Copper Peptide Studies on Skin Renewal, Wound Healing and Hair Growth

I discovered GHK-Cu in human plasma during experiments that tried to reverse certain effects of aging in animals. Since then, GHK-Cu and certain other copper-peptides, have been found to accelerate the regeneration and repair of many types of mammalian tissues. Studies, at numerous universities and research institutes, have found copper-peptides to:

- Reverse aging effects on skin (humans) - thickens skin, reduces small and large wrinkles, reduces blotchiness and blemishes, improves elasticity, and increases keratinocyte and fibroblast production, thickens the subcutaneous fat layer

- GHK analogs with fatty residue analogs increase hair follicle size, stimulate hair growth and reduce hair loss (humans, mice, rats). Increases hair growth after chemotherapy (rats)

- Have profound anti-inflammatory actions (reduces release of oxidizing iron from ferritin, TNF-alpha, TGF-beta-1, IL-1). The actions on TNF-alpha (tumor necrosis factor alpha) are especially important since it is considered to the the "master inflammatory molecule" that plays a causative role in chronic inflammatory conditions (rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, acute alcoholic hepatitis) and possibily atherosclerosis, Alzheimer’s disease, and neuropathies. Drugs that block TNF-alpha also block the other cytokines involved in the inflammation.

SRCPs are copper peptides that repair and remodel skin. While all tissue remodeling copper peptides may be similar, most information at this time is about skin repair and renewal.

First generation SRCPs are GHK-copper and chemical analogs that I developed from 1969 to 1991. The second generation are the breakdown resistant, highly adhesive copper peptides that I am currently developing at Skin Biology.

As we age, a number of skin changes occur which require different types of skin care and treatments. These are four basic problems as we age.

Problem 1. The rate of skin cell replacement is reduced producing a thinner, more fragile skin. Skin is replaced every three weeks at age 20 but this increases to every nine weeks by age 50.

Problem 2. Damaged protein accumulates in the skin. This damage can be the results of scars, sun damage, oxidative damage, and the cross-linking of skin proteins by sugars.

Problem 3. After age 25, skin oil production starts to drop. This reduces acne but produces a dryer skin. This drop in oil production becomes more serious after age 45.

Problem 4. The biosynthesis and breakdown of collagen, elastin, and water-holding GAGs exists in a dynamic balance in young healthy skin. However after age 25, the skin's production of collagen, elastin, the water-holding GAGs is reduced while the rate of breakdown of these factors is increased and starts wrinkle formation and loss of elasticity. The problem become progressively more serious with passing years.

Skin Remodeling is the process that removes proteins and older cells from the skin thus removing scars, lesions, and wrinkles while smoothing the skin. Increasing skin remodeling is the key to producing a biologically younger skin.

Skin remodeling is very active in young people but declines rapidly as humans pass age 20. At Skin Biology we are told by our customers of many successes using copper-peptides to increase skin remodeling as evidenced by reduction or total removal of sun damage marks, skin tags, certain types of moles and age spots, old stretch marks of pregnancy, and burn scars. Usually these are reduced with the combination of copper-peptide creams and glycolic acid creams but often the copper peptides are effective alone. The method produces no skin irritation and results are usually evident in one month although maximum effects often require four months of treatment with SRCPs. As skin molecules are broken down, certain peptides are liberated that constitute new signals for the surrounding skin cells. An actual regulation loop exists in connective tissue, constituted by peptides generated by ECM degradation and connective tissue cells. They propose the term of "matrikine" to designate such ECM-derived copper-binding peptides are created and these serve as signals for skin rebuilding.

During aging, skin proteins accumulate damage from many causes - sun damage, cross-linking by sugars, free radical injuries and so on. SRCPs activate both the systems that remove damaged proteins plus the synthesis of new skin proteins. Damaged collagen and elastin are replaced with new elastic and firm collagen and elastin. This removes scar tissue and restores skin elasticity and reduces wrinkles. The activation of such proteins may explain the potent scar reduction properties of such creams.

SRCPs produce a better skin renewal in combination with exfoliation or various controlled skin damage methods. SRCPs help stimulate new capillary formation (angiogenesis), increasing the biosynthesis of collagen, elastin and the water-holding proteoglycans and glycosaminoglycans.

The application of SRCPs to the skin's surface creates an environment that helps the skin tighten its barrier and increase its collagen and elastin density. The photo on the left is an ultrasound scan of the skin of a women aged 59 before treatment with the a cream containing SRCPs. On the right is the same skin after one month of treatment with the complexes. The white-yellow colored areas are the ultrasonic reflection from skin areas that are more dense because of closer cellular binding and increased amounts of collagen and elastin. This is an effect that is opposite to the usual thinning and loosening of skin during aging.

SRCP creams can be used in combination with (1) exfoliating agents such as alpha hydroxy acids, beta-hydroxy acids, and retinoic acid or by (2) methods that mildly damage skin such as by laser re-surfacing or chemical peels.

SRCPs, in combination with the use of exfoliating hydroxy acids, appear to be an excellent method for the reduction of scars and other skin lesions (keratoses, skin tags, some types of moles). 2% salicylic acid works well other hydroxy acids also work. Skin Biology's new breakdown-resistant SRCPs and hydroxy acids are rubbed into the scar once or twice daily. A reduction in scar appearance should be evident in a month but 6 to 8 months of such applications may be necessary to reach a satisfactory appearance in very old scars (30 to 50 year old scars or stretch marks). While such a method is slow, it is low-cost and will not cause further scarring.This method depends on the hydroxy acids to dissolve a very small amount of the scar, stretch mark, skin tag, or mole daily while the copper peptides enhance subsequent skin repair and possibly also act by activation of metalloproteinases and suppression of the production of the scar producing growth factor TGF-beta1 (references are above). It appears that, under proper stimuli, normal skin will replace the slowly-dissolved skin lesions. See www.scar-reduction.com

