suitable pharmaceutically acceptable carriers for parenteral application, such as intravenous, subcutaneous or intramuscular injection, include sterile water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol) and phosphate-buffered saline. Compositions of the present invention may be administered either orally or nasally, by suppository, or by injection either intravenously, subcutaneously, intramuscularly, or intradermally.

The balance of the compositions or pharmaceutical preparations comprises an inert, physiological acceptable carrier. This carrier should not interact with the active ingredients nor reduce the effectiveness of the copper(II) compounds. Suitable carriers include, but are not limited to, water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol), and phosphate buffered saline. An effective dosage of compositions or pharmaceutical preparations of the present invention delivers approximately 0.01 to 20 mg of copper(H) containing compound per kg body weight. The required dosage will vary according to the particular condition to be treated, the severity of the condition, and the duration of the treatment. The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES To summarize the examples that follow, Examples 1-9 illustrate the synthesis of representative copper(H) containing compounds of the present invention; Examples 10-17 illustrate the stimulation of wound healing by representative copper(II) containing compounds of the present invention.

Specifically, Example 1 illustrates the preparation of copper(H) peptide complexes. Example 2 illustrates the synthesis of glycyl-L-histidyl-L-lysine n-octyl ester copper(II). Example 3 illustrates the synthesis of glycyl-L-histidyl-Lr lysine n-octyl amide. Example 4 illustrates the synthesis of glycyl-I iistidyl-Lr lysyl-L-valyl-L-phenylalanyl-L-valine. Example 5 illustrates the synthesis of h- alanyl-L-histidyl-L- lysine. Example 6 illustrates the synthesis of L-lysyl-L-histidyl- L-glycine. Example 7 illustrates the synthesis of I_^lysyl-L-histidyl-L-glycyl-L-valyl- L-phenylalanyl-L-valine. Example 8 illustrates the synthesis of gfycyl-L-histidy-I_ caprolactam. Example 9 illustrates the synthesis of L-histidyl-glycyl-L-lysine. Example 10 illustrates the stimulation of wound healing by intramuscular (I.M.) injection of glycyl-I__-histidyl-L-lysine:copper(II). Example 11 illustrates the stimulation of wound healing in healing impaired rats by I.M. injection of glycyl-I^histidyl-L-lysine:copper(II). Example 12 illustrates the stimulation of wound healing by I.M. injection of glycyl-L-histidyl-L-lysyl-L-valyl- L-phenylalanyl-L-vaUne:copper(II). Example 13 illustrates the stimulation of wound healing in healing impaired animals by I.M. injection of copper(II) compounds. Example 14 illustrates the stimulation of wound healing in mice by I.M. injection of glycyl-L-histidyl-L-lysine:copper(II). Example 15 illustrates the stimulation of wound healing in pigs by I.M. injection of glycyl-L-histidyl-L- lysine:copper(II). Example 16 illustrates the stimulation of wound healing by hydrophobic and albumin binding derivatives of glycyl-L-histidyl-L- lysine:copper(II). Example 17 illustrates the stimulation of healing by sequence variations of glycyl-L-histidyl-L-lysine:copper(II).

Source of Chemicals

Chemicals and peptide intermediates utilized in the following examples may be purchased from a number of suppliers, for example: Sigma Chemical So., St. Louis, Missouri; Peninsula Laboratories, San Carlos, California; Aldrich Chemical Company, Milwaukee, Wisconsin; Vega Biochemicals, Tucson, Arizona; Pierce Chemical Co., Rockford, Illinois; Research Biochemicals, Cleveland, Ohio; Van Waters and Rogers, South San Francisco, California; and Bachem, Inc., Torrance, California.

EXAMPLE 1

PREPARATION OF OOPPER(ir_:PEPTIDE COMPLEXES

The copper(H):peptide complexes of the present invention may be synthesized by dissolving the peptide in distilled water, followed by the addition of purified copper(II) chloride and a then adjusting the pH of the solution. For example, copper(II) complexes of glycyl-L-histidyl-L ysine ("GHL") with a molar ratio of peptide to copper(II) of 1:1, 2:1, or greater (e.g., 3:1), may be prepared by dissolving a given weight of GHL in distilled water (e.g., 50 mg/ml), and adding the desired molar amount of purified copper(II) chloride. The pH of the resulting peptide solution is then adjusted to about 7.0 by the addition of a sodium hydroxide solution. Alternatively, copper(II) salts other than the copper(II) chloride may be utilized, such as copper(II) acetate or copper(II) sulfate. EXAMPLE 2

SYNTHESIS OF GLYCYL-L-HISTIDYL-L-LYSINE OCTYL ESTER:COPPER (U.

A mixture of N^e-ben2yloxycarbonyl-L-Lysine, n-octanol, benzene, and p-toluenesulfonic acid monohydrate was refluxed overnight using a Dean- Stark trap to remove water. After cooling, dry ethyl ether was added. The solution was then allowed to precipitate at 0°C overnight A portion of the precipitate solid was added to 50 ml of potassium carbonate solution and 50 ml of dichloromethane. After extraction, the layers were separated and the organic phase was washed with water and brine, then dried with anhydrous magnesium sulfate. Filtration, evaporation and purification by flash column chromatography gave n-octyl N^e-benzyloxycarbonyl-L-lysinate.

