Wound Healing and Skin Repair Studies Using Copper Peptides
|US Patents describing methods using GHK-Cu stimulating wound healing of wound and methods for enhancing wound healing through an animal||US Patent 4,665,054 New glycyl-L-histidyl-L-lysine copper derivatives of improved resistance to proteolytic enzymes and better fat solubility for use in inhibiting thromboxane production and enhancing wound healing. Pickart US Patent 4,760,051 Compositions containing glycyl-1-histidyl-1-lysine copper(II) enhance the wound healing process without evoking an antigenic response Pickart US Patent 4,810,693 Copper glycyl-L-histidyl-L-lysine complexes enhance the healing of wounds and sores. Pickart US Patent 4,877,770 New glycyl-histidyl-lysine ester copper complex compounds with anti-inflammatory and superoxide dismutase activity useful for enhancing wound healing
Pickart US Patent 4,937,230 Method for healing wounds in horses using a copper complex of Glycyl-L-Histidyl-L-lysine or derivatives on the affected area. Pickart US Patent 5,164,367 Compositions for accelerating wound healing in mammals containing cupric salt or complexes with amino acid or peptide
|Wound healing in rats||
The healing of surgical wound in 10 control rats and 10 GHK-Cu treated rats was investigated.
The application of GHK-Cu markedly stimulated wound closure
|Acceleration of wound healing using glycyl-histidyl-lysine copper (II) Downey, Larrabee, Voci and Pickart (Virginia Mason Research Center) Surg. Forum 573-575, 1985|
|Healing of rat wounds Chemoattraction of capillary endothelial cells||Anti-oxidant actions GHK-Cu was tested on the healing of rat wounds, capillary cell chemoattraction, and superoxide dismutase-like activity. GHK-Cu accelerated the healing of rat wounds and acted as a chemoattractant for capillary endothelial cells at 10exp (-12) M. GHK-Cu also possesses significant superoxide dismutase-like activity.||Gly-l-his-l-lys copper(II) - A human growth factor with superoxide dismutase-like and wound healing activities Pickart, Downey, Lovejoy and Weinstein (University of Washington) In: Superoxide and Superoxide Dismutase (Elsevier, 1986) pp.555-558|
|Wound healing and skin transplantation in mice||GHK-Cu was tested by injection for improving the healing of surgical wounds and the take of skin grafts GHK-Cu accelerated the healing of surgical wound and improve the take of skin grafts||Iamin: A human growth factor with multiple wound healing properties Pickart In: Biology of Copper Complexes Plenum Press 1987, pp.273-282|
|Healing of wounds in mice||GHK-Cu was tested for improving the closure of surgical wounds GHK-Cu accelerated the healing of surgical wounds in mice||Biological activity of human plasma copper-binding growth factor glycyl-L-histidyl-L-lysine. Pickart and Lovejoy Methods Enzymol 1987;147:314-28|
|Wound healing in pigs||Periphery of wound injected with 50 micrograms of GHK-Cu in 1% DMSO. Control wounds received 1% DMSO only. By 17 days post wounding, GHK-Cu accelerated healing and induced a "star" pattern of strong wound contraction.||Pickart, 1984, Unpublished|
|Wound healing in pigs||The healing of punch biopsies wound in pigs was determined GHK-Cu markedly stimulated wound healing and collagen synthesis. The effect is highly localized to the immediate skin area that is treated||Counts, D, Hill E, Turner-Beatty M, Grotewiel M, Fosha-Thomas S, Pickart L. Effect of lamin on full thickness wound healing. Fed Am Soc Exp Biol 1992; A1636|
|Paw healing in dogs||Tested GHK-Cu on paw healing in dogs. Tripeptide-copper complex improved the healing of pad wounds in 12 mature English Pointers. Collagen production was significantly greater in treated pads. Healing was best with light bandaging. Wet bandages nullified the tripeptide-Cu effect.||
Effect of locally injected medications on healing of pad wounds in dogs. Swaim SF, Vaughn DM, Kincaid SA, Morrison NE, Murray SS, Woodhead MA, Hoffman C.E, Wright JC, Kammerman JR, College of Veterinary Medicine, Auburn University, AL, USA, Am J Vet Res 1996 Vol. 57, 394-9
|Dog surgical wounds||Effect of GHK-Cu and GHKF-Cu on wound healing of surgical wounds in dogs. GHK-Cu and GHKF-Cu increased wound closure and contraction and markedly increase the production of granulation tissue.||
