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Validação Cientifica - Eficácia dos Peptídeos (GHK) na Regeneração Moduladora de Folículos Capilares e da Pele +

Skin Biology, Research & Development Department, 4122 Factoria Boulevard, SE Suite No. 200 Bellevue, WA 98006, USA

Received 21 November 2014; Revised 17 March 2015; Accepted 9 April 2015

Academic Editor: May Griffith

Copyright © 2015 Loren Pickart et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

GHK (glycyl-L-histidyl-L-lysine) is present in human plasma, saliva, and urine but declines with age. It is proposed that GHK functions as a complex with copper 2+ which accelerates wound healing and skin repair. GHK stimulates both synthesis and breakdown of collagen and glycosaminoglycans and modulates the activity of both metalloproteinases and their inhibitors. It stimulates collagen, dermatan sulfate, chondroitin sulfate, and the small proteoglycan, decorin. It also restores replicative vitality to fibroblasts after radiation therapy. The molecule attracts immune and endothelial cells to the site of an injury. It accelerates wound-healing of the skin, hair follicles, gastrointestinal tract, boney tissue, and foot pads of dogs. It also induces systemic wound healing in rats, mice, and pigs. In cosmetic products, it has been found to tighten loose skin and improve elasticity, skin density, and firmness, reduce fine lines and wrinkles, reduce photodamage, and hyperpigmentation, and increase keratinocyte proliferation. GHK has been proposed as a therapeutic agent for skin inflammation, chronic obstructive pulmonary disease, and metastatic colon cancer. It is capable of up- and downregulating at least 4,000 human genes, essentially resetting DNA to a healthier state. The present review revisits GHK’s role in skin regeneration in the light of recent discoveries.

1. Introduction

GHK is a tripeptide with the amino acid sequence glycyl-histidyl-lysine. It naturally occurs in human plasma, saliva, and urine. In plasma the level of GHK is about 200 ng/mL (10−7 M) at age 20, but declines to 80 ng/mL by age 60. This decline in the GHK-level coincides with the noticeable decrease in regenerative capacity of an organism. The human peptide GHK-Cu was isolated in 1973 by Pickart as an activity in human albumin that caused old human liver tissue to synthesize proteins like younger tissue [1]. Subsequent studies established this activity as a tripeptide with an amino acid sequence glycyl-L-histidyl-L-lysine with a strong affinity for copper that readily formed the complex GHK-Cu. It was proposed that GHK-Cu functions as a complex with copper 2+ [2]. Pickart et al. have established that GHK-Cu accelerates wound healing and contraction, improves the take of transplanted skin, and also possesses antiinflammatory actions [35].

Subsequent studies directed by Borel and Maquart et al. demonstrated that GHK-Cu at a very low, nontoxic concentration (1–10 nanomolar) stimulated both synthesis and breakdown of collagen and glycosaminoglycans [6]. GHK modulated an activity of both metalloproteinases and their inhibitors (TIMP-1 and TIMP-2), acting as a main regulator of wound healing and skin remodeling processes [78]. GHK-Cu stimulated collagen, dermatan sulfate, chondroitin sulfate, and a small proteoglycan, decorin [9]. In 2001 McCormack et al. established that GHK-Cu restored replicative vitality to fibroblasts from patients after anticancer radiation therapy that damages cellular DNA [10]. GHK was also found to attract immune and endothelial cells to the site of an injury [11].

Wound healing activity of GHK-Cu was confirmed in animal experiments. GHK-Cu accelerated wound healing and increased blood vessel formation and the level of antioxidant enzymes in rabbits. This molecule also induced systemic wound healing in rats, mice, and pigs. It improved the healing of diabetic and ischemic wounds in rats, decreasing the level of TNF-alpha and stimulating collagen synthesis. It also facilitated healing of pad wounds in dogs [1217]. Such well-documented skin regeneration activity prompted widespread use of GHK in antiaging cosmetic products [18].

Recently, GHK-Cu has been gaining publicity as a prospective therapeutic agent for chronic obstructive pulmonary disease (COPD), skin inflammation, and metastatic colon cancer [1921]. It has been established that it is capable of up- and downregulating at least 4,000 genes in the human genome, essentially resetting DNA back to a healthier state [22]. These studies shed new light on the skin remodeling activity of the GHK-Cu peptide.

The present review revisits GHK-Cu’s role in skin regeneration in the light of recent discoveries.

2. GHK Restores TGF-Beta Pathway in COPD Lungs

A collaborative study conducted by scientists from Boston University, University of Groningen, University of British Columbia, and University of Pennsylvania established that the GHK peptide reverses the gene expression signature of COPD, which is manifested by emphysema, inflammation, lung tissue destruction, and significant reduction of lung capacity.

The researchers identified 127 genes whose expression was significantly associated with emphysema severity. Among those genes, whose expression was upregulated in COPD patients, were genes involved in inflammation, while expression of genes involved in tissue remodeling and repair was markedly downregulated. Among the genes displaying decreased activity were genes involved in the TGF-beta pathway. Using the Connectivity Map, a software gene profiling tool developed by the Broad Institute, the researchers identified GHK as a compound which reversed changes in gene expression associated with emphysematous destruction. In particular, patients with COPD displayed a decreased activity of genes involved in the TGF-beta pathway. GHK reversed the gene expression pattern so it became consistent with the activation of the TGF-beta pathway.

In vitro studies confirmed that treating lung fibroblasts with GHK reversed negative changes associated with decreased TGF-beta activity. It has been established that lung fibroblasts derived from COPD patients had certain defects, which impaired their ability to contract and remodel collagen gel. When such fibroblasts were treated with either GHK or TGF-beta, the contraction and remodeling of collagen gel was restored and became comparable to fibroblasts derived from lungs of exsmokers without COPD. After GHK treatment, the lung fibroblasts derived from COPD patients were able to remodel collagen gel into fibrils. They also had an elevated expression of integrin beta 1. These findings indicate that GHK may be able to improve tissue regeneration by restoring activity of genes involved in the TGF-beta pathway [23].

It is known that skin regeneration requires the participation of multiple cytokines and growth factors. Rather than work separately, they engage in a crosstalk, which involves interaction of different cellular pathways. For example, cellular pathways regulated by TGF-beta and integrins seem to be connected [24]. GHK’s ability to restore the contraction and remodeling of collagen gel in the COPD study demonstrate that GHK is capable of activating both TGF-beta and integrin beta 1 pathways during tissue regeneration. Even though the exact mechanism of GHK’s action is yet to be elucidated, it becomes apparent that the diverse and multiple effects of GHK in skin regeneration can be better understood through its ability to reset the gene pattern back to a healthier state, thereby leading to the activation or deactivation of various cellular pathways.

3. Cancer Metastasis Genes and Skin Remodeling

Hong et al. used genome-wide profiling to identify genetic biomarkers for metastasis prone colorectal cancer as well as their perturbagens, substances that modulated their expression. The search out of 1309 bioactive compounds yielded only two substances that were able to effectively downregulate expression of “metastatic” genes, GHK and the plant alkaloid, securinine. GHK suppressed RNA production in 70% of 54 human genes overexpressed in patients with an aggressive metastatic form of colon cancer. GHK produced the result at a low non-toxic 1 micromolar concentration and securinine at 18 micromolar. The authors point out that both GHK and securinine are well-known skin remodeling agents. Securinine activates macrophages and is a component of traditional African and Chinese medicines for skin injuries [25]. The 54 genes whose expression was reversed by GHK included “node molecules” YWHAB, MAP3K5, LMNA, APP, GNAQ, F3, NFATC2, and TGM2, all of which are involved in regulation of multiple biological functions through a complex molecular network [20]. The fact that GHK was able to suppress 70% of genes involved in the development of an aggressive metastatic form of colon cancer indicates that GHK is capable of the regulation of various biochemical pathways on a gene level and it seems to be resetting the gene activity back to health, which leads to the improvement of tissue repair.

In vitro studies by Matalka et al. found that when three lines of human cancer cells (SH-SY5Y neuroblastoma cells, U937 histolytic cells, breast cancer cells) were incubated in culture with 1 to 10 nanomolar GHK, the programmed cell death system (apoptosis) was reactivated and cell growth inhibited [26].

Pickart et al., using the Broad Institute data, found that GHK induces anti-cancer expression of numerous caspase, growth regulatory, and DNA repair genes. The combination of ascorbic acid and GHK-Cu strongly inhibited the growth of sarcoma-180 in mice [27].

4. Recovery of Skin Stem Cells

Skin regeneration depends on viability and proliferative potential of stem cells. Skin proliferation starts in the basal layer of keratinocytes, which are attached to the basal membrane. When a cell leaves the basal layer, it undergoes terminal differentiation. Stem cells have unlimited self-renewal capacity; however, their proliferative potential declines with age. GHK-Cu, in concentrations of 0.1–10 micromolar, increased expression of epidermal stem cell markers such as integrins and p63 in basal keratinocytes in dermal skin equivalents, which according to the authors indicate increased stemness and proliferative potential of basal keratinocytes. Therefore, restoration of gene pattern characteristic of healthy stem cells, which leads to activation of integrins and p63 cellular pathways may be another target of GHK’s gene modulatory activity relevant to skin regeneration [2829].

A recent study has established that pretreatment of human mesenchymal stem/stromal cells (MSC) with GHK presented in a biodegradable carrier (alginate gel) produced a dose-dependent increase in secretion of proangiogenic factors such as the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. When pretreated with antibodies to integrins alpha 1 and beta 1, MSC failed to produce an increase of VEGF, which indicated that the effects of GHK on secretion of trophic factors by MSC involve the integrin cellular pathway [30].

5. GHK and IL-6 in Skin Repair

Wound healing and skin repair involves inflammation, cell proliferation and migration and dermal matrix remodeling. Excessive inflammation may delay healing and lead to scar formation. The copper complexes of the peptides Gly-Gly-His (GGH), Gly-His-Lys (GHK) reduced TNF-alpha induced secretion of proinflammatory cytokine IL-6, in normal human dermal fibroblasts, while saccharomyces/copper ferment (OligolidesA Copper) had no effect. The authors propose that GHK and GHK-Cu can be used as a topical agent in treatment of inflammatory skin conditions instead of corticosteroids [21].

6. GHK and DNA Repair

GHK was able to restore viability of irradiated fibroblasts. The researchers used cultured human fibroblasts obtained from cervical skin that was either intact or exposed to radioactive treatment (5000 rad). GHK (10−9 M) was added in a serum free medium directly to the cell culture. An equivalent amount of plain serum-free medium was added to control cells. Although irradiated fibroblasts survived and replicated in the cell culture, their growth dynamics were markedly different from that of intact cells. The growth of the irradiated cells was especially delayed at 24 and 48 hour measurements. However, the irradiated fibroblasts treated with GHK showed much faster growth that was similar to the normal (non-irradiated control cells). In addition, GHK-treated irradiated fibroblasts showed higher production of growth factors, which are essential for wound healing [31].

Fibroblasts are central cells in both wound healing and tissue renewal processes. They not only synthesize different components of dermal matrix, but also produce a number of growth factors that are involved in a multitude of cellular pathways regulating cell migration and proliferation, angiogenesis, epithelialization, and so forth. Radiation damages cell DNA, thus impairing their function. Since GHK was able to restore function of irradiated fibroblasts, it has to have effects on DNA repair.

Studies using the Broad Institute’s Connectivity map found that GHK significantly increased the expression of DNA repair genes with 47 genes stimulated and 5 genes suppressed (more than or equal to a 50% increase or decrease) [22].

7. Facial Studies

A number of placebo-controlled clinical studies found GHK-Cu to improve skin quality in women around the age of 50. A study of collagen production determined by studying skin biopsy samples using immunohistological techniques found that after applying creams to the thighs for one month, GHK-peptides had a significant effect on collagen production. Increases were found in 70% of the women treated with GHK-Cu, in contrast to 50% treated with the vitamin C cream, and 40% treated with retinoic acid [32].

A GHK-Cu facial cream reduced visible signs of aging after 12 weeks of application to the facial skin of 71 women with mild to advanced signs of photoaging. The cream improved skin laxity, clarity, and appearance, reduced fine lines and the depth of wrinkles, and increased skin density and thickness [33].

A GHK-Cu eye cream, tested on 41 women for twelve weeks with mild to advanced photodamage, was compared to a placebo control and an eye cream containing vitamin K. The GHK-Cu cream performed better than both controls in terms of reducing lines and wrinkles, improving overall appearance, and increasing skin density and thickness [34].

In another 12-week facial study of 67 women between 50 and 59 years with mild to advanced photodamage, a GHK-Cu cream was applied twice daily and improved skin laxity, clarity, firmness and appearance, reduced fine lines, coarse wrinkles and mottled pigmentation, and increased skin density, and thickness. The cream also strongly stimulated dermal keratinocyte proliferation as determined by the histological analysis of biopsies [35].

These placebo-controlled studies demonstrated that GHK-Cu skin creams had the following effects:(1)Tighten loose skin and improve elasticity.(2)Improve skin density and firmness.(3)Reduce fine lines and deep wrinkles.(4)Improve skin clarity.(5)Reduce photodamage and mottled hyper-pigmentation.(6)Strongly increase keratinocyte proliferation.

8. Formulation and Delivery

GHK-Cu appears to pass the skin’s horny layer (stratum corneum) in quantities sufficient to activate regenerative events. The permeability coefficients of copper complexes increase with increasing pH. It was proved that only the tripeptide GHK and its complexes with copper: GHK-Cu and (GHK)(2)-Cu are able to migrate through the membrane model of the stratum corneum [3638]. Yet, because of its susceptibility to the actions of proteolytic enzymes it is important to ensure its sustained delivery in bioactive concentrations. Arul et al. proposed the use of biotinylated peptide incorporated collagen matrix (Boc-GHK) for dermal wound healing. They observed improved wound contraction and increased cell proliferation and a high expression of antioxidant enzymes in wounds treated with Boc-GHK compared to the control [39].

A recent study investigated formulation requirements for GHK. It has been established that the peptide was prone to hydrolytic cleavage when subjected to oxidative stressors. It was stable in water in the pH range 4.5–7.4 buffers for at least two weeks at 60°C. The distribution coefficients in octanol-phosphate buffered saline indicated the highly hydrophilic nature of GHK-Cu with log  values between −2.38 and −2.49 at a pH range of 4.5–7.4. GHK-Cu can be incorporated into Span 60 based niosomes. It is less stable in the presence of the negatively charged lipid diacetyl phosphate [40].

Glycyl-L-histidyl-L-lysine-Cu(II) (GHK-Cu(2+))-loaded Zn-pectinate microparticles in the form of hydroxypropyl cellulose (HPC) compression-coated tablets were developed for colon delivery of GHK. The release of GHK-Cu(2+) from Zn-pectinate microparticles was strongly affected by the cross-linking agent concentration and drug amount, but not by surfactant amount. The microparticles released 50–80% of their drug load within 4 hours. The optimum microparticle formulation (F8) coated with a relatively hydrophobic polymer HPC presented a colonic delivery system. This study indicates a possibility of including GHK into a delivery system for internal use. It should be possible to incorporate GHK into a dietary supplement with many health promoting properties and no side effects. Such formulations can be used to improve dermal healing in addition to topical delivery [41].

9. Breakdown Resistant Mixed Copper Peptide Complexes

A major problem in the treatment of human skin wounds and ulcers is dealing with infected wounds. The powerful bacteria in such wounds secrete proteases that can rapidly breakdown GHK and other types of healing growth factors.

GHK itself is formed during protein breakdown in the company of a large number of other small peptides. When we added copper 2+ to the entire mixture of small peptides formed during breakdown, we found that such a mixture had significant wound healing activity. Moreover, such peptides were resistant to further breakdown. The details of preparing these mixed copper peptide complexes and their incorporation into wound healing creams are given in the referenced US patents [4243].

Howard Maibach’s group later tested the mixed copper peptide complexes using four human wound healing systems.

These skin damaging methods were(1)removal of skin lipids with acetone,(2)irritation of skin with strong sodium lauryl sulfate,(3)irritation of skin by tape stripping,(4)activation of an allergic response in patients with nickel allergies.In all four studies, there was a more rapid healing with creams containing the mixed copper peptide complexes than with the control creams without the complexes. There was also a more rapid reduction in the erythema (redness) in the nickel allergy patients [4447].

10. Biochemistry of GHK-Cu

The molecular structure of the GHK copper complex (GHK-Cu) has been extensively studied using X-ray crystallography, EPR spectroscopy, X-ray absorption spectroscopy, and PMR spectroscopy as well as other methods such as titration. In the GHK-Cu complex, the Cu (II) ion is coordinated by the nitrogen from the imidazole side chain of the histidine, another nitrogen from the alpha-amino group of glycine, and the deprotonated amide nitrogen of the glycine-histidine peptide bond (Figure 1).

Lau and Sarkar found that at the physiological pH, GHK-Cu complexes can form binary and ternary structures which may involve the amino acid histidine and/or the copper binding region of the albumin molecule. They also observed that GHK can easily obtain copper 2+ bound to the high affinity copper transport site on plasma albumin (albumin’s binding constant of log10 = 16.2 versus GHK’s binding constant of log10 = 16.44). It has been established that the copper (II) redox activity is silenced when copper ions that are bound to the GHK tripeptide, which allows the delivery of nontoxic copper into the cell [4850].

The most distinctive feature of GHK is its ability to form complexes with copper (II) [51]. This is very important since copper is required for more than a dozen vital enzymes in the human body and skin including those that participate in connective tissue formation, antioxidant defense, and cellular respiration. Copper also exhibits signaling function and can influence cell behavior and metabolism. For example, sufficient copper is required for stem cells to start proliferating and repairing tissues. GHK also helps to reduce the level of free ionic copper thus preventing the possibility of oxidative damage.

Apart from being able to bind with copper, GHK can also quench some toxins, in particular those that are generated during lipid peroxidation [52]. This makes GHK a quite efficient antioxidant.

Finally, GHK has been shown to be able to serve as a cell adhesion molecule, which means that it helps cells to attach themselves to the extracellular matrix. This facilitates migration, proliferation, and differentiation of repair cells in the skin [5354].

