Published Studies on Tissue and Skin Remodeling Copper-Peptides

by Loren Pickart PhD

Tissue Remodeling Copper Peptides Induce Regeneration of Diverse Organs
The Copper Switch Explained
Skin Renewal Cycle - Biochemistry
SRCPs (Skin Remodeling Copper Peptides)
Cosmetic Skin Renewal and Aging Reversal
Laser and LED Lights
Stimulation of Hair Growth
Reduce Hair Loss After Chemotherapy
GHK, Copper, Regeneration, & Stem Cells
Wound Healing
GHK Suppresses Cancer Metastasis Genes
Skin and Hair Transplantation
Liver Regeneration
Bone Healing
Anti-Oxidant and Anti-Inflammatory Actions
Intestinal and Stomach Healing
SRCP Biochemistry Related to Tissue Regeneration
Stimulation of Fingernail Growth
Improving Suntanning and Reducing Skin Peeling
Background of SRCPs
GHK-Cu - Function in Human Body
The Need for Improved Skin Regenerative Copper Peptides
General References on Chemistry and Biology of GHK
The Aging Reversal Experiments and GHK
Future Uses of Remodeling Copper Peptides


 

Tissue Remodeling Copper Peptides Induce Regeneration of Diverse Organs  

 

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:       

- Accelerate wound repair (humans, mice, rats, guinea pigs, pigs, dogs)       

- Increase skin re-epithelialization (humans, animals)       

- 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       

- Improve skin graft transplant success (pigs)       

- Improve hair transplant success (humans)       

- 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)       

- Stimulate bone healing (guinea pigs, pigs, rabbits)       

- Heal injured intestinal linings (humans, rats)       

- Heal stomach ulcers (rats)       

- Block oxidative injury in tissues.

- Restore normal function in lives damaged by toxins (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.


CuSwitch

 


Skin Renewal Cycle - Biochemistry

Skin Renewal Cycle

 


SRCPs (Skin Remodeling Copper Peptides)

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 breakdown resistant, highly adhesive copper peptides.

Skin Renewal and Aging Reversal       

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.


SRCPs - Skin Remodeling Remodeling Copper Peptides

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. Many individuals report success 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 right is an ultrasound scan of the skin of a women aged 59 before treatment with the a cream containing SRCPs. On the far 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 Used for Scar Reduction

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. The 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  (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 Hair Follicles

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)




 
 

 

 
 
 
 Top Figure. Hair follicle before remodeling signal.
 
 
 
 Middle Figure. The remodeling signal increases the size of the hair follicles.
 
 
 
 Bottom Figure. The enlarged hair follicle begins to produce new skin cells that migrate into the surrounding skin and rebuild the skin.
 
 
 
 
 
 
 
 
 

 
 
 



 

 


 
 

 

 

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

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.

 

 

 

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.

GHK-Cu Analogs and Stimulation of Hair Growth  

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 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 Tricomin and GraftCyte which are based on Pickart's earlier work (from ProCyte Corporation).

More follicular cell growth and less programmed cell death (apoptosis)

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 copper peptides. The result is a much more rapid hair growth (the three circular patches of hair) in the three spots treated with copper peptides. 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.

 


 
 
 
 

 

 

Reducing Hair Loss After Chemotherapy/ Reducing Hair Loss During Chemotherapy

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.

Rats were pretreated with SRCPs then exposed to chemotherapeutic drugs. This reduced hair loss.

If the rats were first given chemotherapeutic drugs, the treated with SRCPs later, the SRCPs speeded hair regrowth.

T. Awa, K. Nogimori and R. Trachey, Hairloss protection by peptide-copper complex in animal models of chemotherapy-induced alopecia. J. Derm. Sci. 10, 99-104 (1995)


GHK, Copper, Regeneration, and Stem Cells

The primary cause of aging is a decline in organ function over time. Up until we reach age 20, tissue and organs are maintained in a fully functional and healthy state. But as we age, repair slows and our organs fail to fulfill their biological role. Adult stem cells in organs create new cells for repair and the key protein in activating and supporting stem cell function appears to be protein P63. Without adequate P63, skin ages rapidly as do other tissue of the body.

GHK-Copper increases protein P63 in addition to all of GHK-Copper's other protective and repair functions. This is the final link in understanding GHK-Copper role in the human body as given in the graphic below.

Recent studies revealed that older animals have as many adult stem cells in their bodies as young animals. However, these stem cells are not differentiated into the types of cells needed to rejuvenate older tissue.

From my previous studies, we know the amount of GHK-Copper needed to activate strong systemic healing of skin throughout the body in mice, rats, and pigs. This should be the same as that needed to increase P63 and activate epithelial stem cells since wound healing proceeds via stem cell actions. GHK-Copper must also activate other types of adult stem cells since it has strong healing actions on on the stomach, intestinal linings and bone tissue. Approximately, 75 milligrams of GHK-Copper injected three times a week should be sufficient to activate human stem cells and improve the function of organs in older individuals. It is possible that GHK-Copper might be effective as an oral supplement enclosed in special liposomes that are taken up by the lymphatic system to avoid possible breakdown by intestinal enzymes.

The 75 milligram level of GHK-Copper should be very safe and is 280-fold below expected negative actions of the molecule caused by its blood pressure lowering actions.

In cell culture studies, GHK-copper increases differentiation of embryonic stem cells. However, in an organ culture system, GHK-copper "activated" adult stem cells which then produced more keratinocytes.