Evaluation of multipeptide copper complex medications on open wound healing in dogs. Swaim, Bradley, Spano, McGuire and Hoffman (College of Veterinary Medicine, Auburn University, AL, USA) J Amer Ani Hos Assoc. 29, 519-525, 1993
|Human - Refractory Venous Stasis Ulcers and Diabetic Ulcers||Effect of GHK-Cu non-ionic cream with minimal preservative was tested on 60 patients GHK-Cu cream accelerated wound re-epithelialization||Effects of the tripeptide glycyl-l-histidyl-l-lysine copper(II) on healing. Clinical and biochemical correlations. Aupaix, Maquart, Salagnac, Pickart, Gillery, Borel and Kalis J Invest Derm 94:390, 1990|
|Human - Acute Surgical Wounds||Tested GHK-Cu cream with benzoyl alcohol as preservative on healing after Moh's surgery. GHK-Cu increased wound healing and skin re-epithelialization||Fish S, Katz I, Hien NR, Briden ME, Johnson JA, Patt, L, Evaluation of glycyl-1-histidyl-1-lysine copper complex in acute wound healing. Wounds 1991, 3:171-177;|
|Healing in immune suppressed rats||
Healing is very impaired in immune suppressed patients and wound contraction is impaired - Rats were immune suppressed with cortisone injections then the effect of GHK-Cu determined.
In immune suppressed rats, collagen synthesis was 23% of that in normal rats. GHK-Cu more than tripled collagen synthesis in these rats, raising it to 77% of normal and restored normal healing. GHK-Cu restored normal wound contraction in suppressed rats
|Stimulation of skin healing in immunosuppressed rats H. P. Ehrlich (Harvard Medical School, Boston, USA) Presented at Symposium on collagen and skin repair Reims, France Sept. 12-13 1991|
|Human - Diabetic skin ulcers||
Tested GHK-Cu cream on healing of skin ulcers.
GHK-Cu increased ulcer re-epithelialization
|Massey P, Patt L, D'Aoust JC, The effects of glycyl-l-histidyl-l-lysine copper chelate on the healing of diabetic ulcers, Wounds 4:21-28, 1992|
|Human - Diabetic skin ulcers||Tested GHK-Cu cream on healing of skin ulcers GHK-Cu increased skin ulcer re-epithelialization||
Enhanced healing of ulcers in patients with diabetes by topical treatment of glycyl-l-histidyl-l-lysine Mulder GD, Patt L, Sanders L, Rosenstock J, Altman MI, Hanley ME, Duncan GW, Wound Rep Reg 2:259-269, 1994
Tested GHK-Cu on the healing of linear incision wounds.
GHK-Cu increased wound tensile strength by 74% and neovascularity by 69%.
|The combined effects of glycyl-l-histidyl-l-lysine copper (II) and Cell-Tak on the healing of linear incision wounds. Schmidt SP, Resser JR, Sims RL, Mullins DL and Smith DJ (University of Akron, Ohio, USA) Wounds 6, 62-67, 1994|
|Wound healing and fibroblast production of collagen in guinea pigs||The effects of GHK-Cu and three synthetic analogues on wound healing of the guinea pig dorsal skin, as well as on cultured fibroblasts, were examined. Hydroxyproline, proteins, DNA and semicarbazide- sensitive amine oxidase, with a high affinity for benzylamine, were measured, and the histology of the wounds was observed after staining with hematoxylin/eosin.GHK-Cu and the analogues caused a decrease of the activity of semicarbazide sensitive amine oxidase, with a high affinity for benzylamine, 4-8 days after surgery, followed by an increase on day 11 that was higher than in the control group. A slower reorganization of the skin and a delayed activation of fibroblasts are observed with these peptides-Cu complexes. The peptides had a direct effect on fibroblasts. The products at a concentration of 10exp (-7) M, decreased cell reproduction and increased collagen expression.||Effect of tripeptide-copper complexes on the process of skin wound healing and on cultured fibroblasts. Buffoni, Pino and Dal Pozzo (Department of Pharmacology, University of Florence, Firenze, Italy) Arch Int Pharmacodyn Ther 1995 330(3):345-60|
|Mice||The use of breakdown resistant copper peptides to stimulate skin repair and wound healing||US Patent 5,382,431 Tissue protective and regenerative compositions Pickart|
|Human skin repair||
Tested 2nd generation SRCPs on repair of human nickel allergy injured skin.