11. GHK: A Built-In Natural Regulator of Dermal Repair

The wound healing process in the skin goes through the following phases: hemostasis (blood clotting), inflammation, granulation, and scar remodeling. Every stage requires well-coordinated cell interaction and therefore is precisely orchestrated by a plethora of biological active molecules coming from different sources.

For example, immediately after injury degranulating platelets release growth factors (such as TGF-beta) that mobilize immune cells and attract them to the site of the injury.

Keratinocytes and fibroblasts also produce a multitude of growth factors. Neutrophils, macrophages, and other immune cells that get recruited to the site of injury produce their share of growth factors and cytokines, as well.

GHK is a rare human sequence in proteins; however, it is more common in proteins of the extracellular matrix (ECM). GHK triplet is present in the alpha 2(I) chain of type I collagen and can be liberated by proteases at the site of a wound [55]. One of the most notable GHK-containing proteins that is always present in sites of remodeling is glycoprotein SPARC. Its proteolytic breakdown after injury generates GHK-Cu [56]. It has long been established that proteolytic breakdown of some proteins and proteoglycans of ECM results in the release of important regulatory molecules, matrikines [57]. These molecules activate and regulate dermal repair processes. The ability of GHK to reset the genome to a healthier gene pattern which leads to a better regulation of various cellular pathways can explain its diverse dermal repair actions. Since GHK is present as an amino acid sequence in proteins of ECM and is released after an injury, it appears to serve as a natural built-in modulator of dermal repair.

12. Conclusion

It has long been accepted that the human copper-binding peptide GHK-Cu enhances healing of dermal wounds and stimulates skin renewal exhibiting a wide range of effects. Cellular pathways involved in dermal repair and skin regeneration form an intricate and finely orchestrated biochemical network, where various regulatory molecules are involved in a cross-talk. When such an interaction is disrupted, the healing is delayed and may result in excessive inflammation and scarring. It appears that GHK is able to restore healthy functioning of essential cellular pathways in dermal repair through resetting the gene pattern to a healthier state.

GHK, abundantly available at low cost in bulk quantities, is a potential treatment for a variety of disease conditions. The molecule is very safe and no issues have ever arisen during its use as a skin cosmetic or in human wound healing studies.

Based on our studies where GHK was injected in a distant part of a body, such as thigh to induce systemic healing, and also on studies where GHK was injected intraperitoneally once daily to induce systemic wound healing throughout the body, we estimate that about 100–200 mgs of GHK will produce therapeutic actions in humans. But even this may overestimate the necessary effective dosage of the molecule [14].

Studies where GHK was used for the healing of bone fractures in rats used a mixture of small molecules (Gly-His-Lys (0.5 μg/kg), dalargin (1.2 μg/kg) (an opioid-like synthetic drug), and the biological peptide thymogen (0.5 μg/kg) (L-glutamyl-L-tryptophan)) to heal bones. The total peptide dosage is about 2.2 micrograms per kilogram or if scaled for the human body, about 140 micrograms per injection with treatments for 10 days [5859]. GHK can be incorporated into topical gels, used in dermal patches, and collagen membranes, as well as being administered orally in liposomes and other carriers. Future research is needed to establish the effective dosage in humans and the best ways of delivery.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

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Fonte: https://www.hindawi.com/journals/bmri/2015/648108/

Validação Cientifica - Eficácia da Estimulação do Crescimento do Folículo Capilar pelos Complexos de Peptídeos de Cobre +
Abstract
Peptide-copper complexes are disclosed which stimulate the growth of hair on warm-blooded animals. In one aspect of this invention, the peptide-copper complexes are dipeptides or tripeptides chelated to copper at a molar ratio ranging from about 1:1 to 3:1, with the second position of the peptide from the amino terminus being histidine, arginine or a derivative thereof. The peptide-copper complexes may be formulated for administration by, for example, topical application or injection. Any affliction associate with hair loss, including hair loss associated with both androgenetic and secondary alopecia, may be treated with the peptide-copper complexes of this invention.
Description

TECHNICAL FIELD

This invention relates generally to peptide-copper complexes and, more specifically, to compositions containing peptide-copper complexes for stimulating hair growth.

BACKGROUND OF THE INVENTION

Hair loss is a prevalent affliction of many humans, the most common being androgenetic alopecia (AGA) where males lose scalp hair as they get older (i.e., male pattern baldness). Other hair loss afflictions include alopecia arcata (AA), female pattern baldness and hair loss secondary to chemotherapy and/or radiation treatment (i.e., secondary alopecia).

Hair is normally divided into two types: "terminal" and "veilus". Terminal hair is coarse, pigmented hair which arises from follicles which are developed deep within the dermis. Vellus hairs are typically thin,-non-pigmented hairs which grow from hair follicles which are smaller and located superficially in the dermis. As alopecia progresses, there is a change from terminal to vellus type hair. Other changes that contribute to alopecia are alterations in the growth cycle of hair. Hair typically progresses through three cycles, anagen (active hair growth), catagen (transition phase), and telogen (resting phase during which the hair shaft is shed prior to new growth). As baldness progresses, there is a shift in the percentages of hair follicles in each phase with the majority shifting from anagen to telogen. The size of hair follicles is also known to decrease while the total number remains relatively constant.

A variety of procedures and drugs have been utilized in an attempt to treat hair loss. A common technique involves hair transplantation. Briefly, plugs of skin containing hair are transplanted from areas of the scalp where hair was growing to bald or balding areas of the scalp. This procedure, however, is time-consuming and relatively painful. Other approaches include ultra-violet radiation and exercise therapy.

More recently, the stimulating hair growth has been achieved, although with limited success, by drug therapy. One of the most well-recognized hair-growth agents is sold under the tradename "Minoxidil", as disclosed in U.S. Pat. No. 4,596,812 assigned to Upjohn. However, while the results generated through the use of Minoxidil have appeared promising, there is still a need in the art for improved compositions capable of stimulating the growth of hair in warm-blooded animals. To this end, certain peptide-copper complexes have been found to be effective hair-growth agents. For example, U.S. Pat. Nos. 5,177,061, 5,120,83 1 and 5,214,032 disclose certain peptide-copper complexes which are effective in stimulating the growth of hair in warm-blooded animals.

While significant progress has been made in the stimulation of hair-growth by drug treatment, there is still a need in the art for compounds which have greater stimulatory effect on hair growth. The present invention fulfills this need, while further providing other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention is directed to peptide-copper complexes, and compositions containing the same, for stimulating the growth of hair in warm-blooded animals. Compositions of this invention include one or more peptide-copper complexes in combination with an acceptable carder or diluent. As used herein, the term "copper" is used to designate copper(II) (i.e., Cu+2).

The peptide-copper complexes of this invention are administered to an animal in need thereof in a manner which results in the application of an effective amount of the peptide-copper complex. As used herein, the term "effective amount" means an amount of the peptide-copper complex which stimulates hair growth associated with a hair-loss affiliations (such as male pattern baldness) or caused by a hair-loss insult (such as radiation or chemotherapy). Thus, the peptide-copper complexes may be used propylactically, as well as therapeutically and cosmetically. Administration of the peptide-copper complexes is preferably by topical application, although other avenues of administration may be employed, such as injection (e.g., intramuscular, intravenous, subcutaneous and intradermal). Typically, the peptide-copper complexes of this invention are formulated as a solution, cream or gel for topical application, or as a solution for injection, and include one or more acceptable carders or diluents.

As used herein, the term "peptide-copper complex" means a peptide having at least two amino acids (or amino acid derivatives) chelated to copper, wherein the second amino acid from the amino terminus of the peptide is histidine, arginine or a derivative thereof. Such peptide-copper complexes have the following general structure A:

A: [R1 -R2 ]:copper(II)

wherein:

R1 is an amino acid or an amino acid derivative; and

R2 is histidine, arginine or a derivative thereof.

The peptide-copper complexes of this invention have a ratio of peptide to copper ranging from about 1:1 to about 3:1, and more preferably from about 1:1 to about 2:1. In short, a component of the peptide occupies at least one coordination site of the copper ion, and multiple peptides may be chelated to a single copper ion.

In a preferred embodiment, the peptide-copper complex comprises a further chemical moiety linked to the R2 moiety of structure A by an amide or peptide bond. (i.e., --C(═O)NH--). In this embodiment, the peptide-copper complex has the following structure B:

B: [R1 -R2 -R3 ]:copper(II)

wherein:

R1 is an amino acid or amino acid derivative;

R2 is histidine, arginine or a derivative thereof, and

R3 is a chemical moiety joined to R2 by an amide bond.

In a further preferred embodiment, R3 of structure B is at least one amino acid joined 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:

R1 is an amino acid or amino acid derivative;

R2 is histidine, arginine or a derivative thereof; and

R3 is an amino acid or amino acid derivative joined to R2 by a peptide bond, with the proviso that R1 is not glycyl, alanyl, seryl or valyl when R2 is histidyl or (3-methyl)histidyl and R3 is lysine, lysyl-prolyl-valyl-phenylalanyl-valine, lysyl-valyl-phenylalanyl-valine, lysyl-tryptophan, or further proviso that R1 is not lysyl when R2 is histidyl or (3 -methyl)histidyl and R3 is glycine, glycyl-prolyl-valyl-phenylalanyl-valine, glycyl-valyl-phenylalanyl-valine, glycyltryptophan, or glycyl-(glycyl)1-2 -tryptophan.

In still a further embodiment of the present invention, an additional chelating agent may be added to the peptide-copper complexes disclosed above to form a ternary peptide-copper-chelating agent complex.

Other aspects of the present invention will become evident upon reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to peptide-copper complexes which stimulate the growth of hair on warm-blooded animals. Such complexes are typically administered as a composition containing acceptable diluents and/or carriers. Administration is preferably by topical 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 may be used to stimulate hair growth in animals (including humans) afflicted with androgenetic alopecia (AGA). Animals afflicted with this condition are usually male, and the condition results in the loss of scalp hair with age (also called "male pattern baldness"). Thus, the peptide-copper complexes may be administered in order to stimulate hair growth, thereby eliminating or reducing the severity of hair loss and/or the speed at which AGA progresses. Other hair loss afflictions include alopecia arcata (AA), female pattern baldness and hair loss secondary to chemotherapy and/or radiation treatment (i.e., secondary alopecia). In the case of secondary alopecia, the peptide-copper complexes may be used in advance of certain hair-loss insults, such as chemotherapy or radiation regiments, to stimulating hair growth prior to the insult and thereby reduce the amount of hair loss resulting therefrom.

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 R1 is an amino acid and R2 is histidyl, or when R1 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 hereinbelow. An amino acid of this invention includes any carboxylic acid having an amino moiety, including (but not limited to) the naturally occurring α-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), lysine (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 γ-carboxyglutamate. In a preferred embodiment, the amino acid is a naturally occurring α-amino acid having an amino moiety (i.e., the --NH2 group, rather than a secondary amine, --NH--, such as present in proline) attached to the α-carbon of the amino acid which, when chelated to copper, occupies a coordination site thereof. As used herein, "hydrophillic amino acids" include (but are not limited to) the 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: ##STR1## wherein R is a derivative of a naturally occurring amino acid side chain. In one embodiment, R1 and R2 in the above structure may be selected from hydrogen, a substituted or unsubstituted, straight chain, branched or cyclic, saturated or unsaturated alkyl moiety containing from 1-20 carbon atoms, and a substituted or unsubstituted aryl moiety containing from 6-20 carbon atoms (including heteroaromatic moieties). In a preferred embodiment, R1 and R2 may be selected from the chemical moieties identified in Table 1 below.

              TABLE 1______________________________________Amino Acid Derivatives ##STR2##______________________________________

Where R2 ═H or the following moieties:

--(CH2)n CH3 where n=1-20

--(CH2)n CH(CH3)(CH2)m CH3 where n, m=0-20 (when n=0, m≠0 or 1 and when n=1, m≠0)

--(CH2)n NH2 where n=1-20 (n≠4)

--(CH2)n CONH2 where n=3-20

--(CH2)n COOH where n=3-20 ##STR3## where n=2-20 ##STR4## where N=2-20 ##STR5## where N=2-20 --(CH2)n SH where n=2-20

--(CH2)n S(CH2)m CH3 where n, m=1-20 (when n=2, m≠0)

--(CH2)n CH2 OH where n=1-20

--(CH2)n CH(CH3)OH where n=1-20

And where R1 ═H or the following moieties:

--(CH2)n CH3 where n=0-20

--(CH2)n CH(CH3)(CH2)m CH3 where n, m=0-20

Histidine derivatives of this invention include compounds having the structure: ##STR6## where n=1-20, and X and Y are independently selected from alkyl moieties containing from 1-12 carbon atoms or an aryl moiety containing from 6-12 carbon atoms. In preferred embodiments, n is 1, X is methyl and Y is H (i.e., 3-methyl histidyl) or X is H and Y is methyl (i.e., 5-methyl histidine).

Similarly, arginine derivatives of this invention include compounds having the structure: ##STR7## 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. (i.e., --C(═O)NH--). The peptide-copper complexes of this embodiment are depicted above as structure B. As used herein, a chemical moiety (i.e., R3) linked to the R2 moiety by an amide bond includes any chemical moiety having an amino group capable of forming an amide linkage with the carboxyl terminus of R2 (i.e., the carboxyl terminus of histidine, arginine, or derivatives thereof). Suitable R3 moieties include (but are not limited to) --NH2, alkylamino moieties having from 1-20 carbon atoms and arylamino moieties having from 6-20 carbon atoms, as well as amino acids and derivatives thereof. As used herein, "alkylamino moieties" include alkyl moieties containing an amino moiety, wherein the alkyl moiety is as defined above, and includes (but is not limited to) octyl amine and propyl amine. Similarly, "arylamino moieties" include aryl moieties containing an amino moiety, wherein the aryl moiety is as defined above, and includes (but is not limited to) benzylamine and benzyl-(CH2)1-14 -amine. Further examples of suitable chemical moieties having amino groups capable of forming an amide linkage with the carboxyl terminus of R2 include polyamines such as spermine and sperimidine.

For example, in structure B when R1 is an amino acid, R2 is histidine or arginine, and R3 is an amino moiety, 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)

Similarly, 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 yet a further embodiment (as represented by structure C above), the R3 moiety of structure B is at least one an amino acid or an amino acid derivative as defined above. In a preferred embodiment, R3 is a naturally occurring α-amino acid joined to R2 by a peptide bond. For example, when R1 and R3 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:

J: [(amino acid)-histidine-(amino acid)]:copper(II)

K: [(amino acid)-arginine-(amino acid)]:copper(II)

It should be understood that while only a single amino acid is depicted in the R3position of structures H and I, other chemical moieties may also be present, including additional amino acids and/or amino acid derivatives. For example, R3 in structures H and I may be a peptide such as phenylalanine-phenylalanine, (glycyl)n -tryptophan where n=1-4, prolyl-X1 -phenylalanyl-X2 or X1 -phenylalanyl-X2 where X1 and X2 are selected from valine, alaninc and glycine.

The peptides of the peptide-copper complexes of this invention may generally be classified as dipeptides (i.e, structure A), dipeptides with a chemical moiety attached to the carboxyl terminus via an amide bond (i.e., structure B) or as tripeptides (i.e., structure C above). In the case of peptide-copper complexes of structures B and C, additional chemical moieties, including amino acids, may be joined to the dipeptide or tripepride to yield peptides containing four or more amino acids. For purpose of illustration, Table 2 presents various representative examples of peptide-copper complexes of this invention.

              TABLE 2______________________________________Representative Peptide-Copper Complexes______________________________________Structure A:glycyl-histidine:copper             alanyl-histidine:copperglycyl-(3-methyl)histidine:             alanyl-(3-methyl)histidine:copper            copperglycyl-(5-methyl)histidine:             alanyl-(5-methyl)histidine:copper            copperglycyl-arginine:copper             alanyl-arginine:copper(N-methyl)glycine-histidine:             (N-methyl)glycine-arginine:copper            copperStructure B:glycyl-histidyl-NH.sub.2 :copper             glycyl-arginyl-NH.sub.2 :copperglycyl-(3-methyl)histidyl-NH.sub.2 :             alanyl-(3-methyl)histidyl-NH.sub.2 :copper            copperglycyl-arginyl-NH.sub.2 :copper             alanyl-arginyl-NH.sub.2 :copper(N-methyl)glycine-histidyl-NH.sub.2 :             (N-methyl)glycine-arginyl-copper            NH.sub.2 :copperglycyl-histidyl-NHoctyl:copper             glycyl-arginyl-NHoctyl:copperStructure C:glycyl-histidyl-lysine:             glycly-arginyl-lysine:coppercopperglycyl-(3-methyl)histidyl-lysine:             glycyl-(5-methyl)histidyl-lysine:copper            copperalanyl-histidyl-lysine:copper             alanyl-arginyl-lysine:copperalanyl-(3-methyl)histidyl-lysine:             alanyl-(5-methyl)histidyl-lysine:copper            copperglycyl-histidyl-phenylalanine:             glycyl-arginyl-phenylalanine:copper            copperglycyl-(3-methyl)histidyl-             glycyl-(5-methyl)histidyl-phenylalanine:copper             phenylalanine:copperalanyl-histidyl-phenylalanine:             alanyl-arginyl-phenylalanine:copper            copperalanyl-(3-methyl)histidyl-             alanyl-(5-methyl)histidyl-phenylalanine:copper             phenylalanine:copperglycyl-histidyl-lysyl-phenyl-             glycyl-arginyl-lysyl-phenyl-alanyl-phenylalanyl:             alanyl-phenylalanyl:copper            copperglycyl-(3-methyl)histidyl-lysyl-             glycyl-(5-methyl)histidyl-lysyl-phenylalanyl-phenylalanyl:             phenylalanyl-phenylalanyl:copper            copper(N-methyl)glycyl-histidyl-lysine:             (N-methyl)glycyl-arginyl-copper            lysine:copper______________________________________

Further examples of peptide-copper complexes of this invention are disclosed in U.S. Pat. Nos. 5,118,665 and 5,164,367, as well as U.S. Pat. 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. Thus, the peptide-copper complexes disclosed in the above U.S. patents may be used to stimulate hair growth in animals (including humans) afflicted with androgenetic alopecia (AGA) or male pattern baldness, thereby eliminating or reducing the severity of hair loss and/or the speed at which AGA progresses. These peptide-copper complexes may also by used to treat other hair loss afflictions, include alopecia areata, female pattern baldness and hair loss secondary to chemotherapy and/or radiation treatment (i.e., secondary alopecia). In the case of secondary alopecia, the peptide-copper complexes may be used to stimulate hair growth prior to a insults which normally result in hair loss, such as chemotherapy or radiation regiments. Thus, the peptide-copper complexes of this invention may be used to prevent hair loss.