The product was dissolved in tetrahydrofuran and mixed with N^a-t- butyloxycarbonyl-N^ιm-benzyloxycarbonyI-L-histidine, isobutyl chloroformate and N-methylmorpholine. After evaporation, water and ethyl acetate were added. The product was extracted into the organic phase, which was dried with anhydrous magnesium sulfate. Filtration, evaporation, and purification by flash column chromatography gave n-octyl N^a-t-butyloxycarbonyl-N^m-benzyloxycarbonyl-L- histidyl-N^e-ben2yloxycarbonyl-I_Λysinate. The product was dissolved in 50% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming n-octyl N¹¹¹¹- benzylo^carbonyl-L-histidyl-N^e-ben2ylo^carbonyl-L-lysinate. This was dissolved in tetrahydrofuran, and isobutyl chlorofoπnates, N-methylmorpholine and benzyloxycarbonylglycine were added to form n-octyl benzyloxycarbonylglycyl- N^ιm-ben2yloxycarbonyl-L-histidyl-N^e-ben^lo^carbonyl-L-lysinate. This was dissolved in glacial acetic acid and hydrogenated overnight in the presence of 10% Pd-C catalyst

The resultant n-octyl ester of glycyl-L-histidyl-I ysine was converted to the copper(π) complex by dissolving water and mixing with equimolar copper(II) acetate. The pH was raised to neutrality with sodium hydroxide. The solution was centrifuged at 20,000 C g for 1 hour at 3°C to remove poorly soluble material. The supernatant solution was lyophilized to obtain glycyl- I--histidyl-L-lysine n-octyl ester:copper(II). EXAMPLE 3

SYNTHESIS OF GLYCYL-L-HISTIDYI_^L-LYSINE N-OCTYL AMIDE

A solution of N^a-t-butyloxycarbonyl-N^e-benzyloxycarbonyl-I ysine in tetrahydrofuran was treated with N-methyl-morpholine, isobutyl chloroformate, and octylamine at -15°C^* The resulting fully protected octyl amide was then treated with 50% trifluoroacetic acid in dichloromethane at room temperature, neutralized with saturated aqueous potassium bicarbonate solution, and extracted into ethyl acetate. Evaporation gave the deblocked lysinamide which was added to a solution prepared from N^a-t-butyloxycarbonyl-N^ιm-benzyloxycarbonyl-L- histidine, N-methylmorpholine, and isobutyl chloroformate in dry tetrahydrofuran at -15°C.

The fully protected dipeptide formed above was deblocked by treatment with 50% trifluoroacetic acid in dichloromethane at room temperature followed by neutralization with saturated aqueous potassium bicarbonate. Extraction into ethyl acetate and evaporation gave the partially deblocked dipeptide, which was added to a solution prepared from benzyloxycarbonyl glycine, N-methylmorpholine, and isobutyl chloroformate in dry tetrahydrofuran at -15°C. The resulting protected tripeptide was deblocked by treatment with hydrogen in the presence of 10% palladium on carbon in glacial acetic acid. Filtration and lyophilization gave glycyl-I ristidyl-I ysine n-octyl amide as its triacetate salt.

EXAMPLE 4 SYNTHESIS OF GLYCYL-L-HISTIDYI_^I^LYSYL-L-VALYL-

I_^PHENYLALANYI^L-VALINE

Glycyl-I^histidyl-I^lysyl-L-valyl-L-phenylalanyl-L-valine was synthesized by standard solution phase method using t-butyloxycarbonyl protecting group for the alpha nitrogen, benzyloxycarbonyl group for side-chain protection and mixed anhydride method for coupling. Briefly stated, L-valine benzyl ester p- toluenesulfonate salt was coupled with t-butyloxycarbonyl-L-phenylalanine using isobutyl chloroformate and N-methylmorpholine as a coupling agent (2 hours at - 20°C, then 1 hour at ambient temperature). The t-butyloxycarbonyl protecting group of the dipeptide was then removed by 30% trifluoroacetic acid in dichloromethane at room temperature for 30 minutes. Blocked amino acids (t- butyloxycarbonyl-L-valine, N^t-butyloxycarbonyl-N^benzyloxycarbonyl-I ysine, N^a-t-butylo^carbonyl-N^un-benzyloxycarbonyl-L-histidine, benzyloxycarbonylglycine) were added in sequential order, and t-butyloxycarbonyl protecting groups were removed to obtain the desired peptide. The final peptide was completely deprotected using hydrogen gas in acetic acid for 5 days in the presence of 10% Pd-C catalyst. The final peptide was lyophilized from water to obtain the tri-acetate salt.