GHK tested for stopping stem cell differentiation

GHK-Cu tested for stimulating stem cell differentiation

GHK reduced the clonogenic potential of stem cells by 78%

GHK-Cu increased cell copper by 2162% above the control value and caused stem cell differentiation

Methods of controlling proliferation and differentiation of stem and progenitor cells, United States Patent:  6,962,698, Peled, Tony, Fibach, Eitan, Treves; Avi, Gamida Cell Ltd. (Jerusalem, IL) and Hadasit Medical Research Services and Development, Ltd. (Jerusalem, IL)

GHK-copper activated adult stem cells by increasing integrins, P63, and PCNA. This increased keratinocyte proliferation.

Arch Dermatol Res. 2009 Apr;301(4):301-6. Copper-GHK increases integrin expression and p63 positivity by keratinocytes. Kang YA, Choi HR, Na JI, Huh CH, Kim MJ, Youn SW, Kim KH, Park KC. Department of Dermatology, Seoul National University College of Medicine, Yeongeon-dong, Jongno-gu, Seoul, Republic of Korea.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.

GHK-copper increases P63. P63 prevents premature aging by promoting adult stem cell maintenance.

Cell Stem Cell. 2009 Jul 2;5(1):64-75. TAp63 prevents premature aging by promoting adult stem cell maintenance. Su X, Paris M, Gi YJ, Tsai KY, Cho MS, Lin YL, Biernaskie JA, Sinha S, Prives C, Pevny LH, Miller FD, Flores ER. Department of Molecular and Cellular Oncology, Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63(-/-)) or in K14-expressing cells in the basal layer of the epidermis (TAp63(fl/fl);K14cre+). TAp63(-/-) mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging.

GHK-copper increases P63. P63 prevents premature aging by promoting adult stem cell maintenance. Cell Cycle. 2006 Feb;5(3):260-5. p63: a new link between senescence and aging. Keyes WM, Mills AA. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Cellular senescence is a distinctive form of cell cycle arrest that has been suggested to modulate the processes of tumor suppression and aging. Though a detailed understanding of the cellular machinery regulating this process is emerging, a more thorough understanding of the key players linking senescence to organismal aging is needed. The recent discovery that loss of the p53-related protein p63 induces cellular senescence and causes features of accelerated aging provides further evidence that cellular senescence is intimately linked with organismal aging, and identifies p63 as a key regulator of both of these processes.

GHK-copper increases P63. P63 prevents premature aging by promoting adult stem cell maintenance. Cell Stem Cell. 2009 Jul 2;5(1):1-2. SKP-ing TAp63: stem cell depletion, senescence, and premature aging. Beaudry VG, Attardi LD. Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The p53 family member p63 comprises multiple isoforms and is critical for stratified epithelial development. In this issue of Cell Stem Cell, by generating isoform-specific knockout mice, Su et al. (2009) reveal pivotal roles for TAp63 in the maintenance of dermal and epidermal precursors, genomic stability, and organismal longevity

GHK-copper increases P63. P63 prevents premature aging by promoting adult stem cell maintenance. Cell. 2007 May 4;129(3):523-36. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Senoo M, Pinto F, Crum CP, McKeon F. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

The distinguishing feature of adult stem cells is their extraordinary capacity to divide prior to the onset of senescence. While stratified epithelia such as skin, prostate, and breast are highly regenerative and account disproportionately for human cancers, genes essential for the proliferative capacity of their stem cells remain unknown. Here we analyze p63, a gene whose deletion in mice results in the catastrophic loss of all stratified epithelia. We demonstrate that p63 is strongly expressed in epithelial cells with high clonogenic and proliferative capacity and that stem cells lacking p63 undergo a premature proliferative rundown. Additionally, we show that p63 is dispensable for both the commitment and differentiation of these stem cells during tissue morphogenesis. Together, these data identify p63 as a key, lineage-specific determinant of the proliferative capacity in stem cells of stratified epithelia.

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  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

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) 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 to the periphery of the skin ulcers has healed the fissured skin and prevented further ulcer development. It should be emphasized that this particular copper peptide cream was designed specifically for "at-risk" skin in the stage of pre-ulcer dermatitis to help prevent further skin breakdown. It was 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.       

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 the copper peptide 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 the copper peptide cream. After 28 days all the skin ulcers were healed. The ulcers never re-occurred and she only used additional product as needed when her skin became excessively irritated and cracked.       

For cracked skin, use a light coating of a copper peptide once daily as needed. For skin ulcers, put the copper peptide around the edge of the ulcers. This aids skin healing from the outer edge of the ulcers. NOTE: Copper peptides are 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)      

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.

Skin and Hair Transplantation  

     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.

 
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

Liver Regeneration

GHK was originally isolated by its actions on increasing the survival of rat liver organ cultures. GHK also increases repair of liver damage in rats.

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

Bone Healing

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.
 

 
 
 

 



Anti-Inflammatory Actions: Damaged Tissue   

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.

 

Anti-Inflammatory Actions: General  

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

 

Anti-Oxidant and Anti-Inflammatory Actions of GHK and GHK-Cu

Human NSAIDs       

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).
 

Similarities between GHK and Anti- Ulcers Pharmaceutics 
Note the 3 main components - 
(1) an N-terminal side change, 
(2) and central imidazole ring, 
(3) a C-terminal - lysine-like - very basic group 

 

Intestinal and Stomach Healing    

GHK-Cu has potent effects on the healing of stomach ulcers and intestinal inflammations. One small human study found a very positive effect of GHK-Cu on the healing of intestinal lesions in persons with refractory inflammatory bowel disease.
 

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
++++

      Likewise, GHK-Cu produced a similar blockage of rat duodenal ulcer formation (cystamine induced).


Healing of Intestinal and Stomach Ulcers 
Study Result Reference
Healing of stomach and intestinal ulcers 

GHK-Cu was tested for healing of experimental stomach ulcers and intestinal damage in rats.