In the practice of this invention, the molar ratio of peptide to copper is greater than zero to one (e.g., 0.1:1, 0.2:1, etc.). The molar ratio of 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 peptide to copper ranges 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 peptide to copper ranges from 1:1 to 2:1, with each tripeptide occupying three coordination sites of the copper. Similarly, with a dipeptide (such as GH:copper), the preferred ratio of peptide to copper ranges from 1:1 to 3:1, with each dipeptide occupying two coordination sites of copper ion.

In another embodiment of this invention, a chelating agent may be added to the peptide-copper complex to form a ternary peptide-copper-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 peptide-copper complex is GHF:copper, histidine may be added to yield the ternary complex GHF:copper:histidine. However, to form such a ternary complex, the molar ratio of copper to peptide to chelating agent must be considered. For example, if the ratio of peptide to copper is 2:1, the addition of a chelating agent to the peptide-copper complex, although possible, is difficult due to site occupancy by the peptide. However, by maintaining the ratio of peptide to copper near 1:1, a chelating group may readily be added to form the ternary complex. Thus, the preferred peptide to copper to chelating agent ratio is about 1:1:1.

While the chiral amino acids of the present invention (particularly the amino acids) have not been specifically designated, the present invention encompasses both the naturally occurring L-form, as well as the D-form. For example, any of the naturally occurring L-amino acids (or amino acid derivatives) disclosed herein may 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 (i.e., R2 of structures A, B and C) is either 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 moiety of the amino acid side chain of histidine, arginine or derivitive thereof. For example, in the case of histidine, an amine group of the histidine imidazole ring occupies a coordination site of the copper (i.e., the residual valencies or unshared electrions 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 coordination site of copper. The binding of R2 to the copper atom is preferably combined with the coordination of an amine group from the R1 moiety of structures A, B and C, to yield the peptide-copper complex. Thus, a peptide of this invention chelates copper by donating the R2 amine group, and preferably both the R1 and R2 amine groups, to the peptide-copper complex. The peptide-copper complexes of structures B and C can further 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 occupy yet a further coordination sites.

As mentioned above, the peptide-copper complexes of this invention have utility as hair growth agents. More particularly, the peptide-copper complexes stimulates hair growth on warm-blooded animals. Thus, the peptide-copper complexes may be used to treat a variety of diseases states associated with hair loss, including (but not limited to) androgenetic alopecia (also know as male pattern baldness), alopecia areata and female pattern baldness. In these instances, the peptide-copper complexes stimulates the growth of hair after the onset of the hair-loss affliction. Alternatively, the peptide-copper complexes may be administered prophylactically for conditions such as secondary alopecia. For example, the complexes may be administered prior to an insult which normally results in hair loss, such as chemotherapy and/or radiation treatment. Thus, the peptide-copper complexes of this invention can be used to prevent hair loss.

Administration of the peptide-copper complexes of the present invention may be accomplished in any manner which will result in the delivery of an effective amount or dose of the peptide-copper complex to the animal, including delivery to the hair follicles. For example, administration may be by topical application directly to the scalp, or other area where hair stimulation is desired (hereinafter "the treatment area"). Alternatively, administration may also be accomplished 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 on or more acceptable carriers or diluents, including formulations which provide for the 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 such instances, such formulations preferably contain one or more peptide-copper complexes of this invention in combination with a suitable vehicle for intradermal injection, with the peptide-copper complex present in the composition at a concentration ranging from 100 μg to 2000 μg per 0.1 ml vehicle (i.e., 1.0 mg/ml to 20 mg/ml). Suitable vehicles for intradermal injection include (but are not limited to) saline and sterile water.

In another embodiment, the peptide-copper complexes are formulated for topical administration. Suitable topical formulations include one or more peptide-copper complexes in the form of a liquid, lotion, cream or and gel. Topical administration may be accomplished by application directly on the treatment area. For example, such application may be accomplished by rubbing the formulation (such as a lotion or gel) onto the skin of the treatment area, or by spray application of a liquid formulation onto the treatment area. Any quantity of the topical formulation sufficient to accelerate the rate of hair growth or prevent subsequent hair loss is effective, and treatment may be repeated as often as the progress of hair growth indicates. Preferable, the topical compositions of this invention contain one or more peptide-copper complexes in an amount ranging from 0.1% to 20% by weight of the composition, and more preferably from 0.1% to 5% by weight of the composition.

In addition to carriers and diluents, the peptide-copper complexes may also be formulated to contain additional ingredients such as penetration enhancement agents and/or surface active agents. For example, topical formulations may contain 0.5% to 10% of one or more surface active agents (also called emulsifying agents). Non-ionic surface active agents and ionic surface active agents may be used for the purposes of the present invention. Examples of suitable non-ionic surface active agents are nonylphenoxypolyethoxy ethanol (Nonoxynol-9), polyoxyethylene oleyl ether (Brij-97), various polyoxyethylene ethers (Tritons), and block copolymers of ethylene oxide and propylene oxide of various molecular weights (such as Plutonit 68). Examples of suitable ionic surface active agents include sodium lauryl sulfate and similar compounds. Penetration enhancing agents may be also be present in topical formulations. Suitable penetration enhancing agents include dimethyl sulfoxide (DMSO), urea and substituted urea compounds. In the case of a liquid formulation for topical administration, the concentration of the penetrating enhancing agent (such as DMSO) may range from 30% to 80% of liquid formulation.

The balance of the topical formulations may include inert, physiologically acceptable carriers or diluents. Suitable carriers or diluents include, but are not limited to, water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol), petrolatum based creams (e.g., USP hydrophilic ointments and similar creams, Unibase, Parke-Davis, for example), various types of pharmaceutically acceptable gels, and short chain alcohols and glycols (e.g., ethyl alcohol and propylene glycol). In another embodiment of the invention, topical formulations may also contain the peptide-copper complex encapsulated in liposomes to aid in the delivery of the peptide-copper complex to the hair follicle. Alternatively, the peptide-copper complex may be formulated in an instrument to deliver the compound via iontophoresis.

The peptide-copper complexes of this invention exhibit superior skin permeability when applied topically. This results in a greater effective dose to the treatment area, and thus correspondingly greater stimulation of hair growth. In the practice of this invention, hydrophobic amino acids or amino acid derivatives are preferably used for administration by injection (such as intradermal injection), while hydrophilic amino acids or amino acid derivatives are used for topical administration. While the use of hydrophobic amino acids or amino acid derivatives generally enhance activity of the copper-peptide complexes of this invention, the use of hydrophilic amino acids or amino acids derivatives for topical administration is preferred due to the enhanced skin permeability associated therewith.

For purpose of illustration, Table 3 presents examples of suitable topical 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 Topical Formulations______________________________________Preparation A:Peptide-Copper Complex  1.0% (w/w)Hydroxy Ethyl Cellulose 3.0% (w/w)Propylene Glycol        20.0% (w/w)Nonoxynol-9             3.0% (w/w)Benzyl Alcohol          2.0% (w/w)Aqueous Phosphate Buffer (0.2N)                   71.0% (w/w)Preparation B:Peptide-Copper Complex  1.0% (w/w)Nonoxynol-9             3.0% (w/w)Ethyl Alcohol           96.0% (w/w)Preparation C:Peptide-Copper Complex  5.0% (w/w)Ethyl Alcohol           47.5% (w/w)Isopropyl Alcohol       4.0% (w/w)Propylene Glycol        20.0% (w/w)Lanoeth-4               1.0% (w/w)Water                   27.5% (w/w)Preparation D:Peptide-Copper Complex  5.0% (w/w)Sterile Water           95.0% (w/w)Preparation E:Peptide-Copper Complex  2.5% (w/w)Hydroxypropyl Cellulose 2.0% (w/w)Glycerine               20.0% (w/w)Nonoxynol-9             3.0% (w/w)Sterile Water           72.5% (w/w)Preparation F:Peptide-Copper Complex  0.5% (w/w)Sterile Water           16.5% (w/w)Propylene Glycol        50.0% (w/w)Ethanol                 30.0% (w/w)Nonoxynol-9             3.0% (w/w)Preparation G:Peptide-Copper Complex  5.0% (w/w)Sterile Water           10.0% (w/w)Hydroxypropyl Cellulose 2.0% (w/w)Propylene Glycol        30.0% (w/w)Ethanol                 50.0% (w/w)Nonoxynol-9             3.0% (w/w)______________________________________

The peptides of the present invention may be synthesized by either solution or solid phase techniques known to one skilled in the art of peptide synthesis. The general procedure involves the stepwise addition of protected amino acids to build up the desired peptide sequence. The resulting peptide may then be complexed to copper (at the desired molar ratio of peptide to copper) by dissolving the peptide in water, followed by the addition of copper chloride and adjusting the pH. A more detailed disclosure directed to the synthesis of the peptide-copper complexes of this invention, as well as the activity certain representative peptide-copper complexes, are 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 discloses the general preparation of peptide-copper complexes of the present invention by chelating a peptide to copper in an aqueous solution. Examples 2-10 disclose the synthesis of peptides which may be chelated to copper to yield peptide-copper complexes. Examples 11-16 disclose the ability of representative peptide-copper complexes of this invention to stimulate hair growth.

Source of Chemicals

Chemicals and peptide intermediates utilized in the following examples may be purchased from a number of suppliers, including: Sigma Chemical So., St. Louis, Mo.; Peninsula Laboratories, San Carlos, Calif.; Aldrich Chemical Company, Milwaukee, Wis.; Vega Biochemicals, Tucson, Ariz.; Pierce Chemical Co., Rockford, Ill.; Research Biochemicals, Cleveland, Ohio; Van Waters and Rogers, South San Francisco, Calif.; and Bachem, Inc., Torrance, Calif.

EXAMPLE 1 Preparation of Peptide-Copper Complex

The peptide-copper complexes of the present invention may be synthesized by dissolving the peptide in distilled water, followed by the addition of copper chloride (e.g., 99.999% available from Chemical Dynamics, N.J.) and then adjusting the pH of the solution to about 7.0. For example, copper complexes of glycyl-L-histidyl-L-phenylalanine (GHF) with a molar ratio of peptide to copper of 1:1, 2:1, or greater (e.g., 3:1), may be prepared by dissolving a given weight of GHF in distilled water (e.g., 50 mg/ml), 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-ε-caprolactam was dissolved in tetrahydrofuran (THF) then coupled with N.sup.α -t-butyloxycarbonyl-Nim -benzyloxycarbonyl-L-histidine (N.sup.α -BOC-im -CBZ-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 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 N.sup.α -BOC-Nim -CBZ-L-histidyl-L-caprolactam.

The above compound was dissolved in 30% trifluoroacetic acid in dichloromethane for 30 minutes, then evaporated, forming Nim -benzyloxycarbonyl-L-histidyl-L-caprolaetam. This was then dissolved in chloroformate, N-methylmorpholine and benzyloxycarbonyl-glycine were added to form benzyloxycarbonyl-glycyl-Nim -benzyloxycarbonyl-L-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-L-caprolactam was lyophilized from water several times, then purified by liquid chromatography on a C-18 reverse-phase column to yield the peptide as a diacetate salt.

EXAMPLE 3 Synthesis of L-Alanyl-L-Histidyl-L-Phenylalanine

To a stirred solution of N.sup.α -BOC-Nim -CBZ-L-histidine (9.74 g, 25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. phenylalanine benzyl ester tosylate (10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3(3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 13.7 g (87%) of the blocked dipeptide as a white semi-solid (Rf =0.75, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (12.9 g, 20.6 mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 13.3 g (ca. 100%+entrained solvent) of the free-amino compound as a white solid: Rf =0.49 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-L-alanine (6.03 g, 27.0 mmol) and N-methylmorpholine (3.3 mL, 3.0 g, 29.7mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.7 mL, 3.9 g, 28.4 mmol). After 2 min. a solution of the suitably protected dipeptide (11.4 g, 21.8 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×100 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), 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% methanol/dichloromethane), which was recrystallized from 95% ethanol to give 12.6 g (79%) of a free-flowing white powder: mp 147°-147.5° C.; Anal. Calcd. for C41 H40 N5 O8 : C, 67.39; H, 5.52; N, 9.58. Found: C, 66.78; H, 5.64; N, 9.24.

To a suspension of the blocked tripeptide (12.6 g, 17.6 mmol) in ethanol (150 mL) was added water, until the mixture became very turbid (about 150 mL). The resulting mixture was shaken with palladium chloride (1.56 g, 8.8 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. The catalyst was removed by filtration through a plug of Celite® and the flitrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 8.30 g of white powder. This material was dissolved in water, filtered through a 0.2 m nylon membrane and lyophilized to give 6.27 g (87%) of the desired tripepride dihydrochlofide as a free-flowing white powder: [a]D 5.1° (c 2.0, water); 1 H NMR (500 MHz, DMSO-d6) d 8.71 (1H, d, J=7.9), 8.49 (1H, d, J=7.8), 8.21 (1H, s), 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-d6) 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 N.sup.α -BOC-Nim -CBZ-L-histidine (9.74 g, 25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. glutantic acid dibenzyl ester tosylate (12.5 g, 25.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3(3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 15.2 g (87%) of the blocked dipeptide as a white semi-solid (Rf =0.74, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (15.1 g, 21.6 mmol) in 35% trifiuoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 14.8 g (ca. 100% +entrained solvent) of the free-amino compound as a white solid: Rf =0.48 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (5.23 g, 25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. a solution of the suitably protected dipeptide (12.9 g, 21.6 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×100 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium surfate, 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 afford 9.93 g (58%) of the blocked tripeptide as a white solid (Rf =0.58, 10% methanol/dichloromethane): mp 114°-116° C. Anal. Calcd. for C43 H43 N5 O10 : C, 65.39; H, 5.49; N, 8.87. Found: C, 64.93; H, 5.56; N, 8.41.

To a suspension of the blocked tripepride (9.6 g, 12.2 mmol) in ethanol (150 mL) was added water, until the mixture became very turbid (about 150 mL). The resulting mixture was shaken with palladium chloride (2.22 g, 12.5 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. The catalyst was removed by filtration through a plug of Celite® and the filtrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 4.72 g of white powder. This material was dissolved in water, filtered through a 0.2 m nylon membrane and lyophilized to give 4.64 g (93%) of the desired tripeptide dihydrochloride as a free-flowing white powder: [a]D -16.6° (c 2.0, water); 1 H NMR (500 MHz, D2 O) d 8.65 (1H, s), 7.35 (1H, s), 4.77 (1H, m), 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, D2 O) 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; Anal. Calcd for C13 H21 Cl2 N5 O6 : 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 N.sup.α -BOC-Nim -CBZ-L-histidine (9.74 g, 25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. phenylalanine benzyl ester tosylate (10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3(3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 13.0 g (83%) of the blocked dipeptide as a white semi-solid (Rf =0.79, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (12.9 g, 20.6 mmol) in 35% tfifiuoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 12.3 g (ca. 100%+entrained solvent) of the free-amino compound as a white solid: Rf =0.50 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (5.23 g, 25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. a solution of the suitably protected dipeptide (10.8 g, 20.6 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×100 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 14.0 g (95%) of the blocked tripepride as a white solid (Rf =0.64, 10% methanol/dichloromethane), which was recrystallized from absolute ethanol to give a free-flowing white powder.

To a suspension of the blocked tripeptide (6.0 g, 8.3 mmol) in ethanol (150 mL) was added water, until the mixture became very turbid (about 150 mL). The resulting mixture was shaken with palladium chloride (1.47 g, 8.3 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. The catalyst was removed by filtration through a plug of Celite® and the flitrate was concentrated to remove volatile organic materials. The remainder was lyophilized to give 1.46 g of white powder. This material was dissolved in water, filtered through a 0.2 m nylon membrane and lyophilized to give 1.38 g (38%) of the desired tripepride dihydrochloride as a free-flowing white powder: [a]D -7.5° (c 1.0, water); 1 H NMR (500 MHz, D2 O) d 8.59 (1H, s), 7.39-7.25 (5H, m), 7.21 (1H, s), 4.70 (1H, br 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); 13 C NMR (125 MHz, DMSO-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 N.sup.α -BOC-Nim -CBZ-L-lysine (9.5 g, 25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.7 mmol). After 2 min. phenylalanine benzyl ester rosylate (10.7 g, 25.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3(3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.76 g (ca. 100%+entrained solvent) of the blocked dipeptide as a white solid (Rf =0.84, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (15.4 g, 25.0 mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×100 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 15.8 g (ca. 100%+entrained solvent) of the free-amino compound as a white semi-solid: Rf =0.55 (10% methanol/dichloromethane).