EXAMPLE 5 SYNTHESIS OF I_^ALANYI_^L-HTSTIDYL-L-LYSINE

N^e-berιzyloxycarbonyl-L-lysine benzyl ester hydrochloride salt was suspended in tetrahydrofuran (THF) and coupled with N^a-t-butyloxycarbonyl- N^m-benzyloxycarbonyl-L-histidine using isobutyl chloroformate and N- methylmorpholine (2 equivalents) in THF. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. The product was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave benzyl N^a-t-butylo^carbonyl-N^ιm-ben2yloxycarbonyl-L-lιistidyl- N^e-benzyloxycarbonyl-L-lysinate.

The product was dissolved in 30% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporate, forming benzyl N¹¹¹¹- ben2ylθ3tycarbonyl-L-histidyl-N^e-ben^lθ3_ycarbonyl-L-lysinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methyl-morpholine and

^{im e} was then dissolved in acetic acid and hydrogenated overnight in the presence of 10% Pd-C catalyst The resultant alanyl-L-histidyl-L-lysine was lyophilized from water several times to yield the desired tripeptide as a diacetate salt

EXAMPLE 6 SYNTHESIS OF L-LYS YL-L-HISΗDYL-GLYCINE

N^a-t-butyloxycarbonyl-N^ιm-ben^loxycarbonyl-L-histidine was dissolved in tetrahydrofuran (THF) and neutralized with one equivalent of N- methylmorpholine. It was then coupled with benzyl glycinate p-toluenesulfonate salt using isobutyl chloroformate and N-methylmorpholine. After two hours at - 20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. The product was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave benzyl N^a-t-butyloxycarbonyl-N^im-ben^loxycarbonyl-I_rhistidyl- glycinate.

This product was dissolved in anhydrous methanolic hydrogen chloride (saturated at 0°C) for 5 minutes, followed by removal of solvent under reduced pressure, forming benzyl N^m-benzyloxycarbonyl-L-histidyl-glycinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N- methylmorpholine and N^a,N^e-dibenzyloxycarbonyl-L-lysine were added to form benzyl N^a,N^e-dibenzyloxycarbonyl-L-lysyl-N^mι-benzyloxycarbonyl-L-histidyl- glycinate (3 hours at -20°C, the 1 hour at ambient temperature). This product was then dissolved in methanol/acetic acid, 1:1 (v/v), and hydrogenated overnight in the presence of 10% Pd-C catalyst. The resultant L-lysyl-L-histidyl-glycine was lyophilized from water several times, then purified by liquid chromatography on a C-18 reverse-phase column to yield the desired tripeptide triacetate salt as a foamy white solid.

EXAMPLE 7

SYNTHESIS OF I_^LYSYL-L-HISTIDYI_^GLYCYL-I_^VALYL- I_^PHENYLALANYL-L-VALINE I_^lysyl-L-histidyl-glycyl-L-valyl-L-phenylalanyl-L-valine was synthesized by standard solution phase method using t-butyloxycarbonyl protecting group for the alpha nitrogen, benzyloxycarbonyl group for side-chain protection and mixed anhydride method for coupling. Briefly stated, L-valine benzyl ester p- toluenesulfonate salt was coupled with t-butyloxycarbonyl-L-phenylalanine using isobutyl chloroformate and N-methylomorpholine as coupling agent (2 hours at - 20°C, then 1 hour at ambient temperature). The t-butyloxycarbonyl protecting group of the dipeptide was then removed by 30% trifluoroacetic acid in dichloromethane at room temperature for 30 minutes. Blocked amino acids (t- butyloxycarbonyl-L-valine, t-butyloxycarbonylglycine, N^t-butyloxycarbonyl-N¹¹¹¹- benzyloxycarbonyl-L-histidine, N^a, N^e-dibenzyloxycarbonyl-L-lysine) were added in sequential order and t-butyloxycarbonyl protecting groups were removed to obtain the desired peptide. The final peptide was completely deprotected using hydrogen gas in glacial acetic acid for five days in the presence of 10% Pd-C catalyst. The final peptide was lyophilized from water and purified by liquid chromatography on a C-18 reverse phase column to produce the desired hexapeptide in multi-gram quantity. The above systematic synthesis proved advantageous over some of the solid phase methods in providing multi-gram quantity of the desired peptide in high purity with minimal purification.

EXAMPLE S

SYNTHESIS OF GLY YL-L-HISTIDYI_^I_^CAPROLACTAM

L(-)-3-amino-e-caprolactam was dissolved in tetrahydrofuran (THF) then coupled with N^a-t-butylo^carbonyl-N^im-benzylojtycarbonyl-L-histidine using isobutyl chloroformate and N-methylmorpholine in THF. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. This produce was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave N^a-t-butyloxycarbonyl-N^im-benxyloxycarbonyl-L-histidyl-L- caprolactam.

The above protected dipeptide was dissolved in 30% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming N¹¹¹¹- benzyloxycarbonyl-L-histi fl-L-caprolactam. This was then dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and benzyloxycarbonylglycine were added to form benzyloxycarbonylglycyl-N^lιn- be:nzyloxycarbonyl-I_ histidyl-L-caprolactam. This product was recrystallized once from ethyl acetate then dissolved in acetic acid and hydrogenated overnight in the presence of 10% Pd-C catalyst The resultant glycyl-L-histidyl-I^caprolactam was lyophilized from water several times, then purified by liquid chromatography on a C-18 reverse-phase column to yield the desired tripeptide as a diacetate salt.