Cosmetic Skin Renewal and Aging Reversal Studies
Study	Result	References
Methods to Reverse Aging Effects in Human Skin	Several open studies gave evidence for wrinkle reduction, blemish reduction, increased skin thickness, elasticity, and improved appearance.	Pickart US Patents, 5,135,913 Skin treatment composition comprises GHL-CU or derivatives for improving skin health, increasing subcutaneous fat, dermal thickness and density, 5,348,943 Cosmetic and skin treatment compositions.
Human Skin Renewal Study	A clinical study which compared the effect on the skin's production of collagen after using creams containing copper-peptides, vitamin C, or retinoic acid (retin-A) Twenty volunteers applied the various creams to their thighs daily for one month. New collagen production was determined by studying skin biopsy samples using immunohistological techniques. The study found, that after one month, copper-peptides had the most significant effect on collagen production. Significant increases in collagen production were found in 70% of the persons treated with copper-peptide creams, 50% of the persons treated with the vitamin C cream, and 40% of the persons treated with retinoic acid.	Effects of topical creams containing vitamin C, a copper-binding peptide cream and melatonin compared with tretinoin on the ultrastructure of normal skin - A pilot clinical, histologic, and ultrastructural study. Abdulghani A.A.; Sherr A.; Shirin S.; Solodkina G.; Tapia E.M.; Wolf.GottliebA.B.; Dermatology, UMDNJ, Robert Wood Johnson Medical School; Disease Management and Clinical Outcomes, 1998, 1:136-141.
Human Skin Renewal Study	GHK-Cu containing liquid foundation tested on skin appearance, skin elasticity and epidermal thickness in an 8 week study. GHK-Cu containing liquid foundation improved skin appearance, and increased skin elasticity and epidermal thickness.	A Clinical Evaluation of a Copper-Peptide Containing Liquid Foundation and Cream Concealer Designed for Improving Skin Condition. Appa Y, Barkovic S, Finkey M B, Neutrogena Corporation, Los Angeles, CA, Stephens, T, TJ Stephens & Associates, Inc, Dallas, TX Abstract P66, American Academy of Dermatology Meeting, February 2002
Reduction of Photo Damage	GHK-Cu containing creams tested for reducing visible signs of aging and increasing skin density. GHK-Cu containing creams reduced visible signs of aging and increased skin density in 8 weeks on facial skin.	The Effect of Tripeptide to Copper Ratio in Two Copper Peptide Creams on Photoaged Facial Skin. Leyden J, University of Pennsylvania, Philadelphia, PA, Grove, G, KGL, Inc/Skin Study Center, Broomall, PA; Barkovic S, Appa Y, Neutrogena Corporation, Los Angeles, CA; Abstract P67, American Academy of Dermatology Meeting, February 2002
Human Skin Renewal Study	GHK-Cu containing creams tested for effect on wrinkles, fine line, skin elasticity, skin density, and thickness. Placebo-controlled study, 71 females, 12 weeks. GHK-Cu containing creams reduced wrinkles and fine lines while increasing skin elasticity, skin density, and thickness.	Skin Care Benefits of Copper Peptide Containing Facial Cream. Leyden J, University of Pennsylvania, Philadelphia, PA Stephens T, Thomas J Stephens & Associates, Inc, Dallas, TX; Finkey MB, Barkovic S, Neutrogena Corporation, Los Angeles, CA; Abstract P68, American Academy of Dermatology Meeting, February 2002
Human Skin Renewal Study	GHK-Cu containing eye creams tested on wrinkles, fine lines and eye appearance in placebo controlled study, 41 females, 12 weeks. In a second placebo controlled, 3 week, half face study, GHK-Cu was significantly better than a vitamin K cream. GHK-Cu containing eye creams reduced wrinkles and fine lines while improving eye appearance in placebo-controlled study. GHK-Cu was significantly better than a vitamin K cream.	Skin Care Benefits of Copper Peptide Containing Eye Creams. Leyden J, University of Pennsylvania, Philadelphia, PA; Stephens T, Thomas J Stephens & Associates, Inc, Dallas, TX; Finkey MB, Barkovic S, Neutrogena Corporation, Los Angeles, CA; Abstract P69, American Academy of Dermatology Meeting, February 2002
Review	Review of skin remodeling	Pickart L, Copper Peptides for Tissue Regeneration, Specialty Chemicals, Oct. 9, 2002, 29-31. 2002
Human Skin Renewal Study	Renewal with GHK-Cu in SPF 20 skin cream	Stephens TJ, Sigler ML, Finkley MB, Appa Y, Skin benefits of a SPF-20 copper peptide containing skin cream, 61th Amer Acad Derm Meeting, San Francisco, 2003
Review	Review of skin remodeling	Pickart L, Copper peptides for aging reversal, Body Language Dermatology, April 2003,
Review	Review of skin remodeling	Pickart, L, Copperceuticals and the skin, Cosmetics & Toiletries 2003, 118:24-28
Theory of Skin Remodeling	Biochemical theory of mechanism of skin remodeling	Pickart L, Skin remodeling copper peptides, Cosmetics & Medicine (Russia) 2004, Number 2, pages 14-29,
Review of Cosmetic Studies on GHK-Cu	A series of placebo-controlled studies found GHK-Cu skin creams to: 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 hyperpigmentation 6. Strongly increase keratinocyte proliferation in women of 50 year old range	Copper Peptide and Skin, M.B. Finkley, Y. Appa, S. Bhandarkar, Cosmeceuticals and Active Cosmetic, 2nd Edition (ISBN: 0-8247-4239-7), 2005, pp 549-563
GHK actions on sebum production	GHK suppresses sebum production in cultured sebocytes but not overall lipid synthesis	Schagen S, Voegeli R, Imfeld D, Schreier T, Zouboulis CC, Lipid regulation in SZ95 sebocytes by glycyl-histidyl-lysine, 16th European Academy of Dermatology and Venereology Congress, 16-20 May 2007
Review of Remodeling	Review of the New Biochemistry and Cell Biology of Skin Remodeling	Pickart L, The human tripeptide GHK and tissue remodeling, "Wound Healing and Technology", J. Biomater, Sci. Polymer Edit. Vol 19, No. 8 pp. 969-988 (2008).
Skin Penetration of Copper Peptides	GHK-Cu penetrates skin via the horny lipid layer of the skin barrier	Mazurowska L, Mojski M, Biological activities of selected peptides: skin penetration ability of copper complexes with peptides, J Cosmet Sci. 2008 Jan-Feb;59(1):59-69.
Update on Remodeling	Presented at Wound Healing Society, April 2008	Pickart L and Pickart F, GHK defines the biochemical reactions of tissue remodeling. Look at http://www.skinbiology.com/copperpeptideregeneration-WHS2008.html

Link Between Hair Follicles and Skin Remodeling
Skin renewal and hair follicles are closely linked. New skin is now thought to arise from the hair follicles. All skin remodeling chemicals or genes that activate skin remodeling also increase hair follicle size. But although skin remodeling improves hair follicle health, it does not create new follicles or grow hair in hairless areas.

Skin Remodeling Chemicals or Methods
Product or Method	Causes skin remodeling	Present in wounds	Anti-inflammatory	Increases hair follicle size?	Reduces TGF-beta and scar production
Retinoic acid (Retin-A)	Yes	Not significant	No, causes redness and irritation	Yes	No, increases TGF-beta production
First Generation Skin Remodeling Copper Peptides GHK-Copper	Yes 1. Rebuilds skin protective barrier 2. Increases collagen and elastin - more than vitamin C 3. Increases water-holding proteoglycans 4. Rebuilds blood microcirculation 5. Activates removal of damaged skin proteins and scars	Yes, this is the human body's natural remodeling signal	Yes, very potent anti-inflammatory. Increase superoxide dismutase production, the skin's main antioxidant protein. Blocks ferritin release of iron that triggers damaging oxidations after injury. Blocks tissue damaging actions of Interleukin-1, TGF-beta-1, TNF-alpha & reactive carbonyl species.	Yes (humans). Also blocks hair loss if given before chemotherapy and accelerates hair growth after chemotherapy (rats)	Yes, reduces TGF-beta-1 and TNF-alpha
Second Generation - Skin Remodeling Copper Peptides (Skin Biology products, being developed to find the optimal copper peptide remodeling system)	Yes, similar to GHK-Copper but not as well studied	Yes	Similar to GHK-Copper, More effective than cortisone on reducing inflammation in nickel allergy patients Yes, potent anti-inflammatory	Yes	Reduces scaring but effect on TGF beta unknown
Increase HEDGEHOG or Wnt Genes in mice, increase production of beta-catenin	Yes	Not applicable	Unknown	Yes	Unknown