To a stirred solution of N.sup.α -BOC-Nim -CBZ-L-histidine (9.74 g, 25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.7 mmol). After 2 min. a solution of the suitably protected dipeptide (12.9 g, 25.0 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 20.58 g (93%) of the blocked tripeptide as a white semi-solid (Rf =0.67, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected tripeptide (20.5 g, 23.1 mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 20.5 g (ca. 100%+entrained solvent) of the free-amino compound as a white solid: Rf =0.51 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-glycine (7.24 g, 34.6 mmol) and N-methylmorpholine (4.2 mL, 3.9 g, 38.1 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (4.7 mL, 5.0 g, 36.3 mmol). After 2 min. a solution of the suitably protected tripeptide (18.2 g, 23.1 mmol) in 1:1 tetrahydrofuran/dimethylformamide (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 21.6 g (95%) of the blocked tetrapeptide as a white solid (Rf =-0.85, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

To a suspension of the blocked tetrapeptide (21.5 g, 21.9 mmol) in ethanol (150 mL) was added water, until the mixture became very turbid (about 125 mL). The resulting mixture was shaken with palladium chloride (3.89 g, 21.9 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. The reaction mixture became clear within about 1/2 h, which may indicate completion of the reaction. The catalyst was removed by filtration and the filtrate was evaporated to give 13.7 g of colorless semi-solid. This material was dissolved in water and lyophilized to give 11.5 g (94%) of the desired tetrapeptide dihydrochloride as a free-flowing white powder: [a]D -12.4° (c 2.0, H2 O); 1 H NMR (500 MHz, D2 O) 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 N.sup.α -BOC-L-phenylalanine (10.6 g, 40.0 mmol) and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (5.5 mL, 5.7 g, 42.0 mmol). After 2 min. a solution prepared by mixing phenylalanine benzyl ester tosylate (17.1 g, 40.0 mmol), tetrahydrofuran (50 mL), and N-methylmorpholine (4.4 mL, 4.08, 40.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 19.8 g (98%) of the blocked dipeptide as a white solid (Rf =0.98, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected dipeptide (19.7 g, 39.2 mmol) in 35% trifiuoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×100 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 19.3 g (ca. 100%+entrained solvent) of the free-amino compound: Rf =0.65 (10% methanol/dichloromethane).

To a stirred solution of N.sup.α -BOC-Nim -CBZ-L-lysine (15.2 g, 40.0 mmol) and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) in tetrahydrofuran (100 mL) at -15° C. was added isobutyl chloroformate (5.5 mL, 5.7 g, 42.0 mmol). After 2 min. the protected dipeptide (15.8 g, 39.2 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29.9 g (98%) of the blocked tripepride as a white solid (Rf =0.84, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tripepride (15.4 g, 25.0 mmol) in 35% tfifiuoroacetic acid/dichloromethane (300 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×100 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.7 g (ca. 100%+entrained solvent) of the free-amino compound as a fluffy white solid: Rf -=0.72 (10% methanol/dichloromethane).

To a stirred solution of N.sup.α -BOC-Nim -CBZ-L-histidine (15.6 g, 40.0 mmol) and N-methylmorpholine (4.8 mL, 4.5 g, 44.0 mmol) in tetrahydrofuran (80 mL) at -15° C. was added isobutyl chloroformate (5.5 mL, 5.7 g, 42.0 mmol). After 2 min. a solution of the suitably protected tripeptide (12.9 g, 25.0 mmol) in dimethylformamide (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 29.1 g (72%) of the blocked tetrapeptide as a white solid (Rf=0.97, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected tetrapeptide (29.1 g, 28.0 mmol) in 35% trifluoroacetic acid/dichloromethane (300 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 28.4 g (ca. 100%+entrained solvent) of the free-amino compound as a white solid.

To a stirred solution of N-CBZ-glycine (7.32 g, 35.0 mmol) and N-methylmorpholine (4.2 mL, 3.9 g, 38.1 mmol) in tetrahydrofuran (100 mL) at -15° C. was added isobutyl chloroformate (4.8 mL, 5.0 g, 36.7 mmol). After 2 min. a solution of the suitably protected tetrapeptide (26.3 g, 28.0 mmol) in 1:1 tetrahydrofuran/dimethylformamide (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 27.3 g (87%) of the blocked pentapeptide as a white solid (Rf =0.95, 10% methanol/dichloromethane).

To a suspension of the blocked pentapeptide (27.3 g, 24.2 mmol) in ethanol (200 mL) was added water, until the mixture became very turbid (about 100 mL). The resulting mixture was shaken with palladium chloride (4.3 g, 24.4 mmol) under an atmosphere of hydrogen (5 atm) for 16 h. The reaction mixture became clear within about 1/2 h, which may indicate completion of the reaction. The catalyst was removed by filtration and the flitrate was evaporated to give 14.6 g (82%) of the desired pentapeptide dihydrochloride as a free-flowing white powder: [a]D-12.1°(c 2.0, methanol).

EXAMPLE 8 Synthesis of Glycyl-L-Arginyl-L-Lysine

To a stirred solution of N.sup.α -BOC-Ng -nitro-L-arginine (8.0 g, 25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. a solution of L-(Nim -CBZ)lysine benzyl ester hydrochloride (10.2 g, 25.0 mmol) and N-methylmorpholine (2.8 mL, 2.5 g, 25.0 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 16.3 g (97%) of the blocked dipeptide as a white solid (Rf =0.57, 10% methanol/dichloromethane).

A solution of the t-butyloxycarbonyl protected dipeptide (16.3 g, 24.3 mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred for 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×100 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 17.0 g (ca. 100%+entrained solvent) ofthe free-amino compound as a white semi-solid: Rf =0.12 (10% methanol/dichloromethane).

To a stirred solution of CBZ-glycine (7.32 g, 35.0 mmol) and N-methylmorpholine (4.2 mL, 4.0 g, 38.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (4.8 mL, 5.0 g, 36.8 mmol). After 2 min. a solution of the protected dipeptide (13.9 g, 24.3 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.7 g (95%) of the blocked tripeptide as a white solid (Rf =0.51, 10% methanol/dichloromethane).

To a suspension of the blocked tripeptide (17.7 g, 23.2 mmol) in ethanol (250 mL) was added water, until the mixture became very turbid (about 100 mL). The resulting mixture was shaken with palladium chloride (4.25 g, 24.0 mmol) under an atmosphere of hydrogen (5 atm) for 18 h. The catalyst was removed by filtration and the filtrate was evaporated to give a white semi-solid. This material was dissolved in water, filtered through 0.45 m nylon syringe filters, and lyophilized to give 10.2 g (ca. 100%) of the desired tripeptide dihydrochloride as a white powder: [a].sub.D -14.6° (c 2, water); 1 H NMR (500 MHz, D2 O) d 8.81(1H, br s), 8.30(1H, br s), 7.92(1H, br s), 4.37(1H, br s), 3.96(1H, 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); 13 C NMR (125 MHz, D2 O) 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 may be obtained as an acetate salt from Bachem Bioscience Inc., Philadephia, Pa. (Catalog No. #1555). Alternatively, AHK may be synthesized as the dihydrochloride salt by the following procedure.

To a stirred solution of N.sup.α -BOC-Nim -CBZ-L-histidine (9.74 g, 25.0 mmol) and N-methylmorpholine (5.8 mL, 5.3 g, 52.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. (N-ε-CBZ)-L-lysine benzyl ester hydrochloride (10.2 g, 25.0 mmol) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), water, and brine. The resulting solution was dried over sodium sulfate, filtered, and evaporated to give 17.2 g (93%) of the blocked dipeptide as a white semi-solid (Rf =0.61, 10% methanol/dichloromethane), which was used in the following transformation without further purification.

A solution of the t-butyloxycarbonyl protected dipeptide (17.2 g, 23.2 mmol) in 35% trifluoroacetic acid/dichloromethane (150 mL) was stirred 1/2 h at room temperature. The resulting solution was concentrated in vacuo and neutralized with 2M aqueous potassium bicarbonate. The product was extracted into ethyl acetate (3×150 mL). The combined extracts were dried over sodium sulfate, filtered, and evaporated to give 16.8 g (ca. 100%+entrained solvent) of the free-amino compound as a white solid: Rf =0.26 (10% methanol/dichloromethane).

To a stirred solution of N-CBZ-L-alanine (6.28 g, 25.0 mmol) and N-methylmorpholine (3.0 mL, 2.8 g, 27.5 mmol) in tetrahydrofuran (50 mL) at -15° C. was added isobutyl chloroformate (3.4 mL, 3.6 g, 26.3 mmol). After 2 min. a solution of the above protected dipeptide (14.9 g, 23.2 mmol) in tetrahydrofuran (50 mL) was added. The reaction mixture was stirred at -15° C. for 1.5 h 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 (3×150 mL). The combined extracts were washed with 1M citric acid (3×100 mL), water, 2M KHCO3 (3×100 mL), 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% ethanol (300 mL). The resulting material was collected on a filter, washed with 95% ethanol and dried to give a white solid: (Rf =0.49, 10% methanol/dichloromethane); mp 151°-153° C.

To a suspension of the blocked tripepride (21.5 g, 21.9 mmol) in ethanol (200 mL) was added water (about 200 mL). The resulting mixture was shaken with palladium chloride (4.25 g, 24.0 mmol) under an atmosphere of hydrogen (5 atm) for 1 h. The resulting mixture, in which the bulk of the material (other than the catalyst) became dissolved, was filtered and the flitrate was concentrated in vacuo to remove volatile organics. The remaining aqueous solution was lyophilized to give 10.88 g of a white solid. This material was dissolved in water, filtered through a 0.2 m nylon membrane, and, again, lyophilized to give 10.50 g (99%) of the desired tripeptide dihydrochloride as a white powder: [a]D -4.43° (c 3, H2 O); 1 H NMR (500 MHz, DMSO-d6) d 8.73 (1H, d, J=7.8), 8.45 (1H, d, J=7.5), 8.09 (1H, s), 7.08 (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, m), 1.41-1.21 (2H, m), 1.32 (3H, d, J=7.0); 13 C NMR (125 MHz, DMSO-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 at Various Molar Ratios of Peptide to Copper A. Peptide-Copper Complex at a 2:1 Molar Ratio

A solution of AHK was prepared by dissolving 2.6954 (0.0065 mole) of the AHK acetate (Bachem Bioscience Inc.) in approximately 10 ml of distilled water. The initial pH of this AHK solution was 6.71. Separately, a solution of copper(II) chloride was prepared by dissolving 0.4479 gm (0.0033 mole) of anhydrous copper(II) chloride in approximately 2.0 ml of distilled water. The copper(II) chloride solution was slowly added to the rapidly stirring AHK solution and the pH was constantly monitored with a pH meter. After all the copper(II) chloride solution was added, the combined solution pH was 3.83. The pH was then adjusted to 7.16 by the slow addition of a solution of 0.5M NaOH, and the final volume was adjusted to 20.0 ml by addition of distilled water. This procedure yielded an aqueous solution containing AHK:Cu at a molar ratio of peptide to copper of 2:1, and at a concentration of 10 mg/ml. The solution was a dark blue-purple and had a characteristic absorption maximum at 563 to 580 nm.

B. peptide-Copper Complex at a 2:1 Molar Ratio

AHK was prepared as the dihydrochloride salt as described in Example 9. A solution of AHK was prepared by dissolving 0.6388 gm (0.00146 mole) of L-alanyl-L-histidyl-L-lysine hydrochloride in approximately 5 ml of distilled water. The initial pH of this AHK solution was 2.45. Separately, a solution of copper(H) chloride was prepared by dissolving 0.0967 gm (0.0007 mole) of anhydrous copper(II) chloride in approximately 1.0 ml of distilled water. The copper(II) chloride solution was slowly added to the rapidly stirring AHK solution and the pH was constantly monitored with a pH meter. After all the copper(II) chloride solution was added, the combined solution pH was 2.36. The pH was then adjusted to 7.05 by the slow addition of a solution of 0.5M NaOH, and the final volume was adjusted to 20.0 ml by addition of distilled water. This procedure yielded an aqueous solution containing AHK:Cu at a molar ratio of peptide to copper of 2:1, and at a concentration of 10 mg/ml. The solution was a dark blue-purple and had a characteristic absorption maximum at 563 to 580 nm.

C. Peptide-Copper Complex at a 1.1:1 Molar Ratio

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

D. Peptide-Copper Complex at a 1:1 Molar Ratio

A solution of AHK was prepared by dissolving 1.3007 gm (0.0007 mole) of AHK acetate (Bathem Biosceince Inc.) in approximately 5 ml of distilled water. The initial pH of this AHK solution was 6.95. Separately, a solution of copper(II) chloride was prepared by dissolving 0.0966 gm (0.0007 mole) of anhydrous copper(II) chloride in approximately 2.0 ml of distilled water. The copper(II) chloride solution was slowly added to the rapidly stirring AHK solution and the pH was constantly monitored with a pH meter. After all the copper(H) chloride solution was added, the combined solution pH was 2.91. The pH was then adjusted to 7.08 by the slow addition of a solution of 0.5M NaOH, and the final volume was adjusted to 15.0 ml by addition of distilled water. This procedure yielded an aqueous solution containing AHK:Cu at a molar ratio of peptide to copper of 1:1, and at a concentration of 10 mg/ml. The solution was a dark blue-purple and had a characteristic absorption maximum at 595 nm, and a broad peak at 584 to 612 nm.

EXAMPLE 11 Stimulation of Hair Growth by Representative Copper-Peptide Complexes

The following example illustrates the stimulation of hair growth in warm-blooded animals after intradermal injection of representative peptide-copper complexes of this invention.

In this experiment, the backs of C3H mice (60 days old, telogen hair growth phase) were closely clipped on day 1 using an electric clipper. A sterile saline solution containing the indicated peptide-copper complex was then injected intradermally (i.e., infiltrated under the skin) at two locations within the clipped areas of the mice. Injection at two locations provided two test locations within the clipped area of each mouse. Each injection (0.1 ml) contained between 0.36 to 0.55 mg of the peptide-copper complex within the sterile saline solution. A group of saline injected mice (0.1 ml) served as controls. Following 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 surrounding the injection site. The size of this region is generally dose dependent, increasing with an increase in dose. The 0.1 ml injections used in this experiment produced a circle of hair growth measuring approximately 0.5 cm2 to 5.0 cm2 in diameter. Active hair growth occurred between 14-20 days following injection, with a maximum effect seen by day 29. Both the number of mice growing hair at the injection site and the diameter of the hair growth region were determined at day 21. A positive response was expressed as the number of mice exhibiting hair growth at the injection sites compared to the total number of mice injected in the study. The results of this experiment are presented in Table 4 below (the day of onset is the day at which hair follicle pigmentation was first observed):

              TABLE 4______________________________________Stimulation of Hair Growth by Peptide-Copper Complexes      Molar     Dose     NumberPeptide-   Ratio     (mg/     of Animals                                  DayCopper     (peptide to                injec-   Growing  ofComplex    copper)   tion)    Hair     Onset______________________________________GHKF:Cu    2:1       0.36 mg  4/5      10PHKF:Cu    2:1       0.43 mg  5/5      10(N-methyl) 2:1       0.55 mg  5/5      10GHKVFV:CuGHKVF:Cu   2:1       0.43 mg  5/5      10SALINE     --        --       0/5      NA______________________________________

EXAMPLE 12 Stimulation of Hair Growth by Representative Peptide-Copper Complexes

The following example illustrates the stimulation of hair growth in warm-blooded animals after intradermal injection of representative peptide-copper complexes of this invention.

As in Example 11 above, the backs of C3H mice (60 days old, telogen hair growth phase) were closely clipped on day 1 using an electric clipper. A sterile saline solution containing the indicated peptide-copper complex was then injected intradermally (i.e., infiltrated under the skin) at two locations within the clipped areas of the mice. Injection at two locations provided two test locations within the clipped area of each mouse. Each injection (0.1 ml) contained between 0.75 to 1.5 mg of the peptide-copper complex within the sterile saline solution. A group of saline injected mice (0.1 ml) served as controls. Following 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 surrounding the injection site. The size of this region is generally dose dependent, increasing with an increase in dose. The 0.1 ml injections used in this experiment produced a circle of hair growth measuring approximately 0.5 cm2 to 5 cm2 in diameter. Active hair growth occurred between 14-20 days following injection, with a maximum effect seen by day 29. Both the number of mice growing hair at the injection site and the diameter of the hair growth region were determined at day 21. A positive response was expressed as the number of mice exhibiting hair growth at the injection sites compared to the total number of mice injected in the study. The results of this experiment are presented in Table 5.

              TABLE 5______________________________________Stimulation of Hair Growth by Peptide-Copper Complexes  Molar     Dose    Number ofPeptide-  Ratio     (mg/    AnimalsCopper (peptide to            injec-  Growing Area of HairComplex  copper)   tion)   Hair    Growth______________________________________PHK:Cu 2:1       1.00    2/5     >1 cm diameterGHL:Cu 2:1       1.50    3/4     >1 cm diameterGHE:Cu 2:1       1.50    2/4     >1 cm diameterPHA:Cu 2:1       1.50    1/4     <1 cm diameterPHF:Cu 2:1       0.75    4/4     >1 cm diameterPBL:Cu 2:1       1.50    2/4     <1 cm diameterAHK:Cu 2:1       0.75    1/4     <1 cm diameterAHK:Cu 2:1       1.50    4/4     >1 cm diameterVHK:CU 2:1       0.75    3/4     <1 cm diameterVHK:CU 2:1       1.50    4/4     >1 cm diameter______________________________________

EXAMPLE 13

Stimulation of Hair Growth 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) utilizing a D-amino acids inplace of the naturally occurring L-amino acid.

In this experiment, the backs of C3H mice (60 days old, telogen hair growth phase) were closely clipped on day 1 using an electric clipper. A sterile saline solution containing AHK:Cu (1.1:1), or AHK:Cu (1.1:1) containing a D-amino acid, was then injected intradermally (i.e., infiltrated under the skin) at two locations within the clipped areas of the mice. Injection at two locations provided two test locations within the clipped area of each mouse. Each injection (0.1 ml) contained either 1.2 or 1.8 μmoles per injection of peptide-copper complex in the sterile saline solution. A group of saline injected mice (0.1 ml) served as controls. Following injection of 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 is generally dose dependent, increasing with an increase in dose. The 0.1 ml injections used in this experiment produced a circle of hair growth measuring approximately 0.5 cm2 to 5 cm2 in diameter. Active hair growth occurred between 14-20 days following injection, with a maximum effect seen by day 29.

The degree of hair growth was determined by measuring the total area of hair growth at the two injection sites. The data from this experiment is presented in Table 6.