GHK-Cu healed experimental stomach ulcers and intestinal inflammation and damage

Pickart US Patent 4,767,753   Copper complexes of histidyl-lysine polypeptide(s) for reducing stomach secretions, increasing stomach mucous and preventing ulcers. US Patent 5,023,237   Use of polypeptide or its copper complex for cytoprotection in treatment of intestinal and stomach ulcers, and to facilitate wound healing. US Patent 5,145,838   Methods and compositions for healing ulcers and peptide derivatives.
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.

Human Study - Inflammatory bowel disease

16 patients with refractory inflammatory bowel disease were treated with rectally administered solutions of GHK-Cu.

After the 12 weeks treatment, there was a 60% reduction in severity bas measured by endoscopy, histopathology, and symptoms

An open study of PC1020 (GHK-Cu) rectal solution in treatment of distal inflammatory bowel disease.  Levine, Patt, Koren  (University of Washington) World Congress of Dermatology, October 1994,  Further details presented at 25th Annual Meeting of DDW,  Levien, Patt, Koren,Joslin (University of Washington)  May 1995

 


SRCP Biochemistry Related to Tissue Regeneration

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 tested for effects on phosphoylase A activation.

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 Gly-His-Lys 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 Gly-His-Lys 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
Review of Copper Peptide Tissue Regeneration - Year 2000 Overview of biological and clinical results. Link to abstract of talk Prague, Czech Republic, May 19, 2000
GHK is a matrikine that stimulates tissue remodeling The term of "matrikine" has been proposed to designate such ECM-derived peptides able to regulate cell activity. GHK is a matrikine and  a potent activator of ECM synthesis and remodeling Regulation de l'activite cellulaire par la matrice extracelulaire: le concept de matrikines. Maquart FX; Simeon A; Pasco S; Monboisse JC. J Soc Biol 1999;193(4-5):423-8
GHK-Cu and reduction of keloid scar formation GHK-Cu was tested for effects on the production of the keloid-producing hormone TGF-beta1 by normal and keloid-producing fibroblasts. GHK-Cu, at 10exp (-9) M reduced the production of TGF-beta1 in both normal and keloid-producing fibroblasts. In contrast, retinoic acid (Retin-A, Tretinoin) increases the production of scar producing TGF-beta1 in these lines.  The effect of copper tripeptide and tretinoin on growth factor production in a serum-free fibroblast model. McCormack, M., Nowak KC, Koch, J.  Arch Facial Plast Surg 2001; 3: 28-32
Review of Copper Peptide Tissue Regeneration - Year 2002 Overview of biological and clinical results Pickart, L.  Copper Peptides for Tissue Regeneration.  Specialty Chemicals Oct. 9, 2002, 29-31. 

Stimulation of Fingernail Growth

During prolonged rainy periods in Washington State, horses often develop severe irritations in their lower extremities – especially where the hair covered skin joins to the hooves. This skin area can develop irritations, infections and bleeding. During experiments to heal such irritated skin with creams containing copper peptides, the application of the copper-peptide cream was often imprecise due to movements of the horses and the cream were generously applied to the lower skin of the leg and part of the upper hooves. While the creams were observed to rapidly heal the skin areas, we also unexpectedly observed that damaged hooves appeared to improve markedly in health. Later, we experimented with more controlled application of the copper peptide creams into cracks in badly damaged hooves. We found that the copper peptide cream usually produced a remarkable healing of the hooves and closure of the cracks.       

Since the hooves of horses and the nails of humans are similar in terms of their biochemistry and cellular biology, we tested the application of such copper peptide creams to damaged human fingernails and toenails. We observed that such treatment produced in humans, as in the horses, a remarkable improvement in nail health and growth. Such types of copper-peptides, when applied to the nail matrix and nail bed area, enhance the process of nail growth resulting in stronger, thicker and smoother nails. Such types of copper-peptides have previously been found to strongly enhance the production of the protein collagen and also accelerate the repair of damaged skin. However, since nails are primarily composed of the hard protein keratin, it was not expected that copper-peptides would increase the production of keratin and nail growth.       

We then studied the actions of a copper-peptide cream in an informal pilot study on nail growth in humans. The Placebo Control nails were treated with a similar cream that contained no copper-peptide. For testing experiments, the fingernail growth rates of the index finger were used as a measurement. In the first set of experiments, the copper peptide was applied to the index fingernail and cuticle on the right hand while the left hand nail was untreated and used as a control. In the second set of experiments with different volunteers, the cream was applied to the index fingernail and cuticle on the left hand while the right hand nail was treated with the placebo cream and used as a control. Nails were treated for four weeks. Nail length was measured from the end of the nail bed to the tip of the nail at its center by pushing a small plastic ruler under the nail and firmly against the nail bed. The nails were also visually inspected to determine that the treated nails appeared to be longer on the treated hand and this was readily apparent in all cases. The effect of nail growth stimulation was approximately similar if either the right hand treated group or the left hand treated group was used.As seen in Table 1, Nails treated with the copper-peptide complex had better nail growth. However, it must be emphasized this was not a blinded study.
 

Table 1 - Effect of Copper Peptide Product on Nail Growth
First Experiment (Sex: M or F)
Finger Nail Growth with Copper Peptide Cream
(Millimeters in four weeks)
Finger Nail Growth with Placebo cream 
(Millimeters in four weeks)
Right Hand
Left Hand
Person 1M
4.2
2.7
Person 2M
3.2
2.1
Person 3F
4.3
3.2
Person 4F
3.8
2.9
Person 5F
3.9
2.6
 
Second Experiment 
Left Hand
Right Hand
Person 6M
2.8
1.5
Person 7M
3.7
2.6
Person 8F
4.1
3.0
Person 9F
4.1
2.6
Person 10F
3.5
2.2

Improving Suntanning and Reducing Skin Peeling Reactive Carbonyl     

This patent contains methods for increasing the efficiency of melanin formation and reducing post-tanning peeling damage. Decreasing the time in sunlight and post-tanning peeling would reduce overall ultraviolet exposure.       