Skin remodeling starts with the hair follicles. The body's signal for remodeling first activate the systems that break down damaged proteins and remove damaged skin lesion. Various proteins (proteolytic enzymes) start braking down scars and damaged tissue . Then the signals enlarge the hair follicles in the skin area to be rebuilt. New skin cells arise from the hair follicle and migrate into the surrounding skin area.This effect was first noted about 50 years ago during World War II. It was observed, in patients with severe burns and burn scars, that if hair follicles began growing at the edge of the burned skin, then this was an excellent prognosis for removal of the scarred skin and its replacement by healthy, normal skin. If the follicles were enlarging, the healing was poor and the burn scars remained. Later, in 1985, I found that copper peptides not only stimulated healing of wounds but also increased the size of the hair follicles near the wounds. The copper peptides were not hair growth stimulators, per se, but did increase the hair follicle size and vitality.In recent years, it has been established that genetic modifications in mice that stimulate skin remodeling also increase hair follicle size (Fuchs 1998). Genes such as sonic hedgehog (Sato et al 2001, Nanba et al 2003, Oro et al 2003. Mill eta al 2003), catinin (Huelsken 2001, Van Mater et al 2003), Wnt (Stenn 2001) and Noggin (Botchkarev 2001) all enhance hair follicles then produce remodeling. Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse, Nanba D, Nakanishi Y, Hieda Y., Dev Growth Differ. 2003 Jun;45(3):231-9(Transient activation of beta -catenin signaling in cutaneous keratinocyte is sufficient to trigger the active growth phase of the hair cycle in mice, Van Mater D, Kolligs FT, Dlugosz AA, Fearon ER., Genes Dev. 2003 May 15;17(10):1219-24; Hair cycle regulation of Hedgehog signal reception., Oro AE, Higgins K., Dev Biol. 2003 Mar 15;255(2):238-48, Establishment of cadherin-based intercellular junctions in the dermal papilla of the developing hair follicle., Nanba D, Nakanishi Y, Hieda Y., Anat Rec. 2003 Feb 1,270A(2):97-102; Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development., Mill P, Mo R, Fu H, Grachtchouk M, Kim PC, Dlugosz AA, Hui CC., Genes Dev. 2003 Jan 15;17(2):282-94; Effect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia., Sato N, Leopold PL, Crystal RG., J Natl Cancer Inst. 2001 Dec 19:93(24):1858-64; Noggin is required for induction of the hair follicle growth phase in postnatal skin., Botchkarev VA, Botchkareva NV, Nakamura M, Huber 0, Funa K, Lauster R, Paus R Gilchrest BA., FASEB J. 2001 Oct; 15(12):2205-14; beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin., Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W., Cell. 2001 May 18;105(4):533-45; Controls of hair follicle cycling, Stenn KS, Paus R, Physiol Rev. 2001 Jan:81(1):449-494; Beauty is skin deep: the fascinating biology of the epidermis and its appendages., Fuchs E., Harvey Lect. 1998-99:94:47-77)

Prof. Jacque Paul Borel, (Universite de Reims) whose team, later headed by Francois Maquart, was the first the suggest that GHK-Cu stimulates skin remodeling.

Photograph: Second from left - Prof. Francois Maquart, (Universite de Reims), whose research team first classified GHK-Cu as a tissue remodeling signal. Extreme left, Francoise Dominique Pickart, my daughter and then a University of Washington student who learned DNA isolation techniques with Maquart's group.

Laser and LED Lights - GHK-Cu Treatments

Stimulation of Hair Growth

Publications on SRCPs and Hair Growth
Study	Result	Reference
Use of GHK-Cu analogs for hair follicle enlargement and stimulation of hair growth	Methods for the design and testing of copper-peptide complexes with hair growth properties are described. A wide variety of GHK-Cu analogs were described that increase hair follicle size and increase hair growth in mice and rats.	US Patent 5,120,831 New metal peptide complexes and derivatives used for stimulating growth of hair in warm-blooded animals, especially humans. Pickart US Patent 5,177,061 Compositions for stimulating hair growth containing cupric complexes of peptide derivatives including. glycyl-l-histidyl-l-lysine n-octyl ester. Pickart US Patent 5,214,032 New glycyl-histidyl-lysyl copper compounds used in stimulating hair growth. Pickart US 5,550,183 Metal-peptide compositions and methods for stimulating hair growth. Pickart
Stimulation of hair growth in mice	GHK analogs with hydrophobic residues were tested and found to stimulate hair growth in rats.	The hair follicle stimulating properties of peptide copper complexes. Results in C3H mice. Fors, Pickart and Uno Ann N Y Acad Sci 1991 26;642:468-9
Stimulation of hair growth in mice and rats	The details of hair stimulation by copper peptides was studied by 1) phototrichogram, 2) folliculogram (micro morphometric analysis), and 3) the rate of DNA synthesis in the follicular cells. The 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 piebald terminal follicles (prevention). A SRCP (PC1020) had the effect of follicular enlargement on the back skin of fuzzy rats, covering the vellus follicles.	Chemical agents and peptides affect hair growth. Uno and Kurata (University of Wisconsin, Madison, USA) J Invest Dermatol 1993 101(1 Suppl):143S-147S
Minimizing hair loss after cancer chemotherapy		Hairloss protection by peptide-copper complex in animal models of chemotherapy-induced alopecia. Awa and Nogimori Journal Of Dermatological Science, Vol: 10, 1995, 99-104
Human hair growth	Stimulation of hair growth in humans with GHK-Cu analogs	Phototrichogram Analysis of Hair Follicle Stimulation: A pilot clinical study with a peptide-copper complex. Patt, Duncan and Kalis (University of Reims, France) Dermatological Research Techniques, (CRC Press), pp-217-226, 1996
Hair growth in rats	Stimulation of hair growth in rats	Quantitative Assessment of Peptide-Copper Complex Induced Hair Follicle Stimulation Using the Fuzzy Rat, Uno, Packard, Patt (University of Wisconsin) Dermatological Research Techniques, (CRC Press), pp-227-239, 1996
Hair growth in rats	Stimulation of hair growth in rats with GHK-Cu analogs	Evaluation of Telogen Hair Follicle Stimulation Using an In Vivo Model: Results with Peptide Copper Complexes. Timpe, Dumwiddie, Patt (Procyte Corp.) Dermatological Research Techniques, (CRC Press), pp-241-254, 1996
Human study of hair growth with GHK-Cu analog	Compared GHK-CU analog in Tricomin with 2% minoxidil. Tricomin 2.5% increased hair count by 97 non-vellus hairs while 2% minoxidil increased count by 73 non-vellus hair after 3 months (non-vellus hair count)	Procyte Corp. press release 1997
Breakdown resistant, long acting copper-peptides used for stimulation of hair growth	Tested copper complexed with protein peptones for hair growth effects in mice. Copper-peptide mixture produced more hair growth in mice than GHK-Cu analogs	Pickart US Patent 5,554,375 Tissue protective and regenerative compositions.
Review	Skin remodeling and hair growth	Pickart L, Effect of copper peptides on hair growth and condition, Body Language Dermatology 2004, Number 7, pages 20-22
Review	Skin remodeling and hair growth	Pickart L, Skin remodeling copper peptides for improving hair growth, Cosmetics & Medicine (Russia) 2004, Number 3, pages 14-29
Study of human follicles in organ culture	AHK-Cu increased follicular cell growth while decreasing progrmmed cell death (apoptosis)	Pyo HK, Yoo HG, Won CH, Lee SH, Kang YJ, Eun HC, Cho KH, Kim KH, The effect of tripeptide-copper complex on human hair growth, Arch Pharm Res 2007, vol 7, 834-839.