              TABLE 6______________________________________Stimulation of Hair Growth by Peptide-CopperComplexes Containing D-Amino Acids     Molar Ratio DosePeptide-Copper     (peptide to (μmoles per                            Area ofComplex   copper)     injection) Hair Growth______________________________________AHK:Cu    1.1:1       1.2        3.07 ± 0.76AHK:Cu    1.1:1       1.8        3.24 ± 1.17AH-(D)K:Cu     1.1:1       1.2        3.30 ± 0.30AH-(D)K:Cu     1.1:1       1.8        3.94 ± 0.35(D)A-HK:Cu     1.1:1       1.2        1.88 ± 0.57(D)A-HK:Cu     1.1:1       1.8        2.68 ± 0.49______________________________________

The table above illustrates that the substitution of D-amino acids for a corresponding L-amino acids dose not effect the hair growth activity of the peptide copper complexes.

EXAMPLE 14 Stimulation of Hair Growth by Topical Application of a Peptide-Copper Complex

This example illustrates the stimulation of hair growth in warm-blooded animals by topical application of a peptide-copper complex. In this experiment, telogen cycle female C3H mice (60-65 days old) were prepared by clipping their posterior dorsal region (i.e., day 1). Topical application of peptide-copper complexes was performed twice per day (Monday-Friday) using a cotton-tipped applicator which delivered approximately 0.1 ml per treatment. The topical formulation used in this experiment contained the following components:

______________________________________Peptide copper Complex               0.1-0.5% (w/w)Sterde Water        16.9-16.5% (w/w)Propylene Glycol    50.0% (w/w)Ethanol             30.0% (w/w)Nonoxynol-9         3.0% (w/w)______________________________________

Topical application of the above formulation continued until the onset of follicle pigmentation, which proceeds the emergence of the hair shall. Measurement of the degree of response was performed using digital image analysis at weekly intervals, beginning at day 14. Data was expressed as the percent treatment area response using the following equation:

% treatment area=(growth area/treatment area)×100

For comparison purposes to illustrate the effect of hydrophobic amino acid residues on hair growth after topical application, AHK:Cu was compared to AHF:Cu. In this experiment, topical formulations containing AHK:Cu (1.1:1) and AHF:Cu (1.1:1) were prepared at a concentration of 0.5% and 0.1% (w/w) as indicated above. Hair growth response (i.e., "Percent Treatment Area") was determined at day 20, day 27 and at day 34. The results of this experiment are presented in Table 7.

              TABLE 7______________________________________Peptide-  Molar Ratio Concen-         PercentCopper (peptide to tration         TreatmentComplex  copper)     (% w/w)   Day   Area______________________________________AHK:Cu 1.1:1       0.1%      20    1.29 ± 1.29AHK:Cu 1.1:1       0.1%      27    23.07 ± 18.84AHK:Cu 1.1:1       0.1%      34    90.14 ± 2.96AHK:Cu 1.1:1       0.5%      20    75.87 ± 7.64AHK:Cu 1.1:1       0.5%      27    100AHK:Cu 1.1:1       0.5%      34    100AHF:Cu 1.1:1       0.1%      20    0.00AHF:Cu 1.1:1       0.1%      27    0.00AHF:Cu 1.1:1       0.1%      34    12.91 ± 12.91AHF:Cu 1.1:1       0.5%      20    55.05 ± 17.44AHF:Cu 1.1:1       0.5%      27    100AHF:Cu 1.1:1       0.5%      34    100______________________________________

The data presented in Table 7 illustrates that peptide-copper complexes containing hydrophilic residues (i.e., lysine amino acid of AHK:Cu) are more active in stimulating hair growth than similar peptides containing hydrophobic amino acid residues (i.e., the phenylalanine amino acid of AHF:Cu) following administration by topical administration. This is in contrast to administration by injection where peptide-copper complexes containing hydrophilic residues are less active than than similar peptides containing hydrophobic amino acid residues.

EXAMPLE 15 Stimulation of Hair Growth by Intraperitoneal Injection of Peptide-Copper Complexes

The following experiment illustrates the maintenance of hair follicle viability (i.e., growth) by intraperitoneal (systemic) injection of the peptide-copper complex GHKVFV:Cu during treatment with the chemotherapeutic agent cytosine arabinoside (Ara-C).

In this experiment, Sprague-Dawley rat pups (age 8 days) were maintained in 4 litters (n=10/litter) for the duration of this study. On day 0, litters received intraperitoneal (IP) injections of GHKVFV:Cu (2:1) in a sterile saline solution, or a saline control (1 injection per animal, 0.1 ml per injection). On day 1, all animals began a series of 7 consecutive daily IP injections with Ara-C (50 mg/kg). On day 8, all animals were evaluated for the extent of hairloss (alopecia) using the following rating scale:

______________________________________Grade           Degree of Alopecia______________________________________0               Normal (no loss of hair)1               Slight thinning2               Moderate thinning3               Sparse hair cover4               Total loss of hair______________________________________

Ara-C injections caused significant hair loss by day 5-6 in most animals. In order to evaluate the effect of GHKVFV:Cu, the degree of hairloss was evaluated daily. Injection of GHKVFV:Cu at a dosage of 50 mg/kg caused a mild retention of hair on the body of the test animals. This was primarily seen on the head, with sparse remaining hair on the body. This was in contrast to the saline control (+Ara-C) group which showed total hair loss. Table 8 presents the results of this experiment as evaluated on day 8 using the previously described rating scale, with the "Degree of Alopecia" being expressed as the average response for all animals.

              TABLE 8______________________________________         Dose per Animal        Degree ofPeptide-Copper         injection                  Dosage        AlopeciaComplex       (mg)     (mg/kg)  n =  (mean)______________________________________Saline Only   --       0.0      10   0.0Saline + Ara-C         --       0.0      10   4.0GHKVFV:Cu + Ara-C         1.00     50       10   3.0______________________________________

The observation of retained hair was confirmed histologically on day 8. Of the animals receiving 50 mg/kg of GHKVFV:Cu, approximately 30-40% of dorsal hair was found to be in anagen, compared to 5-10% for animals receiving saline+Ara-C alone. Saline control animals not receiving Ara-C had 100% anagen follicles.

EXAMPLE 16 Stimulation of Hair Growth by Intradermal Injection of Peptide-Copper Complexes

The following experiment illustrates the localized maintenance of hair follicle viability (i.e., growth) by intradermal (local) injection of the peptide-copper complex AHK:Cu during treatment with the chemotherapeutic agent cytosine arabinoside (Ara-C).

In this experiment, Sprague-Dawley rat pups (age 8 days) were maintained in 5 litters (n=10-11/litter) for the duration of this study. On day 0, litters received intradermal (ID) injections of AHK:Cu (1:1) in a sterile saline solution, or a saline control (1 injection per animal, 0.05 ml per injection). Each litter contained 2 normal control animals where no AHK:Cu or Ara-C was administered (i.e., saline only). On day 1, designated animals began a series of 7 consecutive daily intraperitoneal (IP) injections with Ara-C (25 mg/kg). On day 10, all animals were evaluated for the extent of hairloss (alopecia) at the injection sites using the rating identified in Example 15.

Ara-C injections caused significant hair loss by day 5-6 in most animals. In order to evaluate the stimulatory effect of AHK:Cu, the degree of hairloss was evaluated at the injection site daily. AHK:Cu injection generally caused a retention of hair in a 0.25 cm radius around the injection site, most notably in the 0.1 to 0.5 mg dose groups. Table 9 presents the results as evaluated on day 10 using the previously described rating scale, with the the "Degree of Alopecia" being expressed as the average response seen at the site of injection.

              TABLE 9______________________________________         Dose per Animal        Degree ofPeptide-Copper         injection                  Dosage        AlopeciaComplex       (mg)     (mg/kg)  n =  (mean)______________________________________Saline Only   --       0.0      8    0.00Saline + Ara-C         0.0      8        4.00AHK:Cu + Ara-C         0.05     3.5      8    3.25AHK:Cu + Ara-C         0.10     7.0      8    2.38AHK:Cu + Ara-C         0.25     17.5     9    1.44AHK:Cu + Ara-C         0.50     35.0     9    1.11______________________________________

The observation of retained hair within the area of AHK:Cu injection was examined histologically. While normal appearing and functioning anagen hair follicles were seen at the injection site of AHK:Cu, follicles located away from the injection were dystrophic and non-functional (disruption of the integrity of inner and outer root sheaths, and displaced hair shafts). These data confirm the gross observations of normal hair follicle function within the site of AHK:Cu injection, and illustrate the stimulatory effect of AHK-Cu on the hair follicle which maintains the active growth cycle during chemotherapy treatment.

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.

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US082614751994-06-17Stimulation of hair growth by peptide copper complexes
JP50248596A1995-06-16Stimulation of hair growth by peptide copper complex
DE19956237541995-06-16Stimulation of hair growth with peptide-copper complex
KR19960707227A1995-06-16, Hair growth stimulating composition comprising a copper complex compound-peptide
DE19956237541995-06-16Stimulation of hair growth with peptide-copper complex
PCT/US1995/0076261995-06-16Stimulation of hair growth by peptide-copper complexes
EP199509239061995-06-16Stimulation of hair growth by peptide-copper complexes
CA 21929441995-06-16Stimulation of hair growth by peptide-copper complexes
ES95923906T1995-06-16Stimulation of hair growth by peptide-copper complexes.
US089963071997-12-23Stimulation of hair growth by peptide copper complexes
JP2006196269A2006-07-18Stimulation of hair growth by peptide copper complex

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Claims (12)

We claim:

1. A method for stimulating hair-growth on an animal in need thereof, comprising administering topically or by injection to the animal an effective amount of a peptide-copper complex having the structure:

[R.sub.1 -R.sub.2 -R.sub.3 ]:copper(II)
wherein R1 is an amino acid or amino acid derivative; R2 is histidine or arginine; and R3 is at least one amino acid or amino acid derivative joined to R2 by a peptide bond, with the proviso that R1 is not glycyl, alanyl, seryl or valyl when R2 is histidyl and R3 is lysine, lysyl-prolyl-valyl-phenylalanyl-valine, lysyl-valyl-phenylalanyl-valine, lysyl-tryptophan, or lysyl-(glycyl)1-2-tryptophan, and with the further proviso that R1 is not lysyl when R2 is histidyl and R3 is glycine, glycyl-prolyl-valyl-phenylalanyl-valine, glycyl-valyl-phenylalanyl-valine, glycyltryptophan, or glycyl-(glycyl)1-2 -tryptophan.
2. The method of claim 1 wherein R1 is an antino acid.
3. The method of claim 1 wherein R2 is histidine.
4. The method of claim 1 wherein R2 is arginine.
5. The method of claim 1 wherein R3 is at least one amino acid.
6. The method of claim 1 wherein R3 is an amino acid.
7. The method of claim 1 wherein administration of the peptide-copper complex is by topical administration.
8. The method of claim 7 wherein R1 is a hydrophilic amino acid.
9. The method of claim 7 wherein R3 is a hydrophilic amino acid.
10. The method of claim 1 wherein the animal has a hair-loss affliction selected from the group consisting of androgenetic alopecia, alopecia areata, female pattern baldness and secondary alopecia.
11. The method of claim 10 wherein the hair-loss affliction is androgenetic alopecia.
12. The method of claim 10 wherein the hair loss affliction is secondary alopecia.

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Validação Cientifica - Eficácia dos Agentes Químicos e Peptídeos no Crescimento do Folículo Capilar +

Hideo Uno and Sotaro Kurata
Wisconsin Regional Primate Research Center and Department of Pathology and Laboratory Medicine, School of Medicine, University
of Wisconsin, Madison, Wisconsin, U.S.A.

During the past decade we have examined both the therapeutic
and the prophylactic effects of several agents on the macaque
model of androgenetic alopecia. Minoxidil and diazoxide,
potent hypotensive agents acting as peripheral
vasodilators, are known to have a hypertrichotic side effect.
Topical use of both agents induced significant hair regrowth
in the bald seal s of macaques. The application of a steroid
Sa-reductase in K ibitor (4MA) in non-bald preadolescent macaques
has prevented baldness, whereas controls developed it
during 2 years of treatment. The effects of hair growth were
determined by 1) phototrichogram, 2) folliculogram (micromor
hometric analysis), and 3) the rate of DNA synthesis in
the P ollicular cells. These effects were essentially a stimulation of the follicular cell proliferation, resulting in an enlargement
of the anagen follicles from vellus to terminal type
(therapy) or a maintenance of the prebald terminal follicles
(prevention). A copper binding peptide (PC1031) had the
effect of follicular enlargement on the back skin of fuzzy rats,
covering the vellus follicles; the effect was similar to that of
topical minoxidil.
Analyzing the quantitative sequences of follicular size and
cyclic phases, we speculate on the effect of agents on follicular
growth. We also discuss the triggering mechanism of
androgen in the follicular epithelial-mesenchymal (dermal
papilla) interaction.] invest Dermatol 101:143S- 147S, 2993

Abnormal or excessive hair growth caused by medication
can occur in two distinct patterns, hirsutism and
hypertrichosis. Hirsutism is due to hormonal drugs
with androgenic action and the hair growth appears
mostly in the body regions furnishing androgen-sensitive
follicles, such as on the beard, mustache areas, and chest.
Hypertrichosis is caused by non-hormonal drugs, and thick hair
growth develops elsewhere on the body ordinarily covered with
vellus hairs such as on the face, forehead, arms, legs, and trunk.
These undesirable hair-growth patterns occur under long-term continuous
medication with antihypertensive drugs - minoxidil and
diazoxide -and antiepileptics - phenytoin (Dilantin) [ 1 - 51. Hirsutism
is also a typical symptom associated with androgen-producing
tumors or adrenal cortical disorders [6]. These phenomena are
reversible within a few months after withdrawal of the drug or
removal of tumors. Essentially, this excessive hair growth is a structural
transformation of the vellus follicles to the size of terminal
follicles, resulting in short fine hairs growing to long, thick hairs.
Recent studies have revealed that a thinning of hair in androgenetic
alopecia is a progressive miniaturization of the scalp hair follicles
in both human baldness and the frontal alo
macaques [7,8]. The post-pubertal elevation o P
ecia of stumptailed
testosterone and its
intrafollicular conversion to dihydrotestosterone appears to trigger
this regressive change of the follicles [9]. Structurally, these transformed
vellus follicles show no abnormalities and they maintain a
P
otentiality for cyclic growth until the advanced stage of baldness
71. As long as the follicles maintain their cyclic growth, the size of
the follicles can either enlarge or diminish by various influences.
The former is known as secondary sexual hair growth, hirsutism,
and hypertrichosis, and the latter is the hair loss of androgenetic
alopecia [5].
During the past decade, an application of these hypertrichotic drugs or antiandrogens for either treatment or prevention of androgenetic
alopecia has been widely challenged by many investigators.
The present review describes the structural dynamics of hair follicular
transformation by minoxidil, d&oxide, and antiandrogen, and a
copper-binding peptide in the frontal alopecia of stumptailed macaques
and fuzzy rats. We also speculate on the androgenic actions
to hair-follicular transformation with special emphasis on epithelial-mesenchymal
(dermal papilla) interaction.

MATERIALS AND METHODS
Stumptailed Macaques The unique development of frontal alopecia
in this old world monkey is a species-specific phenomenon
and it occurs in nearly 100% of post-adolescent animals in both
sexes. The elevation of serum testosterone (T) and dihydrotestosterone
(DHT) appears around four years of age when a thinning of
hairs begins to show and slowly progresses with age [8,9]. Our
earlier study revealed that baldness was simply due to a progressive
miniaturization of the hair follicleper se and it appeared to be androgen
(DHT) dependent [lo].
Fuzzy Rats A genetic mutant between the hairless and the haired
albino rat exhibits progressive thinning of hair in the body after 2
months of age [ 1 I]. The back of the adult rat is covered with short
vellus hairs and sporadic long kinky hairs are present during young
adulthood.
Chemical Agents
Minowidil: A total of 24 adolescent and adult stumptailed macaques
were used for a series of studies of hair growth during the past
decade. Either 5% or 2% minoxidil in vehicle solution (0.2 ml) was
applied topically on the frontal scalp, over an approximately 50-cm2
area, once a day, 5 d per week. The total
was 10 months for 2% and 2-4 years P or 5% minoxidil. Gross
eriod of the experiment
photographic recordings were performed once a month. Skin biopsies
(4-mm punch) were taken from the frontal scalp of all animals
every 3 to 6 months, prepared for serial paraffin sections (10 microns thick), and used for a folliculogram study [12]. Phototrichogram
studies were done for 3 to 6 months with applications of either
5% minoxidil or its vehicle. Some skin specimens were used for an
autoradiographic study of DNA synthesis involving in vitro uptake
of [3H]thymidine [11,13].
Diazoxide: Using 10 adult macaques, topical application of a 5%
solution was given on the frontal scalp for one year [14].
Antiundrogen: Six periadolescent stumptailed macaques received
topical application of 4-MA (N,N-diethyl-4-methyl-3-oxo-4-azaSa-androstane-I7P-carboximide),
7 mg/O.5 ml dimethylsulfoxide
(DMSO), for a period of 27 months. Three of the six animals were
treated with 0.5 ml DMSO alone. The rate of hair growth was
assessed by the weight of hairs shaved every 2 months from a defined
area of the frontal scalp. Changes in the hair follicles were
analyzed by folliculograms of biopsied frontal scalp skins taken at
pre-treatment and at 2 years post-treatment. The activity of Scr-reductase
in the frontal scalp skin as well as serum levels of androgens
and their metabolites were measured every 2-4 months. Detailed
methods were provided in our previous reports [lo].
PC1 031 (Peptide;ghkvfv-copper, 2: 1 complex): Topical application
of this peptide solution was made upon the backs of fuzzy rats from
30 to I20 d of age.*