Placebo-controlled double-blinded study found an accelerated the recovery of skin after injury and plus potent anti-inflammatory action.
|In vivo nickel contact dermatitis: human model for topical therapeutics. Zhai, Chang, Singh, and Maibach (University of California, San Francisco, USA) Contact Dermatitis Vol. 40, pp. 205-208, 1999|
|Human skin repair||
Tested 2nd generation SRCPs on repair human tape stripped damaged skin.
Placebo-controlled double-blinded study found an accelerated the rate of skin repair
|Stripped skin model to predict irritation potential of topical agents in vivo in man. Zhai, Poblete, and Maibach (University of California, San Francisco, USA) International Journal of Dermatology, Volume 37, pages 386-389, 1998|
|Human skin repair||
Tested 2nd generation SRCPs on repair of human 24-hour detergent damaged skin.
Placebo-controlled double-blinded study found accelerated the rate of skin repair
|Sodium lauryl sulfate damaged skin in vivo in man: a water barrier repair model. Zhai, Leow, and Maibach (University of California, San Francisco, USA) Skin Research and Technology, Volume 4, pages 24-27, 1998|
|Human skin repair||
Tested 2nd generation SRCPs on repair of human acetone damaged skin.
Placebo-controlled double-blinded study found an accelerated the rate of skin repair
|Human barrier recovery after acute acetone perturbation: an irritant dermatitis model. Zhai, Leow, and Maibach (University of California, San Francisco, USA) Clinical and Experimental Dermatology, Volume 23, pages 11-13, 1998|
|Effect of GHK-Cu on healing of ischemic open wounds in rats.||GHK-Cu caused a significant decrease in wound area (64.5% GHK-Cu vs 28.2% control) by day 13. GHK-Cu also significantly lowered concentrations of TNF-alpha and MMP-2 and MMP-9.||Canapp SO Jr, Farese JP, Schultz GS, Gowda S, Ishak A.M, Swaim SF, Vangilder J, Lee-Ambrose L, Martin FG, The effect of topical tripeptide-copper complex on healing of ischemic open wounds, Vet Surg. 2003 Nov-Dec;32(6):515-23|
|Rat dermal skin repair||Biotin was attached to GHK then bound to collagen films. This gave increased wound contraction, increased cell proliferation, and produced a high expression of the antioxidant superoxide dismutase. Tissue copper levels were increased 9-fold.||Arul V, Gopinath D, Gomathi K, Jayakumar R. Biotinylated GHK peptide incorporated collagenous matrix: A novel biomaterial for dermal wound healing in rats. Biomed Mater Res B Appl Biomater. 2005 May;73(2):383-91|
|Compare GHK-Cu and zinc oxide creams for wound healing||The unhealed wound area was smaller and wound contraction was higher in the GHK-Cu than in the zinc oxide group and the control. Median time for the coverage of the wound bed with granulation tissue was significantly shorter in the GHK-Cu group than in the other groups. Filling of the open wound with granulation tissue to skin level was significantly slower in the control group than in the other two groups. Neovascularization was best observed in the GHK-Cu group. The authors suggest that GHK-Cu is a better choice in the treatment protocols of open wounds than zinc oxide.||
Cangul IT, Gul NY, Topal A, Yilmaz R, Evaluation of the effects of topical tripeptide-copper complex and zinc oxide on open-wound healing in rabbits. Vet Dermatol. 2006 Dec;17(6):417-23.