EXAMPLE 9 SYNTHESIS OF L-HISTIDYI_^GLYCYL-L-LYSINE

N^e-benxyloxycarbonyl-L-lysine benzyl ester hydrochloride salt was suspended in tetrahydrofuran (THF) and coupled with N^a-t-butyloxycarbonyl- glycine using isobutyl chloroformate and N-methylmorpholine in THF. After two hours at -20°C and an additional hour at ambient temperature, the reaction was quenched with 2 N aqueous potassium bicarbonate. The produce was extracted into ethyl acetate, washed with 1 M aqueous citric acid, and saturated sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate. Filtration and evaporation gave benzyl N^t-butyloxycarbonyl-glycyl-N⁶- benzyloxycarbonyl-L-lysinate. The product was dissolved in 30% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming benzyl glycyl-N^e- benzyloxycarbonyl-I ysinate. This was dissolved in tetrahydrofuran, and isobutyl chloroformate, N-methylmorpholine and N^benzyloxycarbonyl-N¹¹¹¹- benzyloxycarbonyl-L-histidine were added to form benzyl N^a-benzylojtycarbonyl- N^im-benzylo^carbonyl-L-histidyl-glycyl-N^e-benzyloxycarbonyl-_ lysinate. This product was then dissolved in acetic acid and hydrogenated overnight in the presence of 10% Pd-C catalyst. The resultant L-histidyl-glycyl-L-lysine was lyophilized from water several times to yield the desired tripeptide as a diacetate salt.

EXAMPLE 10

SΉMULAΉON OF WOUND HEALING BY I.M. INJECTION OF GLYCYL-HISΗDYL-LYSINE:COPPERrm The subcutaneous implantation of stainless steel wound chambers in rats provides a model for the healing of open cavity wounds. This model in many ways mimics the early events in the formation of granulation tissue found in many types of wounds and surgical defects. Implantation of these chambers triggers a series of responses which reflect the series of phases involved in wound healing - fibrin clot formation, infiltration of white cells, collagen synthesis, and new blood vessel formation.

The assay involves the implantation of two Stainless steel chambers (1 x 2.5 cm cylindrical 312 SS, 20 mesh, with Teflon end caps ) on each side of the dorsal mid-line of rats. After one week to allow for encapsulation of the chambers, the animal was injected with a solution containing a copper(H) compound of the present invention. Controls consist of chambers injected with the same volume of saline. Injections were made on days 5,7,8,12,13,15,19. The chambers were removed at day 15 & 29.

The chambers were lyophylized and the interior contents removed for biochemical analysis. The biochemical parameters examined include the total dry weight, protein content, angiogenesis (Alkaline Phosphatase activity), and collagen content (Hydroxyproline content after acid hydrolysis). Significant increases have been found in all the biochemical parameters tested.

The protein was determined by the method of Lowry (J. Biol. Chem.. 122:265-275, 1951) using Bovine Serum Albumin (BSA) as a standard. Angiogenesis was determined by measuring the amount of Alkaline Phosphatase using p-nitrophenyl phosphate as a substrate (G. Lyles et al., Biochem. Pharm.. 21:2569-2574, 1984). The collagen content was determined by acid hydrolysis and a colormetric assay for hydroxyproline (I. Bergman, Clin. Chim. Acta. 22:347-349, 1970), an amino acid specific for collagen.

The total protein is expressed as mg protein (relative to BSA) per chamber. Angiogenesis is expressed as alkaline phosphatase (AP) Units per chamber, where one Unit = the amount of homogenate which causes an increase in absorbance at 405 nm of 1.0 per min.. The hydroxyproline ( HP, Collagen

Content ) is expressed as μg HP per chamber.

After allowing for encapsulation of the chambers, the rats were injected LM. with 0.1 ml of either a saline solution containing 10 mg/ml glycyl-Lr histidyl-L-lysine:copper(II) (2:1 molar ratio) or saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described above. The LM. injection of this compound significantly increased the biochemical healing parameters in the rats as summarized in Table 1.

TABLE 1. EFFECT OF LM. GHL_^Cu ON WOUND HEALING IN RATS

COMPOUND DOSE DRY WEIGHT PROTEIN COLLAGEN ANGIOGENESIS mg/injection mg mg/chamber μg HP/chamber Units AP/chamber

STIMULATION OF WOUND HEALING IN HEALING IMPAIRED RATS BY LM. INJECTION OF GLYCYL-fflSTIDYL-LYSINE:COPPER(Tn

Groups of rate had wound chambers implanted as described in

Example 10. After implantation of the chambers, the rats were subsequently injected with Cortisone Acetate (10 mg LM. daily, Cortone Acetate, Merck) to impair the healing response. After allowing for encapsulation of the chambers, the rats were injected LM. (in the opposite leg from the cortisone injection) with 0.1 ml of either a saline solution containing 10 mg/ml of glycyl-L-histidyl-Lr lysine:copper(II) (2:1 molar ratio) or saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described in Example 10. 5 The LM. injection of glycyl-L-histidyl-L-lysine:copper(II) in the cortisone treated animals increased the level of all the biochemical parameters examined compared to the level found in the control animals. Moreover, LM. treatment with the GHL-Cu increased the healing response in the healing impaired animals to the level found in the normal animals. The results of this l o experiment are presented in Table 2.