The entire approach to suntanning and sun protection is in a state of change. Many chemical sunscreens are being banned because of estrogenic actions and other toxicities. The combination of skin repair creams plus ultraviolet reflectors such as zinc oxide or titanium dioxide may be a better approach to sun protection.

 

 

Table 9. Improving Suntanning and Reducing UV Damage to Skin Cells

Improved suntanning methods Compositions and methods for skin tanning and protection.  US Patent 5,698,184 Pickart
Blocks lethal ultraviolet radiation damage and inhibits the damaging glycation GHK blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehye, and glyoxal. See microphotographs below which are courtesy of Lipotec Corporation (www.lipotec.com) . This protection was found at a relatively high level of GHK, 20 mg/ml or 0.2%, but this concentration can be easily added to protective sunscreens. Current sunscreens are built around molecules, such as oxybenzone, avobenzene, menthyl anthranilate, octyl methoxycinnamate, that strongly absorb ultraviolet radiation; they later shed the energy by releasing it as free radicals. Such free radical absorbing chemicals prevent sunburn but many of these chemicals have strong estrogenic activities and there is concern over their accumulation in human tissues. GHK also inhibits the damaging glycation of copper, zinc-superoxide dismutase caused by fructose. GHK is more active than carnosine in this respect. J. Cebrian, A. Messeguer, R.M. Facino and J.M. Garcia-Anton, Inter. J. Cosm. Sci. 27, 271-278 (2005)

 

CONTROL

UVB radiation + HNE (4-Hydroxynonenal)

Cell Death

GHK + UVB + HNE (4-Hydroxynonenal)

Cells Protected


Background of SRCPs

GHK-Cu emerged during my attempts to reverse certain changes that occur during human aging. The goal was to suppress the synthesis of the blood fibrinogen, a protein that rises with age and rises even more after myocardial infraction. Its blood concentration is an excellent predictor of mortality. Elevated fibrinogen levels increase blood coagulation and decrease tissue perfusion, by increasing the thixotropic properties of blood in the microcirculation.       

I found that the albumin fraction of human blood plasma has a suppressive action on fibrinogen synthesis and also improved the survival of the cultured liver cells that produce fibrinogen. Further isolations found these activities concentrated in a low molecular weight fraction that contained GHK-Cu. Subsequent work defined the three dimensional solution structure of GHK-Cu and the binding affinities between GHK and copper (II).  My colleagues at the University of Washington (Seattle) and I used the structure of GHK-Cu to create analogs that were very potent cell growth inhibitors, inhibiting fibroblast replication at concentrations equivalent to chemotherapeutic drugs such as cisplatin and bleomycin. During surgical procedures to test these inhibitors on the suppression of tumor growth in mice, GHK-Cu was used as a control substance. It became apparent that GHK-Cu was rapidly healing the surgical incisions.       

Later research found that Iamin is generated during tissue damage and suggests that, after tissue damage, GHK-Cu serves as a human feedback signal that has potent tissue protective properties and stimulates tissue remodeling after the initial phase of wound healing. It is postulated that a localized generation of GHK-Cu after tissue damage causes an influx of skin repair cells called macrophages which initiate skin repair mechanisms. The decrease in the blood concentrations of GHK-Cu during human aging may be a factor in the decreased tissue repair and subsequent increased organ failure that occurs during aging.       


GHK-Cu - Function in Human Body     

A number of names have been used in the scientific literature for GHK.
 

  Names and identifications used for GHK
Systematic name  N(2)-(N-Glycyl-L-histidyl)-L-lysine 
CAS Registration number  49557-75-7 
Molecular Formula C14-H24-N6-O4
General name  Glycyl-histidyl-lysine 
Synonyms Copper glycyl-histidyl-lysine 
Copper(II)ghk
Glycylhistidyllysine 
Gly-his-lys 
Iamin 
NSC 379527
Prezatide (or Prezatide Copper as a complex)

 

What is the function of GHK-Cu in the human body? The molecule is present blood plasma, saliva, and urine in physiologically active quantities. Evidence suggests that it is a biochemical feedback signal that is generated after tissue injury. It appears to have two main functions: (1) first as a potent tissue protective, anti-inflammatory agent that limits oxidative damage after tissue injury, and (2) as a signal that activates tissue remodeling, that is, the processes for removal of damaged protein and scar tissue and their replacement by normal tissue. Thus, GHK-Cu links the processes of removal of damaged scar tissue and deposition of new tissue.       

GHK, which is generated in damaged tissues, directly accelerates many healing associated properties at concentration of around 10exp(-10)M. Some of the GHK stimulated effects seem to be directly mediated by GHK or GHK-Cu after it obtains copper (II) from albumin. Other actions of GHK and GHK-Cu are likely to arise from GHK-Cu's attraction of wound macrophages and other healing-associated immune cells which, in turn, release families of growth factor proteins appropriate to the repair of the damaged tissue.       

Francois Maquart and colleagues at Reims have argued that GHK-Cu acts on the second phase of healing as an inducer of tissue remodeling processes. Further support for this concept is that the molecular weight of collagen fragments induced by GHK-Cu are much smaller than those produced in the early phase of wound repair. This suggests that, with the copper complex, collagen synthesis and degradation are simultaneously occurring. Also, in cell culture, GHK-Cu reduces the secretion of the scar producing protein, TGF-beta-1 by normal fibroblasts and keloid-producing fibroblasts. This, combined with GHK-Cu's healing activities, suggests that scar-free healing needs both, an activation of metalloproteinases, and a reduction in TGF-beta-1 production.