Certain analogs of GHK-Cu have the property of enlarging hair follicles and stimulating hair growth. These analogs have more fat-like character than GHK-Cu. This increase in fat-like properties is obtained by either chemically synthesizing fatty acids into the GHK molecule or attaching amino acid residues such as alanine, phenylalanine or leucine to the basic GHK structure.

These analogs originally arose in an attempt to create GHK-Cu analogs which would be retained in body tissues for longer periods of time. However, it was noted that - while such analogs were superior wound healing agents - they also markedly increased hair growth around the periphery of experimental wounds in mice.

These hair stimulating analogs were created by Drs. Steven Lovejoy, Loren Pickart and Boris Weinstein. Skin repair and hair growth enhancement effects are closely linked. New skin appears to arise from the hair follicle. Certain products based on Iamin can be used to both repair skin, increase hair follicle size, and stimulate hair growth. As a person ages, our hair follicles get smaller, producing thinner hair shafts. A major cause of hair follicle miniaturization appears to be due to the development of striking changes in capillaries surrounding the hair follicles. Comprehensive surveys of the male scalp from birth to senescence find very significant changes in the structure of the blood vessels of the scalp. The number of the blood capillary loops supplying the hair follicle is greatly diminished. The inadequate subepidermal circulation that can develop as males age does not provide a rich nutrition for the follicle. Strong hair growth requires a large flow of nutrients such as such as vitamins, minerals and amino acids so that the follicle can actively synthesize new hair.

Blood flow impairments to the follicle, and their reversal, may explain why the administration of copper peptides (such as Folligen and Tricomin) to the scalp increases hair growth and increase the size of hair shafts. It has long been known that certain copper-peptide complexes strongly stimulate angiogenesis or new blood vessel formation. The increase in hair follicle size and the rate of hair growth caused by the administration of copper-peptides may be due to their causing blood flow changes that provide adequate nutrients to the follicle, producing faster growing hair with thicker hair shafts. Copper ion complexed with certain peptides has both skin repair and hair growth enhancement effects. Examples of this are Folligen (from Skin Biology), and Tricomin and GraftCyte which are based on Pickart's earlier work (from ProCyte Corporation).

During aging in men and women, there is a progressive decrease in hair follicle size. This produces thinner hair and in time stops new hair growth.

Pyo et al (refererence above) propose, based on studies of human hair follicles, that the actions of copper peptides increase cell growth in cultured hair follicles while decreasing programmed cell death or apoptosis. Copper pepides also decrease the Bax protein which increases apoptosis. Their studies use Ala-His-Lys-copper, as close analog of GHK, that I had found to stimulate hair growth many years ago.

So, the copper peptides may function by slowing the rate of programmed cell death in human hair follicles that ultimately stops human hair growth.

The skin of the mouse to the left was shaved, then treated in three spots with Folligen. The result is a much more rapid hair growth (the three circular patches of hair) in the three spots treated with Folligen. While human hair growth will not respond nearly as dramatically as in mice, skin health and hair follicle function are closely interrelated. New skin appears to arise from the hair follicle. As a person ages, our hair follicles get smaller, producing thinner hair shafts. The blood circulation that supplies nutrients and oxygen to the hair follicle send fewer blood vessels to the hair follicle, thus inhibiting the vital flow of nutrients to the hair follicle. Copper-peptide complexes improve skin health and a more healthy skin increases the blood vessel network to the hair follicles resulting in larger follicles that grow hair faster with thicker hair shafts.

In the microscopic images to the left, the magnifications are identical. The top photo is mouse skin untreated with copper-peptides. The bottom photo is mouse skin treated with copper-peptides. Note the larger hair follicles (the elongated purple columns) in the lower photo, the increased content of subcutaneous fat in the skin (the white material in the center of the skin), and the increased thickness of the skin. When we are young, we have a layer of fat under the skin (part of "baby fat") which is greatly reduced as we age. Hair researchers have noted the accumulation of this fat around healthy follicles that are vigorously growing hair, and its relative lack around dormant follicles, have postulated that these cells serve a supportive function for the hair follicle. It must be emphasized that effects in humans on hair follicle health are not as dramatic.

New Hair Follicle Formation?

At times, SRCPs can apparently induce a proliferation of hair follicles, although this phenomena is difficult to reproduce on a consistent basis. The photograph on the top is a microscopic field of mouse hair follicles in an animal treated only with saline. The photograph on the bottom is a similar area of mouse skin treated with copper-peptides and which has a much higher density of hair follicles. Individual experiments on hair follicle multiplication are consistent, that is, the effect is actual when it occurs, but repeated results are difficult to obtain. The variability may be due to different timing in the hair growth cycle or slight changes in the type of, or formulation of, the copper-peptide preparations. Such experiments strongly suggest that, under certain circumstances, new hair follicle formation can be induced in adult animals.

The hair loss caused by chemotherapy drugs used for cancer treatment can be minimized with copper peptides. Awa and Nogimori found that application of copper peptides minimize hair loss after chemotherapy and accelerated new hair growth in rats.

Copper is intimately involved in stem stem production and differentiation. Low cellular copper retards stem cell differentiation while high cellular copper causes the differentiation of stem cells. This may explain the diverse regenerative actions of GHK.

GHK is a very unique peptide that has the precise binding affinity to obtain copper from its transport site on human albumin. As GHK is generated after tissue damage, it will bind available copper and slow the use of available stem cells, presumably allowing them to build up in concentration. But then about 5 to 20% of the GHK is slowly converted into GHK-Cu and this pushes the stem cells to differentiate.

Wound Healing and Skin Repair Studies Using SRCPs
Study	Result	Reference
	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 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, Patt, Sanders, Rosenstock, Altman, Hanley, Duncan Wound Rep Reg 2:259-269, 1994
Dogs	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) on the healing of linear incision wounds. Schmidt, Resser, Sims, Mullins and Smith (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.

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.

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.

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.

SRCP 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 SRCPs. 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 clinicians 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 10exp (-12) M and peak at about 10exp (-8) M, clinical 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)

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.

GHK-Cu improved the transplants of skin grafts in pigs, mice, and humans. For pig skin grafts, the best results were obtained by using GHK-Cu dissolved in a small amount of DMSO (dimethylsulfoxide). Normally in skin grafts, most of the grafted skin dies off, and new skin grows outward from the surviving core of the skin graft. However, the addition of DMSO often so improved the graft "take" that the final graft "take" area exceeded the actual size of the skin graft.