TABLE 2

EFFECT OF LM. GHI^Cu ON WOUND HEALING IN HEALING IMPAIRED RATS

15

20 HEALING IMPAIRED RATS

SALINE ~ 20 ± 12 11 ± 8 187 ± 101 0.5 ± 03

GHL-Cu 1.0 35 + 13 25 + 9 366 ± 74 IS + 1.6

25

NOTES TO TABLE 1: GHL:Cu = glycyl-L-histidyl-L-lysine:copper(II) (2:1 Molar Complex)

30 EXAMPLE 12.

STIMULATION OF WOUND HEALING BY LM. INJECTION OF

GLYCYL-mSΗDYL-LYSINE-VALINE-PHENYLALANYI_^

VALINE:COPPERQD

Groups of rats had wound chambers implanted as described in

35 Example 10. After implantation of the chambers, the rate were subsequently injected with Cortisone Acetate (10 mg LM. daily, Cortone Acetate, Merck) to impair the healing response. After allowing for encapsulation of the chambers, the rats were injected LM. (in the opposite leg from the cortisone injection) with

0.1 ml of either a saline solution containing 19 mg/ml of glycyl-L-histidyl-L-lysyl-

40 valyl-phenylalanly-valine:copper(II) or saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described in Example 10.

The LM. injection of glycyl-L-histidyl-L-lysyl-valyl-phenylalanly- valine:copper(II) in the cortisone treated animals increased the level of all the biochemical parameters examined. This experiment is summarized in Table 3.

NOTES TO TABLE 1: GHL:Cu = glycyl-L-histidyl-L-lysine:copper(II) (2:1 Molar Complex) GHLVFV:Cu = glycyl-I^histidyl-L-lysyl-L-valyl-L-phenylalanyl-L-valine:copper(II) (2:1 Molar Complex)

EXAMPLE 13.

STIMULATION OF WOUND HEALING IN HEALING IMPAIRED RATS BY LM. INJECTION OF COPPER COMPOUNDS

Groups of rats had wound chambers implanted as described in

Example 10. After implantation of the chambers, the rats were subsequently injected with Cortisone Acetate (10 mg LM. daily, Cortone Acetate, Merck) to impair the healing response. After allowing for encapsulation of the chambers, the rate were injected LM. (in the opposite leg from the cortisone injection) with 0.1 ml of the copper(H) compounds listed at the dosage specified in Table 4, or saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described in Example 10.

The LM. injection of these compounds in the cortisone treated animals increased the level of collagen (a major component of granulation tissue) accumulation above the control level found in the saline injected animals. TABLE 4

EFFECTS OF COPPER COMPOUNDS ON WOUND HEALING IN HEALING IMPAIRED RATS

NOTES TO TABLE 4 GGG:Cu = glycyl-gIycyl-glycine:copper(II) (2:1 Molar complex) CuS0 = cupric sulfate solution CARN:Cu = carnosine:coppper(II) (2:1 Molar complex)

CΓΓRATE:CU = citrate:copper(II) (2:1 Molar complex)

EXAMPLE 14 STIMULATION OF WOUND HEALING BY LM. INJECTION OF

GLYCYL-HISTIDYL-LYSINE:COPPER(π) IN MICE

The ability of copper(II) containing compounds to accelerate wound healing in mice is demonstrated in a full thickness defect model. Following an acclimation period, mice are anesthetized with I.P. pentobarbital and prepped for surgery. The hair is plucked from a wide area of the mid-back. An ink stamp bearing a 15mm diameter circle is used to mark the skin for subsequent full- thickness excision. Tissue is excised to the fascia underlying the paniculus camosus muscle. Following excision, hemostasis is achieved through irrigation and the use of sterile gauze pads. Mice received a first LM. treatment of GHL-Cu following hemostasis, Day 0. Injections consisted of 0.1 mg of GHI_^Cu in a volume of 0.1 ml. Mice used as controls received injections of saline. Injections were repeated on days 1, 2, 3, 6, 7, 8, 9, 10, and 13. The residual wound surface area is measured on days 1, 7, 10 and 14. The final comparison is performed on Day 14. Table 5 below illustrates that LM. injection of GHL-Cu resulted is an approximately 75% smaller residual wound size at 14 days.