In brief, the biochemical actions that suggest that GHK-Cu functions a a feedback signal for tissue protection and repair/remodeling after tissue injury are: 1. GHK is a very rare sequence in human proteins and exists mainly in inflammation associated proteins such as collagen, thrombospondin, fibrin a-chain, prokininogen, complement C1q, interleukin 4, skin collagenase, coagulation factor XI, and SPARC. Biologically effective levels of (10exp (-10) M) of GHK and GHK-Cu are generated by proteolysis after tissue damage. 2. Human plasma levels are significant but highly variable - averaging about 200 nanograms/milliliter at age 20 (10exp (-7) M) but dropping to about 80 nanograms/milliliter at age 70. Tissue regeneration also drops similarly with age. GHK-Cu appears to be bound to the plasma protein albumin. There are relatively few other small peptides in human plasma. GHK is found in lower levels in saliva and urine. 3. In human plasma and damaged tissues, GHK is likely to exist as a mixture of GHK and GHK-Cu. GHK has high binding affinity for copper (II) - pK=16.2 - very close to the affinity for copper (II) of albumin. GHK effectively competes with albumin for copper. However, under physiological conditions only about 5% to 20% of GHK molecules would be expected to exist as GHK-Cu complexes with copper (II). 4.  GHK is a extremely potent chemoattractant (active at 10exp (-12) M) for mast cells, wound macrophages and polymorphonuclear cells, all of which release protein growth factor proteins that stimulate tissue repair. 5. GHK-Cu links the processes of removal of damaged scar tissue and deposition of new tissue. In cell culture systems, and in vivo in rats and mice, GHK-Cu, acts directly, in a dose dependent manner, at approximately (10exp (-9) M), on fibroblasts by increasing the production of m-RNA for, and protein synthesis of, collagen, elastin, proteoglycans, glycosaminoglycans and decorin. In addition, GHK-Cu simultaneously stimulates the m-RNA production of, and synthesis of, certain metalloproteases and protease inhibitors (TIMP-1 and TIMP-2) felt to function in the removal of damaged protein. 6. Interestingly, the molecular weight of collagen induced by GHK-Cu is lower than that produced during healing after injury. H. Paul Ehrlich observed that GHK-Cu appears to simultaneously stimulate both collagen synthesis and breakdown. 7. GHK-Cu, at concentrations of 10exp (-9) M, also reduces fibroblast secretion of TGF-beta (transforming growth factor beta-1). This effect occurs with normal fibroblasts and keloid-producing fibroblasts. TGF-beta1 is a growth factor that increases wound healing but also induces scar formation. This, combined with GHK-Cu's healing activities, suggests that scar-free physiological healing needs a both an activation of metalloproteinases (Item 5 above) plus a reduction in TGF-beta1 production. 8. GHK-Cu increases collagen synthesis by bone chondrocytes (chick). GHK-Cu Increases growth of human marrow stromal cells and promotes the attachment of human osteoblastic cells. 9. GHK-Cu induces angiogenesis and a family of 6 proteins from 35K to 66K. Tissue areas deficient in copper (II) will not support the ingrowth of new blood vessels or angiogenesis. 10. GHK increases differentiation, viability and axon outgrowth in cultured chick and rat neurons at 10exp (-7) M to 10exp (-8) M. 10 GHK-Cu blocks ferritin channels and the release of tissue damaging free (oxidative) iron after tissue injury, thus blocking iron catalyzed lipid peroxidation that occurs after injury. 11. GHK-Cu blocks interleukin-1 damage to pancreatic islet cells at 10exp (-10) M. 12. GHK-Cu blocks low density lipoproteins (LDL) oxidation by copper. 13. The three dimensional structure of GHK-Cu is striking similar to many pharmaceutical anti-ulcer (stomach) medicines. Also virtually all pharmaceutical  non steroidal anti-inflammatories (NSAIDs)s have a high affinity for copper (II).


Proposed Mechanism Copper-Peptide GHK-Cu Actions After Tissue Injury

Information from a variety of sources allows us to propose a mechanism for GHK-Cu effects.The sequence of events of GHK-Cu induced effects appear to be as follows:

1. Initially after tissue damage, the first stage of wound healing processes is activated. These include localized blood coagulation, an early neutrophil invasion that secretes sterilizing oxygen radicals, and later an induction by growth factors, such as TGF-beta-1, of copious amounts of scar-forming collagen to form a protective covering over the injury.

2. A second stage of healing begins to be activated as disrupted cells release proteases that generate a population of peptides that include Gly-His-Lys and His-Gly-His-Lys, both of which have a very high affinity for copper (+2) ion.

3. The Gly-His-Lys and His-Gly-His-Lys begin to accumulate copper (+2) ion from albumin and form GHK-Cu and HGHK-Cu.

4. The accumulation of peptide-bound copper ion produces multiple anti-inflammatory effects that help to stop the actions of sterilizing oxygen radicals and permit the initiation of healing events. GHK-Cu blocks ferritin channels and the release of free (oxidative) iron, thus blocking iron catalyzed lipid peroxidation that occurs after injury. GHK-Cu blocks also interleukin-1 damage to tissue cells.

5. GHK-Cu released into the blood stream raises the body's production of, and circulating blood concentration of, wound macrophages that enhance repair.