Rat liver organ culture	GHK-Cu stimulated the survival of normal rat liver cells	Tripeptide in human serum which prolongs survival of normal liver cells and stimulates growth in neoplastic liver, Pickart L, Thaler M, Nature New Biol,1973, 243:85-7
Rats - Effect of GHK on DNA synthesis and immune status	Ten intraperitoneal injections of tripeptide Gly-His-Lys in doses of 1.5, 5, 50, 150, and 450 mg/kg stimulated mitotic activity of hepatocytes and dose-dependently suppressed immune reactivity (number of antibody-producing cells and delayed-type hypersensitivity reaction).	Smakhtin, MY, Sever’yanova LA, Konoplya AI, Shveinov IA. Tripeptide Gly-His-Lys is a hepatotropic and immunosuppressor. Bull. Exp. Biol. Med. 2002 Jun; 133(6):586-8
Rats- Regeneration of hepatic damage after poison	10-day intraperitoneal administration of Gly-His-Lys in doses 2.5 and 150 mu/kg raises of mitotic index of hepatocytes in acute toxic damage to the liver by dichloromethane (carbon tetrachloride). In the dose 1.5 mu/kg this peptide corrects both functional activity of hepatocytes and immunological responsiveness. In a dose of 150 mu/kg the peptide has a more potent immunosuppressive action and deteriorates biochemical indices of blood serum as well as dystrophic changes in the liver.	Smakhtin MI, Konoplia AI, Sever’ianova LA, Sheveinov IA. Pharmacological correction of immuno-metabolic disorders with the peptide Gly-His-Lys in hepatic damage induced by tetrachloromethane. Patol Fiziol Eksp Ter. 2003 Apr-Jun; (2):19-21

Study	Result	Reference
Bone Healing GHK-Cu was tested for healing of bone tissue	GHK-Cu markedly stimulated bone repair	Pickart L, US Patent 5,059,588 New and known copper peptide complexes for bone healing containing glycyl-histidyl-lysine and lysyl-histidyl-glycine.
Functions of bone forming cells. Effect of GHK-Cu on bone forming cells	GHK-Cu increases the number of human marrow stromal cells and promotes the attachment of human osteoblastic cells.	Effects of the tripeptide glycyl-L-histidyl-L-lysine copper complex on osteoblastic cell spreading, attachment and phenotype. Godet and Marie (INSERM, Cell and Molecular Biology of Bone and Cartilage, Lariboisiere Hospital, Paris, France) Cell Mol Biol (Noisy-le-grand) 1995 41(8):1081-91
Chick Bone forming cells Growth of bone chondrocytes	GHK-CU increased chondrocyte growth and their synthetic rate of bone collagen. Authors suggests GHK-Cu may be useful in the preparation of cartilage implants.	Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes. Pesakova, Novotna, and Adam (Institute of Rheumatology, Postgraduate Medical School, Czech Republic) Biomaterials 1995 16(12):911-5
Bone Healing The effect of GHK-Cu on the stimulation of new bone production in guinea pigs was studied.The authors prepared 7.5% and 12.5% collagen gels, supplemented with the tripeptide GHK-Cu, perfloxacine and hypersulphated glycosaminoglycan (HSGAG).	The gels were tested in guinea pigs for filling artificially created bone defects in diaphyses of femurs, and with cementless endoprostheses. Bone healing process was followed by means of RTG and NMR, and histologically. The slowest healing process was found in unfilled bone defects. Defects filled with Colladel without GHK-Cu healed more quickly, and the most accelerated healing was with the gels with GHK-Cu. The optimum gel, when used with cementless endoprostheses, produced vivid osteogenic activity at the interface of trabecular bone and metal stem.	Morphological features of bone healing under the effect of collagen-graft-glycosaminoglycan copolymer supplemented with the tripeptide Gly-His-Lys. Pohunkova, Stehlik, Vachal, Cech and Adam Institute of Rheumatology, Charles University, Prague, Czech Republic. Biomaterials 1996, 17(16):1567-74

GHK-Cu stimulates bone healing in animals and the functions of bone repair cells in culture. The development of GHK-Cu for clinical use is being conducted under the direction of Prof. Milan Adam (University of Prague, Photograph - on the left) . Adam developed a collagen-graft-glycosaminoglycan copolymer supplemented with GHK-Cu for bone healing.

The anti-oxidant actions of GHK and GHK-Cu that help to protect injured tissue appear to have multiple actions. These are (1) a direct anti-inflammatory of the copper-peptide complex, (2) an activity that blocks the release of free iron from ferritin molecules, (3) an ability to block tissue damage caused by interleukin-1 at a GHK-Cu concentration of about 10exp(-10) M, and (4) an ability to block the oxidation of low density lipoproteins (LDL) by free copper.