TABLE 5

WOUND HEALING ACTIVITY OF GHL-Cu IN MICE AFTER LM. INJECTION

TREATMENT RESIDUAL WOUND SURFACE AREA (SQ MM)

DAY 14

SALINE 1.78 + 2.56

GHL:CU 0.42 ± 0.60

NOTES TO TABLE 5: GHL:Cu = gly<yl-L-I_istidyl-L-lysine:copper(II) (2:1 Molar Complex)

EXAMPLE 15

STIMULATION OF WOUND HEALING IN PIGS BY M. INJECTION OF GLYCYL-HISTIDYL-LYSINE:COPPER( The systemic application of GHL-Cu increases the formation of granulation tissue in pigs. Two domestic pigs approximately 29-31 pounds, about 8 weeks old, were anesthetized with Ketamine (30 mg/Kg) and Rompum (5 mg/Kg). A series of full thickness skin defects measuring 2.0 x 2.0 cm were created on the backs of each pig and bandaged with gauze and adhesive tape. One pig was treated with LM. injections of sterile saline and the other with a solution of GHL-Cu (30 mg/ml). Each pig received 1.0 ml injections of either the saline or GHL-Cu solution on days 0, 1, 2, 5, 6, 7, 8, 9, 12, and 13.

Punch biopsy samples were taken from the centers of the healing wounds on day 7 and day 12 and analyzed for the biochemical parameters of granulation tissue and described in Example 10. The resulte show that there was a significant increase in the weight, collagen content, and angiogenesis in the biopsies from the GHL-Cu treated pig. The data from this experiment is summarized in Table 6 below. In addition, visual examination showed that the granulation tissue in the GHI_^Cu treated pig was raised above the level of the surrounding skin, compared to a level below the skin for the saline treated animal. TABLE 6

EFFECT OF LM. GHI_^Cu ON WOUND HEALING

IN PIGS

TREATMENT DOSE WET WEIGHT COLLAGEN ANGIOGENESIS mg/injection mg μg HP/chamber Units AP/chamber

SALINE — 121 ± 7 628 ± 182 0.4 ± 0.1

GHL:Cu 30 148 ± 11 855 ± 20 2.2 ± 12

NOTES TO TABLE 1: GHL:Cu = glycyl-L-histidyl-L-lysme:copper(II) (2:1 Molar Complex)

EXAMPLE 16.

SΗMULATION OF HEALING BY HYDROPHOBIC AND ALBUMIN BINDING DERIVATIVES OF GHL-Cu Groups of rate had wound chambers implanted as described in

Example 10. After allowing for encapsulation of the chambers, the compounds to be tested were injected (0.2 ml at the dosage specified) directly through the rat skin and into the wound chambers. Controls received injections of saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described in Example 10.

As shown in Table 7, the modification of GHL-Cu by the addition of hydrophobic moieties stimulates the healing parameters in rats.

TABLE 7 EFFECT OF HYDROPHOBIC DERIVATIVES OF GHI^Cu

ON WOUND HEALING

NOTES TO TABLE 7: GHLW:Cu = glycyl-L-histidyl-L-lysyl-L-tryptophan:copper(II) (2:1 Molar complex) GHL-Octyl EsteπCu = glycyl-L-histidyl-L-fysine octyl ester:copper(II) (2:1 Molar Complex) GHI )ctylAmide:Cu = glycyl-Lz-hisύdyl-L-lysine octyl amide:copper(II) (2:1 Molar Complex)

EXAMPLE 17.

STIMULATION OF WOUND HEALING BY SEQUENCE VARIATIONS OF GHI_^Cu

Groups of rats had wound chambers implanted as described in

Example 10. After allowing for encapsulation of the chambers, the compounds were injected (0.2 ml at the dosage specified) directly through the rat skin and into the wound chambers. Controls received injections of saline. The chambers were harvested and the biochemical parameters of granulation tissue formation examined as described in Example 10. The resulte are summarized in Table 8.

TABLE 8

EFFECT OF SEQUENCE MODIFICATIONS OF GHI_^Cu ON WOUND HEALING

COMPOUND DOSE WET WEIGHT PROTEIN COLLAGEN ANGIOGENESIS mg/injection mg mg/chamber μg HP/chamber Units AP/chamber

NOTES TO TABLE 8: G(3-Me)HL:Cu = glycyl-L-(3-methyl)histidyl-L-lysine:copper(π) (2:1 Molar complex) GHCaprolactam:Cu = glycyl-L-histidyl-caprolactam:copper(II) (2:1 Molar complex) AHL:Cu = alanyl-L-histidyl-L-lysine:copper(II) (2:1 Molar complex) HGL:Cu = histidyl-glycyl-L-lysine:copper(π) (2:1 Molar complex)

LHGVFV:Cu = lysyl-L-histidyl-glycyl-L-valyl-L-phenylalanyl-L-valine:copper(π) (2:1 Molar Complex)

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims

1. A composition comprising: R :copper H), wherein Ri is a naturally occurring amino acid or a derivative of a naturally occurring amino acid, for use as an active therapeutic substance.

2. A composition comprising: Ri:copper(II), wherein Ri is a naturally occurring amino acid or a derivative of a naturally occurring amino acid, for use in accelerating wound healing in warm-blooded animals.

3. Use of a composition comprising: Ri:copper(II), wherein Ri is a naturally occurring amino acid or a derivative of a naturally occurring amino acid, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

4. A composition comprising: Rl-R2:copper(II), wherein Ri and R2 are naturally occurring amino acids or derivatives of naturally occurring amino acids, for use as an active therapeutic substance.