6. GHK-Cu suppresses the synthesis of scar development by repressing fibroblast production of TGF-beta-1.

7. GHK-Cu also chemoattracts wound macrophages to the wound area. These macrophages act directly to stimulate healing by removing cellular debris and secreting a family of approximately 20 growth factor proteins.

8. GHK-Cu acts directly on fibroblasts to stimulate m-RNAs for collagen, elastin, proteoglycans, metalloproteinases, and TIMP-1 and TIMP-2. This in turn raises the levels of these proteins. This results in a condition whereby protein synthesis and deposition is occurring concomitant with protein breakdown that removes scar tissue and cellular debris remaining from the tissue disruption. Thus, GHK-Cu links scar reduction and the rebuilding of tissues.

9. GHK-Cu induces angiogenesis by serving as a chemoattractant to direct new blood capillaries to the wound area and by inducing the production of several protein essential for angiogenesis.

10. GHK-Cu induces neuronal outgrowth and re-innervation of the damaged tissues.

11. This mechanism of copper-peptide induced tissue repair appears to function for skin, hair follicles, the stomach lining, the intestinal lining, bone tissue, and hooves and fingernails.

12. This copper-peptide regeneration mechanism is different from most known biochemical hormonal response patterns such as the insulin-glucose system or the erythropoetin-red blood cell system. With these systems, a small change in the concentrations of glucose or red blood cells results in a precisely controlled release of the hormones to re-establish normal glucose or red blood cell levels.

13. The copper-peptide tissue remodeling is a much looser stimulus-response system - somewhat like a "fuzzy logic" response. Traumatic tissue damage is an inherently messy business - damage many be slight or massive. The bodily repair systems do not always receive rapid and clear information as to the extent of tissue damage - damages may be sudden and acute - or the result of a slow degenerative disease. This may explain why dermal scars and lesions last so long in adults; the body just does not recognize the need to remove the imperfections.

The Need for Improved Skin Regenerative Copper Peptides

The first generation products designed around GHK-Copper performed well in many controlled tests, however, the products failed in FDA clinical trials on the healing of very difficult-to-heal human wounds (as have many other approaches). The fragility and rapid breakdown of GHK and similar peptides is the major problem in developing products for clinical and cosmetic use. In the human body, the GHK-Cu complex can be constantly generated. However, when used as a single dose therapy, its fragility leads to rapid breakdown, clearance from the dermis, and a loss of effectiveness.       

In 1975, during attempts to isolate GHK from human blood, we found that the molecule was especially vulnerable to carboxypeptidases and was rapidly degraded by blood enzymes.  Intradermal injections of GHK are cleared from the skin in approximately 30 seconds. If added to blood, GHK is rapidly degraded into constituent amino acids by blood enzymes. Endo, Miyagi, and Ujie also reported that GHK was rapidly degraded by blood plasma and rapidly eliminated from rats. (Reference, Endo, Miyagi, and Ujie, Kissei Pharmaceutical Co., 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.J Chromatogr B Biomed Sci Appl 1997 Apr 25;692(1):37-42)       

This fragility and rapid breakdown of GHK and other simple copper peptide complexes is the major problem in developing products for clinical and cosmetic use. In the human body, the GHK-Cu complex can be generated constantly. However, when used as a single dose therapy, its fragility leads to rapid breakdown, clearance from the dermis, and a loss of effectiveness. A variety of chemical modifications to GHK have produced bioactive copper complexes with enhanced breakdown resistance. The problem with this classical organic chemistry approach is that each new chemical becomes, in FDA regulatory terms, a new chemical entity. This increases the possibility of undesirable side effects and much slower regulatory approvals.

Current Development of Improved Copper Peptides       

Currently more effective copper peptides with tissue regenerative actions have been developed. Several hundred copper-peptide complexes were evaluated but none were significantly better than GHK-Cu. Some complexes, such as f-Met-Leu-Phe-Cu actually produced more scar formation.  The principal defects of single peptides were a lack of stability and poor adhesion to the skin's surface.       

We then tested fractions of peptides remaining after enzymatic digestion of various proteins. These peptides, being the end products of enzymatic digestion, proved to be very resistant to further enzymatic digestion. Further testing found the most promising peptides were a fraction of peptide fragments that remained after partial proteolysis of soy proteins. Such soy peptides have a very low antigencity and long history of safe use in cosmetic products and in solutions used clinically for intravenous alimentation. To increase adherence to skin, a peptide fraction with a significant percentage of sugar residues was used. Peptide digests with attached sugars have mucus-like properties and peptide fragments from collagen have been used as glues such as the "Le Pages" glues once used by school children. This helps adhere the active components of the cream to the skin or to wet wounds.       

Similar mixed peptide digests of proteins were used to attract and concentrate wound macrophages in the 1930s. The digest was injected intraperitoneally into rats and 24 hours later the macrophages concentrated in the intraperitoneal cavity and were harvested.       

When copper (II) is chelated to this peptide fraction, this produces copper peptides with very strong skin repair properties. In studies persons with nickel allergy, these copper peptides were also found to have strong anti-inflammatory actions, approximately equal to that observed for cortisone. Because of their breakdown resistance, such copper peptides can be used with skin exfoliating hydroxy acids for more rapid skin renewal and for scar reduction. These peptides have enhanced potency, breakdown resistance, a longer duration of action and very high adherence to skin.       

In veterinary studies, creams made from these new copper complexes produced rapid and scar-free healing in dogs after spaying operations, and in young horses after leg-straightening operations. This allowed the dogs to be returned to their owners in four days instead of the usual five, while the foals were returned in five days instead of seven. In humans, four small, placebo-controlled studies found faster skin healing after skin injuries induced by tape stripping, acetone burns (removal of skin lipids), 24-hour detergent irritation, and nickel allergy inflammation.