|Biotinylated GHK peptide (BioGHK) incorporated collagen biomaterial (PIC) was tested for wound healing in diabetic rats.||In diabetic rats treated with BioGHK collagen, healing was hastened with an increased rate of wound contraction. Glutathione (GSH) and ascorbic acid levels in the skin of streptozotocin-induced diabetic rats were higher in the PIC group as compared to control (Untreated) and collagen (Collagen Film--CF) treated groups. Superoxide dismutase (SOD) and catalase (CAT) activity was altered in all the groups. Fibroblast cell culture studies suggest that PIC promotes fibroblast growth, revealed epithelialization, increased synthesis of collagen and activation of fibroblasts and mast cells in the PIC group. BioGHK incorporated collagen may be an approach to enhance diabetic wound healing.||
Arul V, Kartha R, Jayakumar R, A therapeutic approach for diabetic wound healing using biotinylated GHK incorporated collagen matrices, Life Sci. 2007 Jan 2;80(4):275-84.
Founding ProCyte for Wound Healing Product Development
In 1985, Barbara Weinstein and I founded ProCyte Corporation to develop GHK-Cu for clinical wound healing uses. William Weinstein, her son and a lawyer, secured the patent rights to the technology for the company. Since GHK-Cu had direct actions on cells important in healing, I named the company ProCyte - Latin words meaning - "for the cell". Dr. John Majnarich gave us laboratory space and supplies.
Since then, GHK-Cu has had numerous successes in experimental wound healing in animal models. However, in human clinical studies it has had a number of limited successes and failed to prove effectiveness in 3rd phase in two clinical trials for drug approval. However, this pattern of successes and failures is similar to other wound growth factors in clinical trials. To date (2005), no successful would healing factor has established itself in the market.
The most successful wound healing agent, Platelet-Derived Growth Factor or PDGF, is sold by Johnson & Johnson as Regranex. But it only improves the closure of diabetic ulcers by about 20%.
Proper dose ranging studies of GHK-Cu in various formulations have never been adequately performed. I designed the first pilot clinical trial in France in 1987-1988 that used a 4% concentration of GHK-Cu in a cream with minimal preservatives since many common preservatives inhibit skin repair. Because of the low preservative level, this cream required refrigeration. This study of 60 patients with diabetic and venous stasis ulcers gave evidence of rapid healing. This apparently successful formulation was never used in later clinical studies, which failed to achieve therapeutic goals in FDA trials for clinical uses.
Bernard Kalis (Universite de Reims) who, along with his colleagues, performed the first successful tests of GHK on the healing of human wounds.
A following study in 1990 designed by Schering Plough Pharmaceutical used a similar cream but with an addition of 1% benzoyl alcohol as additional preservative. In retrospect, the addition of benzoyl alcohol was a mistake since it is now known that benzoyl alcohol inhibits the function of wound fibroblasts about 100-fold more than it inhibits bacteria growth. The GHK-Cu cream improved the healing of wounds caused by Moh's Surgery. However, it did not accelerate the healing of venous stasis ulcers, which may be a poor model for testing wound healing formulations since other growth factors such as PDGF, TGF, FGF, and EGF have failed to demonstrate statistical healing of venous stasis ulcers.
Nonetheless, following this other small human studies gave evidence of wound healing. A well controlled study published in 1993 of 120 diabetic patients in a single medical center found evidence of accelerated healing of diabetic ulcers. A larger double-blinded study in 1995 failed to demonstrate improved closure but did find GHK-Cu to improve re-epithelialization of the ulcers.
This study used 505 patients in 33 medical centers with an average of 17 patients per medical center. Given the variance in treatment procedures at various medical centers, this may have increased the statistical variance and degraded the data. The GHK-Cu concentrations used (2.0% and 0.5%) appear too low for effective healing. Also, benzoyl alcohol, that was known that time to inhibit wound fibroblasts, was inexplicably still used as a preservative.
Because of rapid breakdown of GHK-Cu, dosages should be in the 4% to 10% range and it is best to apply the GHK-Cu in a non-ionic cream that slowly releases the GHK-Cu into the wounded tissue.
Example - Healing of Pre-Ulcer Dermatitis
One of the best uses of SRCPs would be the healing of pre-ulcer dermatitis before the damaged skin develops open sores. The photographs at left are an example of healing "at-risk" skin with pre-ulcer dermatitis. In the top photo, the patient has two open skin ulcers visible in left top and bottom of the photo. On the right of the photo, there are reddish fissures developing into skin ulcers. In the bottom photo, the application of a copper peptide cream (BioHeal) to the periphery of the skin ulcers has healed the fissured skin and prevented further ulcer development. It should be emphasize that BioHeal is only to be used for "at-risk" skin in the stage of pre-ulcer dermatitis to help prevent further skin breakdown. It is not approved for the treatment of open skin ulcers.