5. A composition comprising: Rl-R2:copper(II), wherein Ri and R2 are naturally occurring amino acids or derivatives of naturally occurring amino acids, for use in accelerating wound healing in warm-blooded animals.

6. Use of a composition comprising: Rl-R2:copper(II), wherein Ri and R2 are naturally occurring amino acids or derivatives of naturally occurring amino acids, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

7. A composition comprising: glycyl-glycyl-glycine:copper(II), for use as an active therapeutic substance.

8. A composition comprising: glycyl-glycyl-glycine:copper(II), for use in accelerating wound healing in warm-blooded animals.

9. Use of a composition comprising: glycyl-glycyl-glycine:copper(H), for the manufacture of a medicament for accelerating wound healing in a warm¬ blooded animal.

10. A composition comprising: [glycyl-I_^histidyl-I--lysine-R]:copper(II), wherein R is an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L- tryptophan, (glycyl)y-L-tιyptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xl-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L- valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n-L- tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

11. A composition comprising: [glycyl-L-histidyl-___rlysine-R]:copper(II), wherein R is an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L- tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xl-L-phenylalanyl-X2, where X and X2 are selected from the group consisting of L- valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_π-L- tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

12. Use of a composition comprising: [glycyl-L-histidyl-L-lysine-R]:copper(II), wherein R is an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L- tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xl-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L- valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n-L- tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

13. A composition comprising: [glycyl-Rι-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl, where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R₂ is -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

14. A composition comprising: [glycyl-Rι-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of l-histidyl, ____-(3-W)-histidyl and L-(5-W)-histidyl, where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, l-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

15. Use of a composition comprising: [glycyl-Rι-R2-R3.:copρer(II), wherein

R2 is -NHCH((CH2)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2_» ^an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_ where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

16. A composition comprising: [glycyl-Rι-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of L-(3-W)-histidyl and L-(5-W)- histidyl where W is an alkyl moiety contaimng from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl; and

R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)_n- I_^tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X )_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

17. A composition comprising: [glycyl-Rι-R2-R3]:copper(π), wherein R is selected from the group consisting of I_^(3-W)-histidyl and L-(5-W)- histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl; and

R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tιyptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where X and X2 are not both L-valine, (X3)n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

18. Use of a composition comprising: [glycyl-Rι-R2-R3]:copper(II), wherein

R is selected from the group consisting of L_^(3-W)-histidyl and L-(5-W)- histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is L-lysyl; and

R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or Xι-L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, and where Xi and X2 are not both L-valine, (X3)n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X )_n> where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

19. A composition comprising: [glycyl-Rι-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of Lrhistidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety contaimng from 6-12 carbon atoms;

R2 is a basic amino acid, a basic amino moiety, or a modified basic amino acid; and R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (gtycyl)y-L-tryptophan, ^nere y ^{= 1_4}» L-prolyl-Xi-L-phenylalanyl-X2 or Xi-Lr phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_> where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

20. A composition comprising: [glycyl-Rι-R2-R3]:copper(II), wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is a basic amino acid, a basic amino moiety, or a modified basic amino acid; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-___rtryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, l-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

21. Use of a composition comprising: [glycyl-Rι-R2-R3]:coρper(II), wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; R2 is a basic amino acid, a basic amino moiety, or a modified basic amino acid; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or

phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_ where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

22. A composition comprising:

[Rl-R2-glycine-R3]:copper(II), wherein

Rl is L-lysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10;

R2 is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and ___r(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl rL-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-Lr phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or - X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

23. A composition comprising:

[Rl-R2-glycine-R3]:copper(ϋ), wherein

Rl is L-lysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; R2 is selected from the group consisting of I iistidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n> where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

24. Use of a composition comprising: [Rl-R2-glycine-R3]:copper(π), wherein

Rl is L-lysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10;

R2 is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, l-alanine and glycine, (X3)_n-I_rtryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

25. A composition comprising: [Rl-R₂-R3-R4]:copper(π), wherein

Rl is L-lysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10; R2 is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R3 is a basic amino acid, a basic amino moiety, or a modified basic amino acid; and

R4 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_ where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

26. A composition comprising: [Rl-R₂-R3-R4]:copper(π), wherein

Rl is L-lysyl or -NHCH((CH₂)nNH₃ ⁺)CO- where n = 5-10;

R2 is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L-(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R4 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_> where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

27. Use of a composition comprising: [Rl-R₂-R3-R4]:copper(π), wherein

Rl is L-lysyl or -NHCH((CH2)nNH₃ ⁺)CO- where n = 5-10;

R4 is hydrogen, -NH2_> ^an --~y* moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl rL-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or X -L- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X )_Q, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

28. A composition comprising: [Rl-glycyl-R₂-R3]:copper(π), wherein

R2 is L-lysyl, -NHCH((CH2)_nNH3⁺)CO-, where n = 5-10, or a basic amino acid, a basic amino moiety, or a modified basic amino acid; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-I_^tιyptoρhan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or X -Lr phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n_ where X4 is a naturally occurring carbohydrate selected form the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