Chemical Synthesis vs Biological Methods for Creation of Long-Acting Copper Peptides
Method to Develop Long Acting Copper Peptides Result Potential Problems Reference
Chemical Synthesis. A partially retro-inverso analogue of GHK was synthesized, in which the -CONH- bond between histidine and lysine was modified as -NHCO-.   The new peptide analogue showed approximately a ten-fold increase in stability versus the parent peptide. This is a new chemical entity -  Would require extensive safety testing. May lose anti-inflammatory activity H-Gly-His psi (NHCO)Lys-OH, partially modified retro-inverso analogue of the growth factor glycyl-L-histidyl-L-lysine with enhanced enzymatic stability. Dalpozzo A, Kanai K, Kereszturi G, Calabrese G.  Int J Pept Protein Res 1993 Jun;41(6):561-6
Chemical Synthesis. Synthetically modified GHK with breakdown resistant groups -

Replaced histidine residue with either a synthetic amino acid, L-spinacine, or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid. Investigated with potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies.All the ligands formed copper complexes having different stoichiometries and stabilities. 

After 3 hours in serum, GHK was degraded but the synthetic compounds showed no significant degradation.

These are new chemical entities -  Would require extensive safety testing. May lose anti-inflammatory activity Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogs: chemical behavior and biological activity,  Conato et al,  Biochim Biophys Acta 1526, 199-210, 2001
Biological Synthesis. Use fraction of soybean protein enzymatic digest - these end result peptides are resistant to further enzymatic breakdown  After 3 hours in serum, copper complexes of the soybean digest retained wound healing and anti-inflammatory activities when compared to native serum. None found - Passed all safety tests. These types of peptides have been extensively used for intravenous feeding, in food supplements, and in shampoos and conditioners  Pickart US Patent 5,382,431   Tissue protective and regenerative compositions US Patent 5,554,375   Tissue protective and regenerative compositions US Patent 5,698,184   Compositions and methods for skin tanning and protection US Patent 5,888,522   Tissue protective and regenerative compositions .

 
 
Howard Maibach directed four studies on human skin repair using these breakdown resistant copper peptides formulated into a non-ionic cream base. These placebo-controlled, double-blinded studies gave statistically significant positive results that the copper-peptide creams markedly accelerated the rate of skin recovery and reduced irritation after severe skin damage. The studies included (1)  Acetone damaged skin has had the fats removed from the skin and produces damage similar to a very dry and cracking skin, (2) Detergent damaged skin has many fats removed plus extensive damage to the outer layer of skin barrier proteins. This is the standard test for contact dermatitis, (3) Nickel allergy damaged skin is produced by applying nickel salts to the skin of sensitized patients. This is similar to the skin damage caused by other allergic responses such as poison ivy, poison oak and other allergens, and (4) Tape stripped skin which is similar to damaging scraps, abrasions, and small cuts on the skin's surface. A strip of tape is put on the skin, then quickly ripped off. This process is repeated about 50 times in the same place, ultimately producing a small wound on the skin.
       

 
The mixed copper-peptides uses a different approach than the single molecule GHK-Cu method. The digest contains millions of variant peptide copper ion complexes, more like the situation that arises after tissue damage and subsequent proteolysis. The physiological mechanism whereby the skin or other organs signal for repair is quite complex. However, for therapeutic use, a much simpler approach often suffices to induce tissue regeneration. In vivo, the active peptides must have a very high affinity for copper (+2) to obtain copper ion and form copper peptide complexes. But exogenously, we can easily complex mixtures of peptides, even with much lower affinity for copper ion, to copper (+2) simply by adding copper salts to a solution of the peptides.The scientific rational for the bioactivity of such copper peptides is that numerous peptide fragments are generated by proteolytic enzymes. A fraction of these peptides will have a high affinity to copper(+2) and a portion may have properties similar to GHK-Cu.

Comparison of attributes of GHK and HGHK vs Enzymatic Protein Digests 
Biological Parameter GHK and HGHK Newer Enzymatic Protein Digests
Wound Macrophage Chemoattraction-  Very High High
Copper Binding Affinity - Very High Moderate, Must add copper ion to mixture
Antigenicity - None None in type currently used / Passed human antigenicity testing and animal tests
Healing activity in animals models - Moderate High
Adherence to skin and wounds-  Low Very high, forms a biological "glue"
Clearance from applied area - Rapid  Slow

 


Importance of Careful Formulation of Copper Peptide Complexes  

With copper-peptide creams, great care must be taken to produce a cream that has minimal interactions with the ionic copper in the cream. Formulators who prepare skin creams usually have only a limited knowledge of chemistry and know virtually nothing about cell biology, hormonal interactions, and general biochemistry. It is easily possible to produce copper-containing creams, lotions, and solutions that neutralize copper-peptide effects by the interaction of various components with ionic copper. It is also possible to create copper-complexes that inhibit cell replication. It should be emphasized that the GHK-Cu work arose from National Cancer Institute funded projects for the development of growth inhibitory analogs of GHK-Cu. During this project, GHK-Cu analogs were synthesized that inhibited fibroblast replication at 10exp (-16) M. Some companies have recently sold cosmetic skin products using EDTA-copper, but this complex inhibits fibroblast function and skin repair.        Any product should be carefully tested for its effect on skin repair.       

For the copper-peptide studies, we formulated a non-ionic, but stable cream. For cream preservation, Germaben II was used which our testing found to have no influence on the wound repair process. This product contains diazolinydinyl urea, methylparaben, and propylparaben and produces self-sterilizing creams with excellent preservation for at least four years at room temperature. A drawback of the preservative system is that some persons are sensitized to parabens but there is no perfect preservative for wound healing products. Benzoyl alcohol should never be used as a preservative for wound healing products, since it inhibits the replication of wound fibroblasts.   