Example - Healing of Diabetic Skin Ulcers
Persons with diabetes often have slow and inadequate skin repair. The skin complications of diabetes lead to skin that is dry, tends to crack, and is slow to heal. Leg sore and foot sores are the leading cause of lower leg amputations and presently approximately 10% of diabetic patients require an amputation during their lifetime. The primary reason for amputation is infections associated with the development of broken and ulcerated skin. Persons with diabetes are at 15 times the average risk of experiencing limb amputations.
Copper peptide creams often produce a rapid improvement in skin health and help prevent the development of cracks and fissures in the skin which may turn into skin ulcers. Rapid healing of broken and cracked skin, before an infection sets in, is very important.
Often skin damage in diabetic patients is relatively easy to heal. The best way is to pre-wash affected skin with 3% hydrogen peroxide followed by the application of creams containing copper peptides. In the photograph to left, a women with diabetes had six skin ulcers on her foot and physicians were recommending immediate amputation. Instead she tried the hydrogen peroxide washes followed by application of BioHeal to the periphery of the skin ulcers. The photographs to the left are either side of the foot before this procedure and those to the right are after 28 days of hydrogen peroxide and BioHeal. After 28 days all the skin ulcers were healed. The ulcers never re-occurred and she only used additional BioHeal as needed when her skin became excessively irritated and cracked.
For cracked skin, use a light coating of BioHeal once daily as needed. For skin ulcers, put the BioHeal around the edge of the ulcers. This aids skin healing from the outer edge of the ulcers. BioHeal is not FDA-approved for use within the ulcer area.
Some cliniciancs recommend against the use of hydrogen peroxide on damaged skin and state that it increases skin damage. However, my reviews of the last 70 years of medical literature have found many reports of improved skin healing after washes of hydrogen peroxide at low concentrations (1% to 10%). However, skin damage is occasionally observed at higher concentration of hydrogen peroxide. So any use of hydrogen peroxide on damaged skin should use concentrations of 1% to 3% and no higher.
GHK-Cu Clinical Dosage Levels and Pharmacokinetics of Rapid Breakdown in Vivo
While GHK and GHK-Cu biological actions start at 10 exp(-12) M and peak at about 10 exp(-8) M, clincal studies have used far higher dosages. There may be explanations for this divergence. When GHK-Cu containing creams are applied to wounds or intact skin, the uptake levels are very low - ranging from 0.05 to 0.15%. Alternatively, when GHK-Cu is injected intradermally or into a wound margin. GHK is rapidly cleared from the area with an over 95% clearance in 1 minute. In mice, it has an approximate 20 minute half-life when injected intraperitoneally. It is quickly broken down into free glycine and lysyl-histidine, which is quickly excreted. (Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization, Endo T; Miyagi M; Ujiie A (Kissei Pharmaceutical Co., Ltd., Minamiazumi, Nagano, Japan) J Chromatogr B Biomed Sci Appl 1997 Apr 25;692(1):37-42).
Enhancement of Systemic Wound Healing with GHK-Cu
GHK-Cu can be used to enhance systemic wound repair throughout an animal. In 2.5 kilogram rabbits, H. Paul Ehrlich found that the injection of 1 milligram of GHK-Cu (formulated at a ratio of 2 molecules peptide to one molecule copper) into muscle tissue markedly stimulated healing in areas distant from the the injection site and increased the concentration of circulating wound macrophages. If this technique were used in humans, the injection of a very small amount of GHK-Cu (approximately 30 milligrams) before a surgical operation would accelerate the tissue repair. The amount of copper contained in 30 milligrams of GHK-Cu is about the daily RDA.
Such a technique would be of great value in difficult operations such as hip transplantation surgery in the elderly and in operations in immune suppressed patients. Experiments with immune suppressed rats by H. Paul Ehrlich have found that GHK-Cu normalized wound repair in such situations.