29. A composition comprising: [Rl-glycyl-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and I^(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is l-tryptophan, (glycyl)yL-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or X -L- phenylalanyl-X2, where X and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n_> where X4 is a naturally occurring carbohydrate selected form the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

30. Use of a composition comprising: [Rl-glycyl-R2-R3]:copper(π), wherein

Rl is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, L-prolyl-Xι-L-phenylalanyl-X2 or Xi-Lr phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, L-alanine and glycine, (X3)_n-L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected form the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

31. A composition comprising: [Lalanyl-R -R2-R3]:copper(II), wherein

Rl is selected from the group consisting of L-histidyl, L(3-W)-histidyl and L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is Llysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is Ltryptophan, (glycyl)y-Ltryptophan, where y = 1-4, Lprolyl-Xi-Lphenylalanyl- X2 or Xι- phenylalanyl-X2, where Xi and X2 are selected from the group consisting of Lvaline, alanine and glycine, and where Xi and X2 are not both Lvaline, (X3)n- Ltryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

32. A composition comprising: [Lalanyl-Rι-R2-R3]:copper(II), wherein

R is selected from the group consisting of histidyl, (3-W)-histidyl and L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R2 is Llysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-Ltryptophan, where y = 1-4, L-prolyl-Xi-Lphenylalanyl- X2 or Xι-Lphenylalanyl-X2, where Xi and X2 are selected from the group consisting of Lvaline, Lalanine and glycine, and where Xi and X2 are not both Lvaline, (X3)n- L-tryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

33. Use of a composition comprising: [Lalanyl-Rι-R2-R3]:copper(π), wherein

R is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms;

R₂ is L-lysyl or -NHCH((CH2)nNH3⁺ )CO- where n = 5-10; and R3 is -NH2, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is Ltryptophan, (glycyl)yL-tryptophan, where y = 1-4, L-prolyl-Xi-L-phenylalanyl- X2 or X -L-phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, Lalanine and glycine, and where Xi and X2 are not both L-valine, (X3)n- Ltryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

34. A composition comprising: [glycyl-Rι-R2-R3]:copper(II), wherein

Rl is L-lysyl or -NHCH((CH₂)_nNH3⁺)CO- where n = 5-10 2 is selected from the group consisting of L-histidyl, L-(3-W)-histidyl and _L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is L-tryptophan, (glycyl)y-L-tryptophan, where y = 1-4, Lprolyl-Xι-L-phenylalanyl-X2 or Xi-Lr phenylalanyl-X2, where Xi and X2 are selected from the group consisting of L-valine, Lalanine and glycine, (X3)_n-Ltryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n. where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use as an active therapeutic substance.

35. A composition comprising: [glycyl-Rι-R2-R3]:coρper(II), wherein

Rl is Llysyl or -NHCH((CH2)_nNH3⁺)CO- where n = 5-10 R2 is selected from the group consisting of Lhistidyl, L(3-W)-histidyl and L(5-W)-histidyl where W is an alkyl moiety containing from 1 to 12 carbons atoms or aryl moiety containing from 6-12 carbon atoms; and

R3 is hydrogen, -NH2, an alkyl moiety containing from 1 to 18 carbon atoms, an aiyl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety containing from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is Ltryptophan, (glycyl)y-Ltryptophan, where y = 1-4, Lprolyl-Xι-Lphenylalanyl-X2 or Xi-L phenylalanyl-X2, where X and X2 are selected from the group consisting of Lvaline, Lalanine and glycine, (X3)_n-Ltryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)n, where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for use in accelerating wound healing in warm-blooded animals.

36. Use of a composition comprising: [glycyl-Rι-R2-R3]:copper(II), wherein

R3 is hydrogen, -NH2, an alkyl moiety contaimng from 1 to 18 carbon atoms, an aryl moiety containing from 6 to 12 carbon atoms, an alkoxy moiety containing from 1 to 18 carbon atoms, an aryloxy moiety contaimng from 6-12 carbon atoms, an aminoalkyl moiety containing from 1 to 18 carbon atoms, or is Ltryptophan, (glycyl)y-Ltryptophaιι, where y = 1-4, Lprolyl-Xι-Lphenylalanyl-X2 or Xi-L phenylalanyl-X2, where Xi and X2 are selected from the group consisting of Lvaline, Lalanine and glycine, (X3)_n-Ltryptophan, where X3 is a -CH2- or -CH(OH)- moiety and n = 4-20, or -(X4)_n. where X4 is a naturally occurring carbohydrate selected from the group consisting of glucose, mannose, galactose, glucosamine and galactosamine and n = 1-5, for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

37. A composition comprising a copper(H) salt for use in accelerating wound healing in warm-blooded animals.

38. Use of a composition comprising a copper(ϋ) salt for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.

39. A composition comprising copper(II) citrate for use in accelerating wound healing in warm-blooded animals.

40. Use of a composition comprising copper(II) citrate for the manufacture of a medicament for accelerating wound healing in a warm-blooded animal.