Anti-Oxidant Actions
Study	Result	Reference
Development of tissue protective analogs of GHK-Cu	GHK-Cu and analogs were tested for anti-oxidant and tissue protective properties GHK-Cu and analogs were found to enhance or restore resistance to oxidative or inflammatory damage. Certain analogs were 100-fold more effective than GHK-Cu.	US Patent 5,118,665 New anti oxidative and anti-inflammatory metal peptide complexes - containing glycine, histidine and lysine residues used to enhance or restore resistance to oxidative or inflammatory damage. Pickart
Blocking of iron oxidation	A study of whether some of the wound healing properties of GHK-Cu are due to an affect on iron metabolism. The presence of iron complexes in damaged tissues is detrimental to wound healing, due to local inflammation, as well as microbial infection mediated by iron. The effects of GHK:Cu(II) on iron catalyzed lipid peroxidation. GHK:Cu(II) inhibited lipid peroxidation if the iron source was ferritin. Whereas GHK:Cu(II) inhibited ferritin iron release it did not exhibit significant superoxide dismutase-like or ceruloplasmin activity. It appears that GHK-Cu binds to the channels of ferritin involved in iron release and physically prevents the release of fee). Thus, a biological effect of GHK:Cu(II), related to wound healing, may be the inhibition of ferritin iron release in damaged tissues, preventing inflammation and microbial infections.	Effects of glycyl-histidyl-lysyl chelated Cu(II) on ferritin dependent lipid peroxidation. Miller, DeSilva, Pickart, Aust. Pickart and Aust (Biotechnology Center, Utah State University, Logan, UT, USA) Adv. Exp Med Biol 1990;264:79-84
Finding of superoxide dismutase and catalase-like activities in GHK Nickel complexes	The reactions between nickel ions and GHK and similar oligopeptides were characterized by spin trapping experiments. GHK-Cu possessed superoxide dismutase and catalase-like activities.	Redox chemistry of complexes of nickel) with some biologically important peptides in the presence of reduced oxygen species. Coterie N; Tremolieres E; Berliner JCL; Cattier JP; Henichart JP (INSERM, Ill, France) J Internat BioPharm 1992 Apr;46(1):7-15
Cytoprotective actions against oxygen free radicals	GHK-Cu markedly inhibited intestinal mucosal tissue from lipid peroxidation by oxygen-derived free radicals.	Alberghina M, Lupo G, La Spina G, Mangiameli A, Gulisano M, Sciotto D, Rizzarelli E, Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa, J Inorg Biochem. 1992 Mar;45(4):245-59.
Anti-oxidant protection of insulin secreting cells after injury. Interleukin beta (IL-1 beta) is released during injuries and after tissue damage. IL-1 inhibits insulin release by pancreatic cells.	The study tested whether GHK-Cu would block the IL-1 damage to insulin secreting pancreatic cells. Rat pancreatic islet cells were incubated with or without 50 U/ml IL-1 beta, in the presence or absence of various concentrations of Cu(II)-GHK or CuSO4 (1-1000 ng/ml). After incubation, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets, basal insulin secretion was 92 +/- 11 ( pg/islet) and glucose-induced release was 2824 +/- 249. In islets pre-exposed to 50 U/ml IL-1 beta, basal insulin release was not significantly affected but glucose- induced insulin release was greatly reduced (841 +/- 76 ). In islets incubated with IL-1 beta and Cu-GHK (0.4 mumol/l, maximal effect) basal secretion was 119.0 +/- 13 and glucose-induced release was 2797 +/- 242. CuSO4 was without protective actions.	Copper addition prevents the inhibitory effects of interleukin 1-beta on rat pancreatic islets, Vinci, Caltabiano, Santoro, Rabuazzo, Buscema, Purrello, Rizzarelli, Vigneri and Purrello (University of Catania Medical Endocrinology, University of Catania Medical School, Italy) Diabetologia 1995 38(1):39-45
Effect of GHK on blocking oxidative damage that produces Alzheimer's disease	Loosely bound copper(II) can produce oxidation of amyloid protein of Alzheimer's disease and cause neurodegeneration Loosely bound copper(II) can produce oxidation of amyloid protein of Alzheimer's disease and cause neurodegeneration	The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Multhaup; Schlicksupp Hesse; Beher; Ruppert Masters; Beyreuther ( ZMBH-Center for Molecular Biology University of Heidelberg, Germany) Science 1996 Mar 8;271(5254):1406-9
Increase superoxide dismutase in wounds	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
GHK detoxifies 4-hydroxy-2-nonenal, a toxic mlecule	GHK blocks the toxic actions of 4-hydroxy-2-nonenal (HNE), a fatty acid decomposition product felt to be causative in the development of diabetes, nephropathy, retinopathy, and neurodegenaerative diseases.	Beretta G, Artali R, Regazzoni L, Panigati M, Facino RM, Glycyl-histidyl-lysine (GHK) is a quencher of alpha,beta-4-hydroxy-trans-2-nonenal: a comparison with carnosine. insights into the mechanism of reaction by electrospray ionization mass spectrometry, 1H NMR, and computational techniques. Chem Res Toxicol. 2007 Sep;20(9):1309-14.
GHK blocks block acrolein production	Acrolein is a toxin created by carbonyl radicals from polyunsaturated fatty acids. GHK detoxifies acrolein. The authors suggest that GHK may be of value in prevention of atherosclerosis, diabetes, neuropathy, and Alzheimer disease.	Beretta G, Arlandini E, Artali R, Anton JM, Maffei Facino R. Acrolein sequestering ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK): Characterization of conjugation products by ESI-MS(n) and theoretical calculations. J Pharm Biomed Anal. 2008 Jul 15;47(3):596-602.

Blocking the oxidation of low density lipoproteins GHK tested on effecting the extent of in vitro Cu(2+)-dependent oxidation of low density lipoproteins (LDL)	GHK blocked the extent of in vitro Cu(2+)-dependent oxidation of low density lipoproteins (LDL). Treatment of LDL with 5 microM of copper (+2) for 18 h in either phosphate buffered saline (PBS) or Ham's F-10 medium resulted in extensive oxidation as determined by the content of thiobarbituric acid reactive substances (TABORS). In PBS, oxidation was entirely blocked by gly-his-lys (GHK). In comparison , superoxide dismutase (SOD) provided only 20% protection.	The influence of medium components on Cu(2+) dependent oxidation of low- density lipoproteins and its sensitivity to superoxide dismutase. Thomas (Marion Merle Dow Research Institute, Cincinnati, Ohio, USA) Biochem Biophys Acta 1992 1128(1):50-7
Angiotensin II appears to incite inflammatory processes that accelerated atheroma development. GHK interacts with the angiotensin II AT1 receptor. The effect of GHK on phosphorylase A was determined. Binding competition experiments using the radioligand [125I][Sar1-Ile8] angiotensin II measured the interaction of GHK with AT1 receptors.	GHK stimulated in dose-dependent fashion the activity of phosphorylase A in isolated rat hepatocytes. This effect was associated with increases in both IP3 production and [Ca++]. These effects of GHK were antagonized by losartan, a nonpeptide angiotensin II receptor antagonist (AT1 selective), which suggested that these receptors were involved in its effect. Binding competition experiments clearly indicated that GHK interacts with AT1 receptors.	Glycyl-histidyl-lysine interacts with the angiotensin II AT1 receptor. Garcia-Sainz JA; Olivares-Reyes JA (Departamento de Bioenergetica, Universidad Nacional Autonoma de Mexico, Mexico D. F). Peptides 1995;16(7):1203-7
GHK-Cu inhibits platelet aggregation	GHK-Cu inhibits platelet aggregation at 10exp (-7) M	Unpublished studies. Savage, Pickart, et al, Hope Heart Institute, Seattle, Washington, USA
GHK-Cu inhibits thromboxane production	Significant inhibition at 10exp (-7) M	Unpublished studies. Savage, Pickart, et al, Hope Heart Institute, Seattle, Washington, USA

GHK and GHK-Cu may function as the circulating human non-steroidal anti-inflammatories (NSAIDs). In human plasma there is about 200 nanograms per milliliters of GHK and GHK-Cu at age 20. This declines to about 80 nanograms per milliliter at age 60 but these levels are highly variable. Given the respective binding constants for copper(+2) between GHK and albumin in human plasma, it is likely that only about 10% of circulating GHK is chelated with copper(+2). In areas of tissue damage, this ratio could be higher because of lowered albumin concentrations. There are very close similarities between the three dimensional chemical structures of GHK-Cu and H2-Receptor antagonists used as anti-ulcer medicines such as cimetidine, ranitidine, famotidine and nizatidine. Since GHK-Cu is a normal component of saliva present at about 40 nanograms/milliliter, it may function a natural protector of gastrointestinal linings. Also, most common anti-ulcer drugs are potent binders of ionic copper (II).There are also similarities, though less obvious, between most Non-Steroidal Anti-Inflammatory Drugs (NAISDs) and GHK. Virtually all NSAIDs avidly bind copper(+2).