Methods for Evaluation of Hair and Fokular Growth
Phototrichogrum: Gross photographs of the frontal scalps of the
macaques and the backs of fuzzy rats were taken every 2 weeks to 1
month. The selected areas of frontal scalps of four macaques from
the minoxidil and vehicle groups were clipped to about 2 mm in
length with an electric clipper and the image of short hairs was
taken by either a photographic camera with a close view lens or a
fiberglass surface scope (Scopeman). The images of hairs printed by
computer were compared to those at pre-treatment and 3 months
and terminal hairs were counted by a computer-image program.
Folliculogrum: For a micromorphometric analysis of hair follicles,
4-mm punch biopsies were taken from the frontal scalps of macaques
or from the backs of fuzzy rats. The specimens were embedded
in paraffin and serial sections (10 micron thick) were stained
with H and E staining. The hair follicles observed in all serial sections
were traced and defined as their cyclic phases (telogen, early to
mid-anagen, late anagen, and catagen) and measured for the length
of each follicle. A histogram, representing the proportional number
of each cyclic phase and expressing the length of each follicle, was
made [7,11].
DNA Synthesis: For detection of S-phase (DNA synthesis) cells in
the hair follicles, we have used 1) in vitro uptake of 3H-thymidine in
fresh skin tissues taken from the macaque scalp [12], and 2) in vivo
uptake of 5-bromodeoxyuridine (BrdU) in rats [ll]. After 24 h
incubation with 3H-thymidine, the tissues were embedded with
glycol methacrylate resin; then the sections were processed with
autoradiography, using NTB, (Kodak) emulsion. BrdU was given
to rats either intraperitoneally (100 mg/kg), intravenously (30 mg/
ml), or by osmotic minipump (10 mg/0.2 ml) and after 2 h and 3,
10, and 14 d, the skins were taken and prepared for thin plastic
sectioning (Immunobed, Polyscience) and for split epidermis. The
sections and hair follicles attached to the epidermal sheet were
stained with immunocytochemistry using a monoclonal BrdU antibody[ 1 I].
RESULTS
Hair and Folkular Growth Minoxidil (5%) induced obvious
thickening of hairs in the bald frontal scalp viewed by unclipped
natural photographs compared to pre- and post-treatment stages
(after 3 months). Although the degree of this initial effect was
slightly weaker, diazoxide (5%) induced an increase in thickening
*Trachy RJZ, Packard S, Uno H: The fuzzy rat, a model of iatrogenic hair
growth (abstr). J Invest Dermatol 96:579, 1991. of hair. Continuous treatment with either minoxidil or diazoxide showed progressive thickening of hair density in the frontal scalp. However, the effect was reversible and the photographs showed obvious thinning of hairs at 3 months after cessation of treatment [11,14].
The phototrichograms showed clearly the conversion of vellus to
terminal hairs after 3 months of minoxidil treatment. In Fig 1, the
terminal hairs, darkly pigmented with large caliber hair shafts, were
spread among many less pigmented and fine vellus hairs in the bald
scalp at the pre-treatment stage. After 3 months of minoxidil(5%)
treatment, the most obvious change was an increased number of
terminal hairs in the same scalp region. Most terminal hairs from the
pre-treatment stage showed no change after treatment, but new,
additional terminal hairs appeared to be converted from vellus hairs.
The average population rate between vellus and terminal hairs was
23 + 2/77 + 3 in the bald scalp of four adult macaques at the pretreatment
stage. After 3 months the rate of the minoxidil group
became 31 + l/69 f 2 and that of the vehicle group accounted for
20 + 2/80 f 3. An approximately 10% conversion of vellus hairs
to terminal size appeared to be sufficient to show increased hair
growth in gross photographs of unclipped scalp hairs.
Folliculogram: Folliculograms of the bald scalp showed a similar
pattern in all adult animals that exhibited moderate to advanced
degrees of baldness. Over 70% of the hair follicles belonged to the
telogen phase; the length ranged from 0.5 to 0.9 mm (mean length
0.65 -0.9), and less than 20% were of the late anagen phase (Tables I
and II). The average length of anagen follicles was approximately
1 mm, and the rest were either mid-anagen or catagen. Compared
to the length in non-bald pre-adolescent animals, follicular size in
bald scalps was much smaller [11,12]. The patterns of the folliculogram
of the bald scalp also suggest that most follicles stay in the
telogen phase and have a very short anagen phase. Thus, the length
of hairs in the bald scalp do not grow long.
Sequential patterns of folliculograms showed that most telogen
follicles in the bald scalp were stimulated to progress in their cyclic
growth. After 3 to 4 months of treatment, about half of the telogen
follicles converted to early to mid-anagen follicles and the population
of late anagen follicles also increased. This cyclic progress was
seen in both drug- and vehicle-treated scalps. However, the increased
size of both telogen and anagen follicles was seen only in the
minoxidil or diazoxide groups, and not in the vehicle group (Fig 2).
Continuous treatment with the drugs induced a gradual increase in
follicular size and progressive patterns of cyclic growth. An increased
anagen population can produce longer hairs. The patterns in
the vehicle group were not consistent, but sometimes the population
of late anagen follicles was substantially increased; this represents
the so-called placebo effect (Table I, vehicle). Quantitative
analysis of follicular growth, mean length of telogen and anagen follicles, and the roportional population of vellus versus terminal
follicles in both te P ogen and anagen phases, with both minoxidil and
diazoxide, are shown in Tables I and II. The rate of follicular enlargement
and the increased population of terminal follicles including
both telogen and anagen phases were most significantly seen in
5% minoxidil cases. The rate of follicular growth in diazoxide
(5%) -treated cases was slightly lower than those of minoxidil(5%)
cases. Interestingly, the withdrawal of both drugs induced a rapid
decrease of terminal follicles as early as I month after the cessation
of treatment. This phenomenon suggests that the essential effects of
both drugs were a stimulation and maintenance of the bulbar cell
proliferation resulting in a prolongation of the anagen phase. Enlargement
of the anagen follicles from the previous vellus follicles
to terminal size and a prolongation of the anagen phase results in a
growth of thicker and longer hairs in the bald scalp. The rate of
follicular growth with 2% minoxidil was smaller than that with 5%
minoxidil, but it was sufficient to maintain follicular size and cyclic
growth.

Analysis of the folliculograms was also performed in the fuzzy rat
and both minoxidil and PC1031 induced progression of the cycle
and elongation of follicular size after 2 to 4 months of treatment
compared to the patterns of vehicle groups [l I].
The patterns of folliculograms in non-bald pre-pubertal macaques showed large follicles in all cyclic phases and the majority of
the follicles belonged to the anagen hase with both the mid and
late phases. This non-bald pattern of P olliculogram was maintained
by either topical application of antiandrogen (4MA) or minoxidil
(5%) in the frontal scalp during the peripubertal age (about 3 to 5
years) [lo,1 l]. In the same age group the maca ues treated with
vehicle showed a conversion of the folliculogram s rom the non-bald
to the bald pattern.
Unlike other rodents, the hair follicles in fuzzy rats exhibited
non-synchronized cyclic growth after the second postnatal cycle. At
the age of two and a half months, the ratio of telogen and anagen
follicles was about SO/SO and all follicles were small; the average
length was 0.3 mm in telogen follicles and 0.75 mm in anagen
follicles. Both minoxidil and PC1031 (5% res ectively) induced an
80% increase in the population of anagen fol F icles and an enlargement
to almost double in size.
DNA Synthesis in Follicular Cells The skins of fuzzy rats were
taken after 2 h of intraperitoneal injection of BrdU every 5 d after
topical application of either minoxidil or vehicle solution. After 5 d
of minoxidil and 10 d of vehicle treatment many telogen follicles
showed growth of the secondary germ and the cells containing a
BrdU-positive nucleus were largely found in the secondary germ.

Figure 2. Hair-follicular growth cycles and folliculograms of vehicle-
(upper row) and minoxidil-treated (lower row) cases represent sequential patterns
of follicular transformation from vellus follicles (small sized T, A,, As
and short length of follicles at each phase) to terminal follicles (large sized T,
AJ, As) with minoxidil treatment. The secondary germs producing primordial
anagen follicles (A, and arrows) appeared much larger and longer in
minoxidil- than those in vehicle-treated cases.

The density of DNA synthesis (S phase) cells and the entire size of
the secondary germ were greater and larger in the follicles of the
minoxidil- than in the vehicle-treated cases (Fig 3a, d). In the midanagen
phase, elongation of the follicular peg and the number of
S-phase cells were more pronounced in minoxidil-treated cases (Fig
3b and e). The rate of follicular cell proliferation was continuously
increased in the remodeling follicular process and the primordial
bulb and follicular sheath of the anagen follicles contained many S
phase cells in minoxidil- compared to those in vehicle-treated cases
(Fig 3c andf).
The increased rate of DNA synthesis cells in the follicular germinal
cells with minoxidil was also found in the follicles of stumptailed
macaques in our earlier work [12,13].
In fuzzy rat skin, a longer exposure time to BrdU (time after
injection or use of a continual supply with a minipump) increased
the number of BrdU-positive cells that belong to either the S or G2
phase. After 14 d exposure to BrdU, BrdU-positive cells were almost
saturated in the epidermal basal layer and in the external root
sheath of hair follicles in the upper and lower portion of anagen
follicles (Fig 4a, b).

DISCUSSION
The trichography, a magnified view of shortly clipped hairs, can
clearly demonstrate a conversion of individual fine vellus hairs to
thick-terminal hairs. Three months of minoxidil (5%) treatment
caused approximately a 10% conversion of hairs from vellus to
terminal size. This rate of conversion appeared to be enough to
show a gross hair regrowth in the bald scalp by photographs of the
unclipped natural view.
Studies of sequential data of folliculograms and DNA synthesis in
follicular cells strongly suggested that hypertrichotic drugs enhanced
the rate of cell proliferation of follicular germ cells. The
drugs a
showe B
peared to have a strong affinity to the follicular cells, but
no effect on epidermal basal cells [13,15].
Shortly after treatment, the secondary germs associated with aggregated
dermal papilla cells in vellus telogen follicles began to
proliferate and their continuous growth and differentiation resulted
in construction of new anagen follicles. Although the vehicles also
induced a proliferation of germinal cells, the rate of cell proliferation
was remarkably higher in the drug than in the vehicle groups.
Our observations in the macaque bald scalp obviously showed 1) a
conversion of short vellus hairs to long terminal hairs (phototrichogram),
2) an enlargement of the follicular size, and a prolongation of

Pigure 3. The number of DNA synthesis cells (arrows,) in early and midanagen
follicles showed a marked increase in minoxidil-treated (d,e,J) compared
to those in vehicle-treated (a,b,c) cases. a,b,d,e, early anagen. c,J midanagen.
Bar, 0.05 mm.
the anagen phase (folliculogram), and 3) an enhanced rate of cell
proliferation in the follicular germinal cells (DNA synthesis studies)
during treatment with minoxidil, diazoxide, and copper-peptide.

Follicular enlargement apparently occurred during the cyclic remodeling
process in which the germinal cells in telogen follicles proliferate
and construct fully grown anagen follicles; the rate of germinal
cell proliferation determines the size of new anagen follicles.
The hair follicles in the scalps of both macaque and human androgenetic
alopecia mostly belonged to vellus telogen follicles. Thus,
hypertrichotic agents readily stimulate these telogen buds and
transform them to larger anagen follicles than those in previous
cycles. This initial conversion from vellus telogen to terminal anagen
follicles took about 3 months at which time a dramatic hair
regrowth was grossly observed. The rate was less and speed of the
follicular enlargement slowed after the initial effect, but they slowly
progressed as long as the treatment continued.
Our recent studies using synchronizing hair cycles in rodents
revealed that long-term labeling of a DNA precursor BrdU showed
a saturation of labeled cells (mostly S- or G2-phase) in all epidermal
basal cells as well as throughout the entire external root sheath of
anagen follicles. These potential mitotic cells distributed in the
follicular sheath can add new sheath and the diameter of the follicle
enlarges. Together with the proliferation of the bulbar cells, all of
the anagen follicles appear to enlarge by themselves. Either a gradual
thickening or thinning of women’s scalp hairs during growth or
old age may be the result of this direct (non-cyclic) transformation
of the anagen follicle per se. Indeed, female alopecia usually lacks

Figure 4. S lit epidermal and hair follicle preparation showing BrdU-positive
nuclei arrows) in nearly 100% of epidermal and follicular cells after P
14 d exposure to BrdU. a: tissue included the epidermal sheet and upper
portion of the anagen follicle. b: mid to lower portion of anagen follicle. Bar,
0.1 mm.

short vellus hairs, which represent the cyclic conversion of hair
from old to new.
Androgens are undoubtedly the triggering hormone for inducing
this epigenetic phenomenon called androgenetic alopecia. Furthermore,
dihydrotestosterone (DHT) converted from circulating testosterone
in the follicles appears to be a potent androgen for hair
follicular transformation in particular body regions such as the
beard, chest, and pubic regions and the scalp follicles of bald trait
men. The epigenetic expression of hair follicles in both men and
women by elevated testosterone exhibit dichotomous effects in such
androgen-sensitive follicles, growth versus regression. Growth represents
secondary sexual hair growth or hirsutism and regression is
androgenetic alopecia.
Thus, the most essential treatment for androgenetic alopecia is a
blocking of androgen action on the hair follicles.
Hypertrichotic agents induced successful regrowth of the transformed
vellus follicles to terminal size. However, androgen is continuously
exerting its regressive effect on the regrown follicles. This
fact explains that despite the significant initial effect of hair regrowth
the growth progression during the following period is
much slower and the increased rate of hair length and follicular size
shows a certain limit. The regrown hairs maintain their length and
thickness, but a complete regrowth to the hairs at the non-bald stage
has not been observed. Recent work with a combination of 5a-reductase
inhibitor (finasteride) and minoxidil produced a relatively
higher effect on hair growth than minoxidil alone [16].
Recent work using the co-culture techniques of dermal papilla
cells and follicular epithelial cells opened a new insight into the
mechanism of androgenic action on hair follicles [17]. Dermal papilla
cells derived from the human beard induced significant proliferation
of the follicular epithelial cells with the addition of testosterone
in the medium and this effect was blocked by cyproterone
acetate. The dermal papilla cells derived from androgen non-sensitive
follicles such as in the occipital scalp showed no such androgenie
effect. These facts strongly suggest that the dermal a illa
cells of the beard have genetic machinery to produce specific fpr 01 ICUlar
growth factor that is triggered in its operation by an elevated
level of testosterone. The dermal papilla cells from androgen nonsensitive
follicles apparently lack this genetic machinery. Dermal
papilla cells in the scalp follicles of bald trait men may have similar
genetic machinery, but the factor produced from this acts rather to
suppress follicular growth, resulting in a regression of the follicles.

Indeed, the block of androgenic action by the use of 5&-reductase
inhibitor has successfully prevented the development of baldness
during the peripubertal age in stumptailed macaques [lo]. Drugs
blocking the androgen receptor may have the same effect, but they
likely have more universal side effects on masculinization.
The most effective and safest treatments for androgenetic alopecia
will be blocking the production of either the suppression factor
described above or dihydrotestosterone in the dermal papilla cells.
Furthermore, using the ubiquitous action of hypertrichotic agents
for an initial regrowth of hair follicles then blocking androgenic
actions by the above factor or Sa-reductase inhibitor will be a rational
approach for treatment of alopecia.
This work was supported by NIH Grant RR001 67, and The Upjohn Company, the
Shiseido Research Center, and the ProCyte Corporation. We acknowledge Adrienne
Cappas, Pam Alsum and Michele Zimbricfor their technical assistance and Mary
Schatzfor secretarial work.

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Fonte: https://www.jidonline.org/article/0022-202X(93)90516-K/pdf

Validação Cientifica - Eficácia e Efeito dos Complexos de Peptídeos de Cobre no Crescimento Capilar Humano +

The Effect of Tripeptide-Copper Complex on Human Hair Growth
In Vitro
Hyun Keol Pyo, Hyeon Gyeong Yoo, Chong Hyun Won, Seung Ho Lee, Yong Jung Kang, Hee Chul Eun,
Kwang Hyun Cho, and Kyu Han Kim
Department of Dermatology, College of Medicine, Seoul National University, 1
Laboratory of Cutaneous Aging and
Hair Research, Clinical Research Institute, Seoul National University Hospital, and 2
Institute of Dermatological Science,
Seoul National University, Seoul 110-744, Korea

(Received November 2, 2006)

The tripeptide-copper complex, described as a growth factor for various kinds of differentiated
cells, stimulates the proliferation of dermal fibroblasts and elevates the production of vascular
endothelial growth factor, but decreased the secretion of transforming growth factor-β1 by dermal
fibroblasts. Dermal papilla cells (DPCs) are specialized fibroblasts, which are important in
the morphogenesis and growth of hair follicles. In the present study, the effects of L-alanyl-Lhistidyl-L-lysine-Cu2+
(AHK-Cu) on human hair growth ex vivo and cultured dermal papilla cells
were evaluated. AHK-Cu (10-12~10-9 M) stimulated the elongation of human hair follicles ex vivo
and the proliferation of DPCs in vitro. Annexin V-fluorescein isothiocyanate/propidium iodide
labeling and flow cytometric analysis showed that 10-9 M AHK-Cu reduced the number of apoptotic
DPCs, but this decrease was not statistically significant. The ratio of Bcl-2/Bax was elevated,
and the levels of the cleaved forms of caspase-3 and PARP were reduced by treatment
with 10-9 M AHK-Cu. The present study proposed that AHK-Cu promotes the growth of human
hair follicles, and this stimulatory effect may occur due to stimulation of the proliferation and the
preclusion of the apoptosis of DPCs.
Key words: Hair follicle growth, Copper complex, Apoptosis, Dermal papilla cells

INTRODUCTION
Glycyl-L-histidyl-L-lysine, a tripeptide with affinity for
copper (II) ions, was initially isolated from human plasma,
and has been described as a growth factor for a variety of
differentiated cells and a modulator of the extracellular
matrix (Maquart et al., 1993). Glycyl-L-histidyl-L-lysine-Cu2+
(GHK-Cu) stimulates the proliferation of dermal fibroblasts
and elevates the production of vascular endothelial
growth factor (VEGF) by dermal fibroblasts (Pollard et al.,
2005). However, GHK-Cu decreased the secretion of
transforming growth factor-β1 (TGF-β1) from dermal
fibroblasts (McCormack et al., 2001). Interestingly, VEGF
promoted hair growth and increased hair follicle and hair
size by improving the vascularity around the hair follicle
(Yano et al., 2001), but androgen-inducible TGF-β1 derived from dermal papilla cells (DPCs) of androgenetic
alopecia was involved in the suppression of epithelial cell
growth (Inui et al., 2003). From these reports, it was our
supposition that the tripeptide-copper complex could have
a stimulating effect on hair growth.
Dermal papilla (DP) consists of a discrete population of
specialized fibroblasts, which are important in the morphogenesis
of hair follicles in the embryo and in the control of
the hair growth cycle in adults (Matsuzaki and Yoshizato,
1998; Jahoda et al., 1993). Dermal papilla cells (DPCs)
are thought to play these pivotal roles in hair formation,
growth and cycling through the interaction with follicular
epithelial cells (Krugluger, 2005). Recently, Han et al.
studied the hair growth promoting effect of minoxidil using
cultured DPCs in vitro, and reported that the stimulatory
effect of minoxidil on hair growth may occur via its
proliferative and anti-apoptotic influence on DPCs.
This study was performed to determine whether AHKCu
affects hair growth, and elucidate the mode of action
of AHK-Cu by investigating its effect on the proliferation
and apoptosis of DPCs.

Correspondence to: Kyu Han Kim, Seoul National University College
of Medicine, Seoul 110-744, Korea
Tel: 82-2-2072-3643, Fax: 82-2-742-7344
E-mail: [email protected]

MATERIALS AND METHODS
Drugs and reagents
AHK-Cu (11% stock solution in water) was obtained
from Procyte Co. (Redmond, WA, U.S.A.), 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide (MTT) from
Sigma (St Louis, MO, U.S.A.), and Annexin V-FITC from
BD PharMingen (San Diego, CA, U.S.A.). Anti-poly ADPribose
polymerase (PARP), anti-procaspase-3, and anticleaved
caspase-3 were purchased from Cell Signal
Technology Inc. (Beverly, MA, U.S.A.). Antibodies to Bcl-2
and Bax were from Dako (Glostrup, Denmark), and the β-
actin antibody was from Santa Cruz Biotech Inc. (Santa
Cruz, CA, U.S.A.).
Isolation of human hair follicles and dermal papillae
Follicles were obtained from the occipital scalp region of
ten healthy volunteers (20-35 years of age) who had not
received any medication for at least 1 month. Hair follicles
were isolated under a stereo-dissecting microscopy, with
the DP microdissected from individually isolated hair
follicles, as previously described (Messenger, 1984). The
study was approved by the Institutional Review Board of
Seoul National University Hospital, and all subjects gave
their written informed consent (IRB: H-0307-106-002).
Ex vivo human hair follicle organ culture
Isolated human scalp hair follicles were cultured ex
vivo, as described previously (Philpott et al., 1996). Hair
follicles were isolated from subcutaneous fat, and cut at
about 2.5 mm from the base of the DP. Follicles were
incubated at 37o
C in 5% CO2 and cultured for 12 days in
48 well-plates containing Williams’ E Medium (Gibco BRL,
Gaithersburg, MD, U.S.A.), supplemented with L-glutamine
(2mM), insulin (10 mg/ml), hydrocortisone (40 ng/ml), and
antibiotics (1%) (Thibaut et al., 2003). Hair follicles were
re-fed three times a week. AHK-Cu was added to the
culture media to final concentrations ranging from 10-13 M
to 10-7 M. Hair follicle elongations were measured directly
after 12 days of culture using an Olympus stereo microscope
(Olympus America Inc., Center Valley, PA, U.S.A.)
and real scale bar. A total of 240 hair follicles from 3
different volunteers were analyzed under each set of
conditions (30 follicles/condition).
Primary culture of DPCs
Human DPCs were cultured, as described previously
(Messenger, 1984), in a 35×10 mm culture dish containing
2 mL of Dulbecco’s modified Eagle’s medium (DMEM)
(Gibco BRL, Gaithersburg, MD, U.S.A.), supplemented
with 20% fetal bovine serum (FBS) (Hyclone, Logan, UT,
U.S.A.), 1×antibiotic antimycotic solution (1000 unit/mL of
penicillin G sodium and 2.5 µg/mL of amphotericin B) and fungizone (2.5 µg/mL), at 37°C in a 5% CO2 incubator.
Fourth-passage DPCs were used.

Cell viabilities
Cell viabilities were determined using the MTT assay
(Yu et al, 2007). DPCs (5.0×103
cells/well) were seeded
into 96-well plates, and cultured for 24 h with AHK-Cu
(10-13 ~10-7 M). 20 mL of MTT (5 mg/mL) was added to
each well, and the cells incubated for 4 h at 37o
C. The
optical densities, not the actual cell numbers, were
compared, with the results expressed as mean percentages
of the control for six cultures.
Flow cytometric assay
Both Annexin V- fluorescein isothiocyanate (FITC) and
propidium iodide (PI) labeling for the detection of apoptotic
cell death was performed according to the manufacturer's
protocol (BD PharMingen, San Diego, CA, U.S.A.). Briefly,
cultured DPCs were treated with AHK-Cu (10-9 M) for 72
h. 1×106
DPCs were washed twice with phosphatebuffered
saline (PBS), and stained with 5 µL of Annexin VFITC
and 10 µL of PI (5 µg/mL) in 1×binding buffer (10
mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) for
15 min at room temperature in the dark. DPCs were
analyzed by flow cytometry (Becton-Dickinson FACScan®,
Mansfield, MA, U.S.A.). Annexin V, a protein that binds to
phosphatidylserine residues, are exposed on the surface
of apoptotic, but not normal cells. This test discriminates
intact cells (Annexin V-/PI-), early apoptotic cells (Annexin
V+/PI-) and late apoptotic necrotic cells (Annexin V+/PI+).
Western blotting
Western blot analysis was performed as follows; briefly,
DPCs were cultured for 24 h in serum-free DMEM, and
then treated for 24 or 72 h with 10-9 M AHK-Cu. Cells
were then washed and scraped into 1×PBS, with proteins
extracted using a buffer containing 50 mM Tris-HCl (pH
7.4), 2 mM EDTA, 100 µg/mL leupeptin, 20 µg/mL
aprotinin and 100 mM NaCl. Soluble extracts were
obtained by centrifuging at 13,000 rpm and 4o
C for 15
min. Supernatants were collected and kept at -70o
C until
required. 50 µg of protein per lane was separated using
7.5 or 12% SDS-polyacrylamide gel electrophoresis and
then blotted onto polyvinylidene fluoride (PVDF) membranes.
The membranes were then washed twice with
TBS containing 0.1% Tween 20 (TBST). After blocking,
with TBS containing 5% nonfat milk, for 60 min, the
membranes were incubated overnight at 4o
C with the
primary antibodies at appropriate dilutions (anti-Bcl-2
monoclonal antibody, 1:1000; anti-Bax monoclonal antibody,
1:1000; anti-actin monoclonal antibody, 1:2000; antiprocaspase-3
monoclonal antibody and anti-cleaved
caspase-3 polyclonal antibody, 1:500; anti-PARP polyclonal antibody, 1:1000) in TBST-5% bovine albumin, and then
washed three times with TBST. The membranes were
probed with anti-mouse IgG-Horseradish peroxidase
(HRP) conjugates (1:2000), anti-rabbit IgG-HRP conjugates,
or anti-goat IgG-HRP conjugates (1:2000) for 1 h at room
temperature, and then washed three times with TBST.
The antibody-antigen complexes were detected using an
ECL plus system (Amersham Bioscience, Buckinghamshire,
UK) and exposed to Kodak X-ray film, with the band
intensities measured using the TINA software (Raytest
Isotopenmeβgerate, Straubenhardt, Germany)

Statistical methods
Data are presented as the means ± S.E. Statistical
comparisons were performed using Student t- and
Wilcoxon-rank sum tests, with p < 0.05 considered to
indicate statistical significance.

RESULTS
AHK-Cu stimulated the elongation of human hair
follicles ex vivo
After 12 days of organ culture, the length of human hair
follicles in the 10-12~10-9 M AHK-Cu treated group were
significantly increased compared with the vehicle-treated
group (Fig. 1). 10-8 and 10-7 M AHK-Cu, however, significantly
inhibited the hair follicle elongation by 14.8 ± 1.2 (2.3 ±
0.18 mm) and 81.5 ± 40.8% (0.5 ± 0.25 mm), respectively,
as compared with the vehicle-treated control.

AHK-Cu induced the proliferation of cultured DPCs
According to the result of MTT assay, AHK-Cu significantly
stimulated the proliferation of cultured DPCs at concentrations
of 10-12~10-9 M versus the vehicle-treated control
(Fig. 2); however, 10-8 M AHK-Cu did not affect the
proliferation of DPCs.
AHK-Cu decreased the number of apoptotic DPCs
Double staining with Annexin V-FITC and propidium
iodide was performed to discriminate viable, apoptotic and
late apoptotic/necrotic cells. The treatment with 10-9 M
AHK-Cu reduced the number of apoptotic DPCs by 3.48
%, as shown by Annexin V+/PI- versus the vehicle-treated
controls (Fig. 3); however, this decrease was not statistically
significant.
AHK-Cu increased Bcl-2 expression and decreased
Bax expression in DPCs
To evaluate any possible association in the changes on
Bcl-2 family proteins, the effects of AHK-Cu on the
expressions of Bcl-2 and Bax protein were investigated.
When the cultured DPCs were treated with 10-9 M AHKCu
for 24 h, the expression of Bcl-2 protein increased
compared with the vehicle-treated control (p < 0.05), but
the expression of Bax protein was decreased (p < 0.05)
(Fig. 4). No remarkable differences were observed in the
expressions of Bcl-2 and Bax between the AHK-Cutreated
group and vehicle-treated control when 10% fetal
bovine serum was treated to both groups.

Fig. 2. Viabilities of human dermal papilla cells (DPCs) treated with
AHK-Cu. DPCs (5.0×103
cells/well) were seeded into 96-well plates,
and cultured for 24 h with AHK-Cu (10-12~10-7 M). 20 µL of 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (5 mg/
mL) was added to each well, with the cells then incubated for 4 h at
37o
C. The optical densities were compared, with the results expressed
as mean percentages of the controls for six cultures. Values are the
means±S.E. from eight wells. *p<0.05, **p<0.001 compared with
vehicle-treated controls.

Fig. 3. Flow cytometric analysis of human dermal papilla cells (DPCs)
treated with AHK-Cu. Cultured human DPCs were treated with AHKCu
(10-9 M) for 72 h, and stained with Annexin V- fluorescein
isothiocyanate (FITC) and propidium iodide (PI). The serum (+) group
is a positive control, where fetal bovine serum was added instead of
AHK-Cu. This group represents the state of normal cell growth. The
apoptotic cells are located in right lower quadrant (Annexin V+ / PI-)
(A). Experiments were repeated in triplicate, with the values expressed
as the means±S.E. (B).

Levels of cleaved caspase-3 and PARP cleavage
fragments in DPCs were reduced by AHK-Cu
treatment
Western blotting was performed to detect the pro

Fig. 4. Effect of AHK-Cu on the expressions of Bax and Bcl-2 in
human dermal papilla cells (DPCs). DPCs were cultured in the
presence or absence of 10-9 M AHK-Cu for 24 h. Cell lysates were
subjected to western blot analysis using the indicated antibodies (A).
Western blotting results of the DPCs cultured under serum-free
conditions are expressed in histogram form as the percentages of the
vehicle-treated controls. Values are the means±S.E. of three independent
experiments (B). *p<0.05 compared with vehicle-treated controls.

caspase-3 and cleaved caspase-3, as well as the 115 and
85 kDa PARP cleavage fragments. Treatment of DPCs for
72 h, with 10-9 M AHK-Cu downregulated the levels of the
cleaved caspase-3 and PARP cleavage fragments by
42.7% (p < 0.05) and 77.5% (p < 0.05), respectively,
compared with the vehicle-treated control (Fig. 5). The
expression of procaspase-3 showed no remarkable
differences between the AHK-Cu-treated group and the
control. When the cultured DPCs were treated with 10%
fetal bovine serum, the AHK-Cu-treated group and
vehicle-treated control showed similar expression levels
of procaspase-3, cleaved caspase-3 and PARP cleavage
fragments.
DISCUSSION
Hair follicle organ cultures have been used to evaluate
the effects of various factors, such as insulin-like growth
factor-1, epidermal growth factor, TGF-β and minoxidil, on
hair growth ex vivo (Philpott et al., 1996; Magerl et al.,

Fig. 5. Effect of AHK-Cu on the expressions of caspase-3 and PARP in
human dermal papilla cells (DPCs). DPCs were cultured in the
presence or absence of 10-9 M AHK-Cu for 72 h. Cell lysates were
subjected to western blot analysis (A). Western blotting results of the
DPCs cultured under serum-free conditions are expressed in histogram
form as the percentages of the vehicle-treated controls. Values are the
means±standard error of three independent experiments (B). *p<0.05
compared with vehicle-treated controls.

2004). In the present study, 10-12 ~ 10-9 M AHK-Cu was
found to stimulate human hair growth ex vivo. It is
generally believed that DPCs are primarily responsible for
the proliferation and differentiation of hair matrix cells in
the hair cycle. DPCs may secrete many kinds of growth
hormones, as well as stimulate the proliferation and
differentiation of the follicular epithelium (Rhee et al.,
2006). Hence, any cause that promotes the survival of
DPCs may ultimately stimulate the proliferation and growth
of hair matrix cells, as well as hair follicle elongation. In
this respect, we supposed that AHK-Cu would have the
proliferative or anti-apoptotic effect on DPCs. At concentrations
between 10-12 and 10-9 M, AHK-Cu induced the
proliferation of DPCs. In the flow cytometric analysis, the
treatment of 10-9 M AHK-Cu did not significantly decrease
the number of apoptotic DPCs. However, the reduction of
apoptotic cells was not be negligible; therefore, Bcl-2,
Bax, caspase-3 and PARP were further evaluated.
Bcl-2 family proteins are well-known regulators of
apoptosis in both directions. Bcl-2 itself is an anti-apoptotic
molecule; whereas, Bax is pro-apoptotic (Nunez and Clarke, 1994). The expression of Bcl-2 in human hair is
dominant during telogen-anagen transition (Soma and
Hibino, 2004). In the present study, AHK-Cu increased the
expression of Bcl-2 in DPCs, decreased that of Bax,
leading to a net sum in the anti-apoptosis arm. Caspase-3
is a critical component molecule in the apoptosis of many
cell types (Sawaya et al., 2002), with poly ADP-ribose
polymerase (PARP) being one of the main cleavage
targets of caspase-3 in vivo (Nicholson et al., 1995). The
apoptosis of DPCs induced by serum starvation was
detected by monitoring the cleaved forms of caspase-3
(19, 17 kDa) and PARP (89 kDa). Our study showed that
AHK-Cu treatment reduced the expressions of the
cleaved forms of caspase-3 and PARP versus those in
the vehicle-treated control.
In summary, the present study has provided strong in
vitro evidence that AHK-Cu may stimulate hair growth by
increasing the proliferation of DPCs, and by preventing
their apoptosis. The effects of AHK-Cu on different cell
types in hair follicles as well as the molecular basis for its
promotion of hair growth both require further investigation.

ACKNOWLEDGEMENTS
This study was partly supported by a grant from the
Korea Health 21 R&D Project, the Ministry of Health &
Welfare, Republic of Korea (03-PJ1-PG1-CH13-0001)
and by a research agreement with AmorePacific Corporation,
Korea.

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Fonte: https://www.researchgate.net/profile/Chong_Hyun_Won/publication/6135527_The_effect_of_tripeptide-copper_complex_on_human_hair_growth_in_vitro/links/0912f5073b9e4ded2a000000/The-effect-of-tripeptide-copper-complex-on-human-hair-growth-in-vitro.pdf?origin=publication_detail

Validação Cientifica - Eficácia da Queratina e Peptídeos nas Propriedades do Folículo Capilar (Hidratação) +

Clara Barba*, Sonya Scott1
, Alisa Roddick-Lanzilotta1
, Rob Kelly2
, Albert M Manich,
Jose Luis Parra, and Luisa Coderch
IIQAB (CSIC), Jordi Girona, Barcelona 08034, Spain
1
Agresearch, Private Bag, Christchurch, New Zealand 2
Keratec Limited, Canterbury
Agricultural and Science Centre, Canterbury, New Zealand
(Received December 23, 2009; Revised May 17, 2010; Accepted July 10, 2010)
Abstract: In this work the effect on hair of two types of keratin samples obtained from wool, was investigated. Fiber surface
changes were evaluated by contact angle measurements. The effectiveness of these keratin ingredients to restore the mechanical
properties and the moisture content of the fibers was also determined. Modifications of hair properties due to some conventional
chemical treatments were demonstrated with lower values of contact angle and detrimental effects on the
mechanical properties. Application of keratin peptides and proteins to pretreated hair improved the fibers’ moisture content
and their mechanical properties.
Keywords: Wool keratins, Hair, Contact angle, Tensile properties, Moisture content

Introduction
Human hair consists principally of proteins, some of
which are a group of insoluble cystine-containing helicoidal
protein complexes, which form more than the 80 % of the
hair by weight and a small amount of lipids [1,2]. Hair fibers
are made of a central assembly of elongated cortical cells,
surrounded by flattened, overlapping cuticle cells. The
cortex accounts for most of the hair shaft and it is
responsible for the tensile strength of the hair [3]. The cuticle
protects the underlaying cortex and acts as a barrier. Each
cuticle cell consists of an inner region of low sulfur content
(the endocuticle), a central sulfur-rich band (the exocuticle),
and an outer layer known as the epicuticle [4]. The disulfide
bridges of cystine contribute to the mechanical strength [5].
The epicuticle is believed to comprise an outer layer of lipids
bonded to an underlying layer of cysteine-rich proteins
through a thioester linkage. The lipid layer forms a
hydrophobic barrier that affects the fiber properties [6].
Chemical treatment of the fiber with bases, oxidants, or
reducing agents strips the lipid layer producing a hydrophilic
surface [6]. Hair fibers have complex structures and the
knowledge of the surface properties of its fibers is important
in the hair-care industry[7].
From a cosmetic point of view, it is the cuticle that gives
hair a healthy or unhealthy look. Most beauty care products
and treatments primarily affect the cuticle layers. Permanent
waving, straightening, or relaxing and bleaching during hair
coloring processes are major causes of hair damage [8,9].
Bleaching is based on an oxidation of melanin and other hair
components are oxidized [10]. Perming consists on breaking
the disulfide linkages, molecular shifting, and joining the pairs of cysteine units together in a new position.
The complex protein chemistry and the way the molecules
assemble together give rise to the unique tensile properties
of hair fibers [11]. An important indicator of hair health and
strength is its resistance to fracture breakage. Day-to-day
grooming and styling processes damage the cuticle layers[12,13]. Therefore, conventional hair treatments can modify
hair structure and compromise some of the natural properties
of the hair. The deterioration of hair properties can be noticed
in the form of poor manageability, dryness, brittleness, loss of
shine, and decrease strength (fiber breakage).
There is a growing consumer trend in natural actives that
can address these negative issues and have the potential to
maintain youthful hair [14,15]. It has been well known for
some time, for example, that proteins, and protein hydrolysates,
are very beneficial to the hair, imparting increased moisturization,
enhancing softness and flexibility [16]. Hydrolyzed
protein and its derivatives have been widely incorporated in
hair-care and skin-care products due to their high biocompatibility
and high performance of imparting smoothness,
luster, softness, elasticity and protective effect [17,18].
Wool proteins are natural, biodegradable, and sustainable
with multiple functionalities and have the potential for use in
the personal care and detergent market. In this work the
effect on hair of two keratin proteins isolated from wool, are
investigated, an intact keratin intermediate filament protein
extract (K-protein, MW by SDS-PAGE of 55kD) and a low
molecular weight keratin peptide from intermediate filament
proteins, which has been enzymatically hydrolyzed to give a
peptide in the range of 6-8 aminoacids (K-peptide, MW by
SDS-PAGE<1000D). Due to the fact that it is isolated intact
and in its natural state, the protein has the ability to form
cohesive films which may have important implications in the improvement of hair properties. Both keratins have cystine present in the active S-sulfonated form. This unique
chemistry enables the keratin peptide and protein to reform
disulfide bonds in damaged hair and directly affect hair
properties.
The main aim of this work is to apply these two different
keratin samples to untreated hair fibers and to hair fibers
subjected to chemical treatments. Fiber surface changes are
evaluated by contact angle measurements. The effectiveness
of these keratin ingredients to affect the mechanical properties
and the moisture content of the fibers is determined.