**Effects of GHK-Cu on gastric acidity, mucous production and the development of ulcers**
(Shay gastric ulcer model (95% ethanol) in rats)
Dosage of GHK-Cu	Stomach pH	Rats with visible gastric	Gastric mucous production
none	2.3	72%	Unobservable
1 milligram	3.8	33%	++
3 milligrams	4.7	none	++++
10 milligrams	6.7	none	++++


Study	Result	Reference
Databank search for source of human GHK - Effect of GHK-Cu on collagen synthesis	Determined possible sources of GHK and effect of GHK-Cu on collagen synthesis in two strains of human fibroblasts and embryonic lung fibroblasts. GHK is a very rare sequence appearing in only 8 human proteins sequences as of 1990. It appears three times in collagen and also exist in several inflammatory proteins - All fibroblast lines stimulated collagen synthesis at 10exp (-12) M and maximally at 10exp (-9) M	Glycyl-l-histidyl-l-lysine, a triplet from the a2 (I) chain of human type I collagen, stimulates collagen synthesis by fibroblast cultures Maquart, Gillery, Monboisse, Pickart, Laurent and Borel Ann. N.Y. Acad. Sci. 580:573-575, 1990
Search for source of GHK	Studies on a skin structural protein called SPARC. GHK and HGHK, two peptides with very high copper-binding affinity are angiogenic. They are generated by the breakdown of SPARC, a structural protein in skin which is rich in cysteine. SPARC may function at several levels to control the progression of neovessels. Proteolysis of SPARC by plasmin results in the release of peptides containing the sequence Gly-His-Lys, which are angiogenic in vitro and in vivo. At later stages of angiogenesis when endothelial cell proliferation ceases, SPARC may exert inhibitory effects on angiogenesis.	Regulation of angiogenesis by extracellular matrix: the growth and the glue. Sage and Vernon (University of Washington School of Medicine, Seattle, WA, USA) J Hypertens Suppl 1994 12(10):S145-52
Effect of GHK on chemoattraction of healing associated immune cells	Compared GHK to known potent chemoattractants and GHK analogs. GHK was most potent chemoattractant tested for mast cells, but Gly-His-Gly, His-Lys, and His-Gly-Gly were inactive.	Stimulation of rat peritoneal mast cell migration by tumor derived peptides. Poole and Zetter (Harvard Medical School) Cancer. Res. 43, 5857-5861, 1983
Effect of GHK on chemoattraction of healing associated immune cells	Compared GHK to known potent chemoattractants and GHK analogs. An implantable device was used for the study of leukocyte chemoattraction in rats for up to 18 days. GHK attracted wound healing immune cells (mast cells, macrophages, polymorphonuclear leukocytes) at about 10exp (-10) M.	An in vivo assay for chemoattractant activity. Zetter, Rasmussen and Brown (Harvard University Medical School, Boston, MA, USA) Lab Invest 1985 53(3):362-8
Heparin is felt to mediate some healing events	GHK-Cu and heparin which is a natural anti-coagulant and a mediator of wound healing. GHK found to bind to heparin in nuclear magnetic resonance spectroscopy studies.	Binding of the growth factor glycyl-L-histidyl-L-lysine by heparin. Rabenstein, Robert and Hari (University of California at Riverside, USA) FEBS Lett 1995, 376, pp. 216-20
Angiogenesis in rabbit cornea model and capillary cell migration	GHK-Cu tested for angiogenic activity. Copper complexes of glycyl-L-histidyl-L-lysine and heparin induced angiogenesis in rabbits. Copper deficient rabbits cannot induce angiogenesis.	Ceruloplasmin, copper ions, and angiogenesis. Raju, Alessandri, Ziche and Gullino (National Cancer Institute, Bethesda, MD, USA) J Natl Cancer Inst 1982 69(5):1183-8
Angiogenesis in rabbit cornea model and capillary cell migration	GHK-Cu tested for angiogenic activity and effect on capillary cell migration. Increased angiogenesis in rabbit cornea model - Increased in vitro migration of capillary cells by 8-fold	Characterization of a chemoattractant for endothelium induced by angiogenesis effectors. Raju, Alessandri, Gullino (National Cancer Institute, Bethesda, MD, USA) Cancer Res. 44:1579-1584, 1984
Nerve Growth - comment	When wound healing is inadequate, the healed area is often devoid of sensory abilities.
Nerve Growth	Studied the effect of GHK on nerve growth. Gly-His-Lys supported chick neuron differentiation and viability in cell culture and increased nerve outgrowth . The optimum concentrations of GHK for neuron function were 100-400 ng/ml.	Effects of synthetic tripeptide on the differentiation of dissociated cerebral hemisphere nerve cells in culture. Sensenbrenner; Jaros; Moonen, Mandel (University of Strausbourg, France) Neurobiology 1975 5(4):207-13
Nerve Growth	Studied the effect of GHK on nerve growth. Gly-His-Lys supported chick neuron differentiation and viability in cell culture of various neurons - chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral tissue. The optimum concentrations of GHK for neuron outgrowth was 10 ng/ml. GHK increased the ratio of neurons to glial cell in culture.	Uber die Wirkung eines synthetischen Tripeptids auf in vitro kultiviertes Nervengewebe (The effect of a synthetic tripeptide nervous tissue cultured in vitro), Lindner, Grosse, Halle and Henklein (Karl Marx University, Berlin, Germany) Z Mikrosk Anat Forsch 1979;93(5):820-8
Nerve Growth	Severed nerves are placed in a collagen tube impregnated with GHK. This caused an increased the production of Nerve Growth Factor and the neurotrophins NT-3 and NT-4	Ahmed MR, Basha SH, Gopinath D, Muthusamy J, Jayakumar RJ, Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes, J Peripher Nerv Syst. 2005, 10:17-30
GHK was tested for maintaining fibroblast viability in serum free medium	Chick fibroblast were maintained in serum free medium after the addition of GHK. The GHK containing culture medium allowed studies of factors affecting collagen metabolism without the complications of the proteins from serum.	An in vitro model of fibroblasia - Simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis. Graham, Diegelmann and Cohen (Medical College of Virginia, Richmond, VA, USA) Proc Soc Exp Bio Med 176, 302-308, 1984
Collagen synthesis in cultured fibroblasts	The effect of GHK-Cu was determined. GHK-Cu stimulated collagen synthesis in cultured fibroblasts. The stimulation began between 10exp (-12) and 10exp (-11) M, maximized at 10exp (-9) M, and was independent of any change in cell number. The presence of a GHK triplet in the alpha 2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects.	Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+. Maquart, Pickart, Laurent, Gillery, Monboisse and Borel (Laboratoire de Biochimie, CNRS URA 84, Faculte de Medecine, Reims, France. FEBS Lett 1988, 238(2):343-6
Collagen synthesis in cultured fibroblasts	The effect of GHK-Cu on collagen production was determined. GHK-Cu increased collagen synthesis at 10exp (-9) M	Requirement of Copper and Tripeptide Glycyl-L-Histidyl-L-Lysine Complex Formation for Collagen Synthesis Activity in Normal Human Dermal Fibroblasts. Oddos, T, Jumeau-Lafond, A ; Johnson & Johnson, Val de Reuil, France, Ries, G, Johnson & Johnson, Dusseldorf, Germany Abstract P72, American Academy of Dermatology Meeting, February 2002
Sulfated glycosaminoglycan (water-holding proteins) synthesis in cultured fibroblasts.	The effect of GHK-Cu on the synthesis of sulfated glycosaminoglycan (water-holding proteins) in cultured fibroblasts was determined. GHK-Cu induced a dose-dependent increase of the synthesis of total GAGs secreted into the culture medium and those associated with the cell layer. The effect of GHK-Cu increased with dosage and was optimal at 10exp (-9) to 10exp (-8) M. Higher concentrations had less effect the rate of synthesis. GHK-Cu preferentially stimulated the synthesis of extracellular dermatan sulfate and cell layer associated heparin sulfate.	Stimulation of sulfated glycosaminoglycans by the tripeptide copper complex glycyl-l-histidyl-l-lysine copper(II). Wegrowski, Maquart, Borel (University de Reims, France) Life Sci. 51, 1049-1056, 1992
The effects of GHK-Cu on the synthesis of glycosaminoglycans and small major proteoglycans was determined.	A study of the effects of GHK- Cu in vivo, using the wound chamber model. Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats.GHK-Cu treated cultured fibroblasts and rat wound chambers had an increase in messenger RNA for decorin by not for biglycan. In both systems, GHK-Cu increased the synthesis of of decorin, dermatin sulfate and chondroitin sulfate.	Expression of glycosaminoglycans and small proteoglycans in wounds: Modulation by the tripeptide copper complex glycyl-histidyl-lysine Cu(II). Simeon, Wegrowski, Bontemps and Maquart J Invest Dermatol 2000 Dec;115(6):962-968
Wound healing events in "wound chambers" were studied in rats	A study of the effects of GHK- Cu in vivo, using the rat wound chamber model. GHK-Cu treated rat wounds had a concentration dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased. An increase of the relative amount of dermatan sulfate was also found.	In vivo stimulation of connective tissue accumulation by the tripeptide- copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds. Maquart, Bellon, Chaqour, Wegrowski, Monboisse, Chastang, Birembaut and Gillery (Universite de Reims, France) J Clin Invest 92: 2368-76, 1993
Metalloproteinases that remove damaged proteins and scar tissue	Metalloproteinases are a family of proteins that remove damaged proteins and scar tissue. The expression and activation of matrix metalloproteinases where investigated in a model of experimental wounds in rats, and their modulation by GHK-Cu. Wound chambers were inserted under the skin of rats and were injected daily with either injections of either 2 mg GHK-Cu or the same volume of saline. The wound fluid and the neosynthetized connective tissue deposited in the chambers were collected and analyzed for matrix metalloproteinase expression and/or activity. Interstitial collagenase increased in the wound fluid throughout the experiment and GHK-Cu did not alter its activity. Matrix metalloproteinase-9 (gelatinase B) and matrix metalloproteinase-2 (gelatinase A) were the two main gelatinolytic activities expressed during the healing process. Pro-matrix metalloproteinase-9 was strongly expressed during the early stages of wound healing (day 3) but decreased rapidly whereas in GHK-Cu treated chambers it persisted until day 22. Pro-matrix metalloproteinase-2 increased progressively until day 7, then decreased until day 18. Activated matrix metalloproteinase-2 increased until day 12, then decreased progressively whereas GHK-Cu increased pro-matrix metalloproteinase-2 and activated matrix metalloproteinase-2 during the later stages of healing. GHK-Cu increased metalloproteinases activity up to 4-fold which may increase the activity of wound remodeling processes which remove damaged protein and scar tissue.	Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine- Cu2+, Simeon; Monier; Emonard; Gillery; Birembaut, Hornebeck and Maquart (Faculte de Medecine, Reims, France) J Invest Dermatol 1999 112(6):957-64
Metalloproteinases and antiproteinases - comment	GHK-Cu increases MMP-2 in cultured fibroblasts but in rat wounds it decreases MMP-2 and MMP-9. GHK-Cu also increases inhibitors of metalloproteinases TIMP-1 and TIMP-2 in cultured fibroblasts. The overall actions would appear to reduce proteolysis but perhaps maintain it at a lower level than in early stage wounds.
Metalloproteinases and antiproteinases that remodel tissue	The effect of GHK-Cu on the induction of metalloproteinases in cultured wound fibroblasts. GHK-Cu at 10exp(-10) M increased MMP-2 mRNA and also inhibitors of metalloproteinases TIMP-1 and TIMP-2. The authors argue this indicate GHK-Cu modulates tissue remodeling.	The tripeptide-copper complex GHK-Cu stimulates matrix metalloproteinases 2 expression by fibroblast cultures. Simeon, Emonard, Hornebeck & Maquart Laboratoire de Biochimie-UPRESA CNRS 6021, Faculte de Medecine, Reims, France. Life Sci 2000 Sep 22;67(18):2257-65
Metalloproteinases and antiproteinases that remodel tissue	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
GHK and cellular energy production GHK was test