Materials and Methods
Materials
Ammonium persulfate (Amresco, Ohio), decane (purum,
Fluka, Germany), hydrogen peroxide 30 % (Merck, Schuchardt,
Germany), keratin peptide (MW<1000D (SDS-PAGE),
Keratec Limited, New Zealand), keratin protein (MW of
55kD (SDS-PAGE), Keratec Limitied, New Zealand),
Natural red hair tresses (with 20 cm length, purchased from
De Meo Brothers Inc., New York), thioglycolic acid (Merck,
Darmstadt, Germany).

Hair Chemical Treatments
Hair was chemically damaged by treatments commonly
used in hair dressing such as:
- Bleaching (B Hair): Hair was placed in a bleaching
solution (9% H2O2, 1% ammonium persulfate, pH 8.3) for
3 h on a rocking table; then it was rinsed with water and
dried in air.
- Perming (P Hair): Hair was placed in a perming solution
(8% thioglycollate, pH 8) for 3 h on a rocking table; after
this it was rinsed with water and placed in a neutralizing
solution (2.5 % H2O2, pH 3) for 30 min. It was then rinsed
again and dried in air.
For comparative study, tresses of virgin hair were kept as
control (UT Hair).

Hair Treatments with Keratin Protein or Peptide
Hair samples (1.5 g) were treated with 1 % aqueous
solution (25 ml) of keratin peptide (K-peptide) or keratin
protein (K-protein) during 1 h. Treatments were performed
twice a day for 5 days being after always rinsed and dried at
room temperature.
Dynamic Wetting Force Measurements of Hair Fibers
Contact angles were calculated from the dynamic wetting
force measurements carried out in an electrobalance KSV
Sigma 70 contact angle meter [19]. A single fiber was
mounted overhanging 2-3 mm from an aluminium support
in order to keep the fiber straight and rigid, avoiding the
buoyant effect and obtaining constant fiber perimeter during
wetting measurements. The aluminium support was suspended
by a hook from the electrobalance. The vessel containing the wetting liquid was raised and lowered using a motorized
platform. Both electrobalance and motorized platform were
connected to a PC for data acquisition and control purposes.
Prior to scanning, the fiber weight was zeroed. The liquid
was advanced very slowly until a distinct perturbation in the
measured force, corresponding to solid-liquid contact, occurred.
The position and time were also zeroed. The fiber was
scanned 1 mm at a velocity of 0.5 mm/min for both the
advancing and receding modes. Two hysteresis cycles were
evaluated for each fiber. Contact angles were calculated
from the dynamic mean wetting force values obtained for the
advancing mode on the first hysteresis. All measurements were
made at room temperature (20 ºC) for both scale cuticular
directions of immersion: against scale (AS) and with scale
(WS).

Determination of the Liquid Surface Tension
The surface tension of wetting liquids used in this work
was measured by the Wilhelmy plate technique with a
platinum plate [20,21].
Determination of the Fiber Perimeter
Perimeters of the scanned fibers were estimated from the
wetting force measured in a total wetting liquid, decane.
Moisture Content Measurements
To measure the moisture content, a thermogravimetric
analysis (TGA) instruments (TG-50, Mettler Toledo) was
employed. Before measuring the moisturizing effect, the hair
samples were kept in a humidity controlled box (22 ºC, 50 %
RH) for 24 h. Samples were transfered to an aluminum
crucible, weighed, and sealed for an elapsed time of 30 s.
The crucible was then placed in the TGA balance where it
was pierced. Measurements were conducted in an atmosphere
of dry a N2, where a purge at 30 ml min-1 was employed.
The hair sample was then heated from 25 to 65 ºC at
20 ºC/min, and a temperature of 65 ºC was maintained for
40 min, which is assumed to be the normal temperature used
by a hair dryer. Thereafter, the temperature was increased
from 65 to 180 ºC at 20 ºC/min, and was kept at 180 ºC for
30 min to evaporate all the water contained in hair [18,19,
22,23].
All hair sample TGA measurements have been done three
times. Mean values and standard deviations for external,
internal and total water content of each sample were
calculated.
Tensile Properties of Hair Fibers
Stress-strain Test
Ten fibers were randomly taken from samples previously
conditioned for 48 h in a standard atmosphere (20 ºC, 65 %
RH) and centrally attached into a pair of cardboard frames
with an internal rectangular cut frame of 50×25 mm
following the longest direction.
Fiber fineness along the 50 mm subjected to testing, was
examined by image analysis, and the minimum diameter was taken as fiber fineness because the breakage is normally
produced at the thinnest (weakest) point.
Samples into the cardboard were attached to the Instron
5500R dynamometer with gauge length of 50 mm. The two
sides of the cardboard were cut before the beginning of the
stress-strain test to enable to be stressed just the fiber under
testing. The test was performed according to the ASTM
Standard D 3822 (1980) with some modifications. Gauge
length 50 mm, rate of strain 30 mm/min and the breaking
stress in MPa and strain in % were recorded. Multiplication
of breaking stress and strain in % gives rise to the evaluation
of the breakage work which is related to the fiber condition.
Stress-relaxation Test
Ten fibers were randomly taken following the same
procedure of the stress-strain test and were also attached to
the Instron 5500R dynamometer to perform the stressrelaxation
test. Fibers were strained 30 % at the same rate of
the stress-strain test and stresses at 0, 2, 5, 10, 15, 30, 45, 60,
120, and 180 s were recorded. Using the results and by the
application of non-linear regression the high-rate, the
medium-rate, the low-rate, and the non relaxed stresses were
estimated.

Data Treatment
The Dixon’s test has been used for detecting outliers,
which were excluded from the data. The ANOVA variance
analyses have been used to determine significant differences
between values obtained from different treatments
(significance level accepted *p<0.1 and **p<0.05) using the
Statgraphics® program.
Results and Discussion
Contact Angle Measurements
The dynamic wetting force was measured for all hair
samples. Wetting properties of a solid surface can change as
a consequence of chemical treatment. There is a relationship
between the molecular structure of a surface and the
macroscopic properties of this surface such as wetting,
adhesion, biocompatibility, corrosion, and lubrication. Wetting
has the advantage of experimental simplicity and applicability
to complex systems [24]. Contact angle determination is a
simple technique for examining the immediate surface of
low-energy solids, such as natural synthetic polymers [25].
Contact angle measurements are attributed to several factors
such as surface chemical heterogeneity, roughness, surface
deformability, surface configuration change, surface polarity,
and adsorption and desorption [25-27].
Contact angle was determined on hair fibers chemically
modified with treatments such as bleaching and perming, as
well as control hair which was untreated. The two hair
treatments employed affect the internal and external keratin
structure of the hair. Bleaching is based on an oxidation
process [10] and perming consists on a reduction followed

by an oxidation process. Values of contact angle for virgin,
bleached and permed hair samples are given in Table 1. It is
clear that the contact angle decreases as a result of perming
or bleaching processes relative to untreated hair. This
decrease can be attributed to the chemical treatments and
was statistically significant for the bleached hair sample.
During bleaching treatment, the components of the hair
fibers surface are oxidized, producing a decrease of the
surface hydrophobicity that is reflected in a marked decrease
of its contact angle.
Contact angle of virgin, bleached and permed hairs treated
with the keratin samples were also evaluated and results are
detailed in Table 1. Very little surface modification occurred
for virgin fibers when treated with the two keratin samples, a
slight hydrophilicity increase was found for K-protein treated
hair, possibly indicating surface absorption. However, the
results showed that the treatment with the keratin peptide
induced an increase of the contact angle value for bleached
and permed hair samples. This change in the contact angle,
reaching similar values than the one of the virgin hair
sample, indicated a possible restoration of the fiber surface
due to the keratin peptide treatment. Moreover, treatment
with the keratin protein also led to an increase of the contact
angle values for bleached and permed hair samples, this
increase being statistically significant for the bleached hair
sample. As in the case of the keratin peptide it can be seen
that treatment with the keratin protein gave rise to an
improvement of the fibers surface with a substantial recovery
of its hydrophobicity. This recovery was very important for
the bleached hair sample reaching higher values of contact
angle than the obtained for the virgin hair sample and would
be expected to lead to noticeably improved softness
characteristics for people with bleached hair.

Moisture Content Measurements
The water content of the different hair samples was
evaluated by a thermogravimetrical analysis with two heat
treatments as described in the experimental part. Following
this methodology it is possible to differentiate between
external and internal water contents of the hair fibers[18,23,28].
The hair samples were heated for the first 40 min at 65 ºC
which is assumed to be the temperature in normal use of a
hair dryer, and for the next 30 min at 180 ºC to evaporate the
whole water contained in hair. The first converging point (A)

was observed between 30 and 40 min after starting the heat,
and the second converging (B) point was observed between
60 and 70 min (Figure 1).
Table 2 shows the results for the total moisture content
(%) of the different hair samples studied. Statistically
differences can be seen between the total moisture content of
virgin, bleached, and permed hair samples. While bleached
hair had the lowest value of total moisture content, permed
hair sample showed an increase of its total moisture content.
If we consider separately the external and the internal water
content of bleached fibers (Figures 2 and 3) it can be seen
that there is a statistically significant decrease of the external
water content for bleached fibers. This decrease can be
attributed to the bleach treatment which affects mainly the
fiber surface and decreases its ability to retain water. If we
consider separately the external and internal water content of
permed hair samples we find a statistically significant
increase on both parameters. As described above, permed
treatment consist of a reorganization of the disulfide bonds
of the fiber, the new bond reorganization may lead to a fiber
with higher capacity to absorb water.
Results for the total moisture content of the different hairs
after being treated with the keratin samples demonstrated a
moisturizing effect of both keratin treatments. The keratin
peptide treatment led to an increase of the water content for
virgin and bleached hair samples, this increase being
statistically significant in the first transpiration content

(Figure 2). In addition, treatment with the keratin protein led
to an important increase of the water content for all hair
samples studied, being this increase statistically significant
for virgin and bleached hair fibers. In particular, a
statistically significant increase of the first transpiration
content was obtained when virgin and bleached hair were
treated with the keratin protein (Figure 2) and an important
increase of the second transpiration content was observed for
bleached and permed samples treated with the keratin
protein (Figure 3).
The results for the increase of water content due to the
wool keratin treatments on untreated and chemically
damaged hair fibers, while the hydrophobicity increases
(demonstrated by the contact angle results) could seem
contradictory. Water sorption studies [29] support an
increase of the water binding sites of hair due to the presence
of the keratin peptide and the keratin proteins. Moreover, the surface hidrophobicity could promote a diminution of water
diffusion also demonstrated in a previous work [29] which
leads to a decrease in water permeability of bleached hair
subjected to the keratin treatments.
From our results it can be deduced that both proteins
induced a high moisture retention of the hair fiber at the
external level for untreated and bleached fibers. In addition,
K-protein increased the water retention at internal level for
the two fibers chemically modified. As described before, the
keratin protein has the ability to form a cohesive film in the
hair surface, this keratin coat may act preventing the hair
moisture loss thus increasing the fiber internal water content.
The overall effect of the K-protein treatment is to restore the
damaged hair to its original moisture levels.

Strength Measurements
Initially, a stress-strain test was performed to all the different
hair samples. Mean values of stress and deformation at break
for the different chemical treated hair samples are given in
Table 3. Breaking stress evaluates the fiber integrity.
Therefore, higher values of this parameter indicate larger
amount of bonds present in the fiber structure. Results
showed that, chemical treatments led to a modification of the
hair fiber integrity with lower values of stress at break with
respect to the virgin one. Besides, results for the deformation
at break indicated an increase of this parameter for the
permed hair sample. This increase in the fiber deformation
can be explained by an increase of the fiber plasticity, which
can be deduced by an increase of its water content. These
results are consistent with the values obtained for the
moisture content measurements.
As explained before, considering stress and deformation at
break values the breakage work can be calculated and the
results for the chemically treated hair fibers are also detailed
in Table 4. Values for the breakage work, which evaluates
total energy to break, show that the fibers with the greatest change in breakage work were the bleached fibers.
The same experiments were done for the keratin treated
hair and results are shown also in Table 3. A decrease of the
stress at break for almost all hair fibers treated with both
keratin samples was obtained. Only for the bleached hair
fibers treated with the keratin peptide an increase of this
parameter was observed. However, a clear increase on the
deformation at break for most of the hair samples treated
with the keratin solutions was found. Furthermore, this
increase was most substantial in all hair samples treated with
the keratin peptide and in particular was statistically
significant when the bleached hair is treated with the keratin
peptide. An increase on the deformation at break indicates
an increase of the fibers plasticity. This increase can be
explained by: (1) an increase of the water content of the
fibers due to the keratin peptide or protein as detailed earlier.
Increased moisture content can increase the fiber ability to
be deformed; (2) binding of the keratin peptide S-sulfonate
groups to cystine within the hair fiber, restoring some of the
disulfuide bonds broken on chemical treatment. This could
also impart greater elasticity to the fiber. When the
deformability is increased the resulting hair fibers are softer
and are more resistant to breakage. The breakage work was
calculated for all the different hair samples and values are
shown in Table 4. These results indicate that bleached fibers
treated with the keratin peptide have an important
improvement of its fibers, reaching values for the breakage
work higher that the obtained for untreated virgin hair fibers.
To obtain additional information of the possible structure
modification produced in the hair fibers a stress-relaxation
test was performed, which is attributed first to the breaking
of various chemical crosslinks on extension and next their
reformation which may occur with the passage of time.
Based on time scale theses bonds are categorized in three
groups: (a) weak bonds (relaxation time<0.1 min) including
hydrogen bonds, salt linkages, van der Waals and electrostatic

forces; (b) bonds of intermediate strength (relaxation time
between 0.1-10 min) due to disengagement of bonds
between matrix and filament components; (c) strong bonds
(relaxation time>10 min) consisting of covalents crosslinks,
mainly disulfide bonds [30]. Table 5 summarizes the
percentages of bonds found in each hair sample evaluated.
The non-relaxed stress corresponds to the strong bonds, the
relaxed stresses at 5.8 s correspond to the weak bonds and
the relaxed stress at 79 s corresponds to the intermediate
bonds.
Comparing values for virgin and chemically treated hair
samples was observed a slight decrease on the weak bonds
much more marked for intermediate bonds, therefore the
contribution of covalent crosslinks was higher. An application
of K-protein slightly increases the virgin and permed weak
bonds and the bleached strong bonds. However K-peptide
induced a much marked increase of strong bonds again more
pronounced for bleached samples.
The substantial increase in covalent crosslinks when virgin
and in particular bleached hair fibers were treated with Kpeptide
is in accordance with the increase in breakage work
of bleached fibers treated with keratin peptide.
This modification indicates an increase on the strong
bonds for the keratin treated hair samples which can be
attributed to an increase of the disulfide bonds due to the
presence of the keratin protein and more over the reactive
keratin peptide in the hair fibers.

Conclusion
Modifications of hairs properties due to conventional
chemical treatments were demonstrated with lower values of
contact angle and detrimental effects on the mechanical
properties. Application of keratin peptides and proteins to
chemically treated hair showed to restore the fiber surface
hydrophobicity with an increase on the value of its contact
angle.
An improvement of the moisture content of virgin and pretreated
hairs due to the application of keratin peptides and
proteins were demonstrated. While the keratin peptide led to
an increase mainly in the external water content, the film
formed by the keratin protein at the fiber surface might act
preventing the loss of the hair moisture thus increasing the
internal water content.

Keratin peptides and proteins demonstrated to restore the
internal strong bonds of chemically damaged fibers, inducing
an increase of the fiber elasticity thus, improving the
mechanical properties of the fibers. This improvement was
most significant when the hair fibers are treated with the Ssulfonated
keratin peptide.

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