Human Laser Treatments	GHK-Cu skin care products placed on CO(2) laser-resurfaced skin offered no significant reduction or resolution of posttreatment erythema or improvement in wrinkles. However, patient satisfaction was significantly higher for those who used GHK-Cu skin care products after CO(2) laser skin resurfacing.	Miller TR, Wagner JD, Baack BR, Eisbach KJ, Effects of topical copper tripeptide complex on CO2 laser-resurfaced skin. Arch Facial Plast Surg. 2006 Jul-Aug;8(4):252-9.
Rabbit Laser Wounds	Healing parameters between treatments by laser or GHK-Cu in rabbits were compared. GHK-Cu filled the open wound with granualtion tissue faster than the laser. GHK-Cu and laser reduced time to wound closure about equally (29.8 days GHK-Cu, 30.2 days laser, 34.6 days control)	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.
Effect of LED lights and GHK-Cu on fibroblasts	Cultured fibroblasts were treated with red LED lights and GHK-Cu. The combination had an additive action increasing cell viability 12.5 fold, beta-FGF 2.3 fold, and also collagen production.	Huang PJ, Huang YC, Su MF, Yang TY, Huang JR, Jiang CP, In vitro observations on the influence of copper peptide aids for the LED photoirradiation of fibroblast collagen synthesis. Photomed Laser Surg. 2007 Jun;25(3):183-90.

Study	Result	Reference
Improvement of skin grafts in pigs	Photograph: Left - Control Graft about 20% of skin graft (at center) established -this is a typical graft "take". Right - Graft soaked in GHK-Cu in liposomes. Original graft overgrew transplant area .	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.
Tested GHK-Cu on skin transplants in mice	Transplanted 1.5 cm diameter full thickness skin grafts in mice. This is considered an "impossible" transplant experiment 40% of full thickness transplants became permanent grafts	Pickart Iamin: A Human Growth Factor with Multiple Wound Healing Properties. in Biology of Copper Complexes, Clifton, NJ, 1987, pp. 273-285.
Human hair transplants	.Studied the effect of GHK-Cu analog on hair transplants. Treated patients saw new hair growth in six weeks, versus the normal 10 to 14 weeks. In most cases, skin crusting after transplantation is reduced from 10 to 14 days to five days. Increased the degree of hair outgrowth from human hair transplants	Perez-Meza et al, (International Journal of Cosmetic Surgery (Vol. 6, 1998, pp 80-84)
Human hair transplants	30 hair transplant patients, found GHK-Cu analog reduced the shedding of transplanted hair from 30 percent with saline to 10 percent. The healing time of the transplanted grafts was cut in half. Regrowth of new hair from the transplants occurred in six to eight weeks with saline and four to six weeks with GHK-CU analog. Patient satisfaction after transplantation rose from 80 percent to 95 percent.	Hitzig,G. Enhanced healing and growth in hair transplantation using copper peptides, Cosmetic Dermatol 2000 (June) ; 13, 18-21