GHK Suppresion of Cancer Metastasis Genes, Decorin, and Growth Inhibitory Analogs of the GHK copper binding region

GHK supresses human cancer metastasis genes but because of the cell culture system used, it is likely that the GHK was converted to GHK-copper. GHK-copper also increases the protein Decorin (Named because it "decorates" the collagen strands). Decorin has a perplexing mixture of effects: Regeneration of muscles and nerves, reduction of scarring, and inhibition of tumor growth and cancer metastasis in animals.

GHL Suppression of Cancer Metastasis

The authors searched for substances that can reverse expression of those genes involved in human colon cancer metastasis.

To determine gene expression of human normal and cancerous cells, they measured RNA produced by cells. To find substances that can reverse expression of those genes involved in metastasis, they used very reliable database developed at the Broad Institute (Massachusetts Institute of Technology and Harvard) of 7000 genome wide expression profiles after treatment of 4 human cell lines with 1309 bioactive substances. Only two substances, GHK and securenine (an alkaloid) from the choice of 1309 were able to quiet down the genes involved in tumor spreading. The authors also mention low toxicity of GHK and the low concentration (1 micromolar) that produces desired effect.

GHK suppresses human cancer metastasis genes

Clin Exp Metastasis. 2010 Feb;27(2):83-90. Epub 2010 Feb 9.

A 'metastasis-prone' signature for early-stage mismatch-repair proficient sporadic colorectal cancer patients and its implications for possible therapeutics.

Hong Y, Downey T, Eu KW, Koh PK, Cheah PY.,Department of Colorectal Surgery, Singapore General Hospital, Singapore, 169608, Singapore.

Abstract

Metastasis is the major cause of cancer mortality. We aimed to find a metastasis-prone signature for early stage mismatch-repair proficient sporadic colorectal cancer (CRC) patients for better prognosis and informed use of adjuvant chemotherapy. The genome-wide expression profiles of 82 age-, ethnicity- and tissue-matched patients and healthy controls were analyzed using the Affymetrix U133 Plus 2 array. Metastasis-negative patients have 5 years or more of follow-up. A 10 x 10 two-level nested cross-validation design was used with several families of classification models to identify the optimal predictor for metastasis. The best classification model yielded a 54 gene-set (74 probe sets) with an estimated prediction accuracy of 71%. The specificity, sensitivity, negative and positive predictive values of the signature are 0.88, 0.58, 0.84 and 0.65, respectively, indicating that the gene-set can improve prognosis for early stage sporadic CRC patients. These 54 genes, including node molecules YWHAB, MAP3K5, LMNA, APP, GNAQ, F3, NFATC2, and TGM2, integrate multiple bio-functions in various compartments into an intricate molecular network, suggesting that cell-wide perturbations are involved in metastasis transformation. Further, querying the; 'Connectivity Map' with a subset (70%) of these genes shows that Gly-His-Lys and securinine could reverse the differential expressions of these genes significantly, suggesting that they have combinatorial therapeutic effect on the metastasis-prone patients. These two perturbagens promote wound-healing, extracellular matrix remodeling and macrophage activation thus highlighting the importance of these pathways in metastasis suppression for early-stage CRC.

GHK-copper increases decorin which suppresses cancer growth and metastasis

 

 

GHK-copper increases Decorin J Invest Dermatol. 2000 Dec;115(6):962-8.
Expression of glycosaminoglycans and small proteoglycans in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu(2+).
Siméon A, Wegrowski Y, Bontemps Y, Maquart FX. Laboratoire de Biochimie Médicale et de Biologie Moléculaire, FRE CNRS 2260, IFR 53-Biomolécules, Faculté de Médecine, Reims, France.

Glycyl-histidyl-lysine-Cu(2+) is a tripeptide-copper complex previously shown to be an activator of wound healing. We have investigated the effects of glycyl-histidyl-lysine-Cu(2+) on the synthesis of glycosaminoglycans and small proteoglycans in a model of rat experimental wounds and in rat dermal fibroblast cultures. Repeated injections of glycyl-histidyl-lysine-Cu(2+) (2 mg per injection) stimulated the wound tissue production, as appreciated by dry weight and total protein measurements. This stimulation was accompanied by an increased production of type I collagen and glycosaminoglycans (assessed, respectively, by hydroxyproline and uronic acid contents of the chamber). Electrophoretic analysis of wound tissue glycosaminoglycans showed an accumulation of chondroitin sulfate and dermatan sulfate in control wound chambers, whereas the proportion of hyaluronic acid decreased with time. The accumulation of chondroitin sulfate and dermatan sulfate was enhanced by glycyl-histidyl-lysine-Cu(2+) treatment. The expression of two small proteoglycans of the dermis, decorin and biglycan, was analyzed by northern blot. The biglycan mRNA steady-state level in the chamber was maximal at day 12, whereas the decorin mRNA increased progressively until the end of the experiment (day 22). Glycyl-histidyl-lysine-Cu(2+) treatment increased the mRNA level of decorin and decreased those of biglycan. In dermal fibroblast cultures, the stimulation of decorin expression by glycyl-histidyl-lysine-Cu(2+) was also found. In contrast, biglycan expression was not modified. These results show that the expression of different proteoglycans in wound tissue are regulated in a different manner during wound healing. The glycyl-histidyl-lysine-Cu(2+) complex is able to modulate the expression of the extracellular matrix macromolecules differently during the wound repair process.
Decorin suppresses growth and metastasis of cancer cells Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Feb;24(1):222-5.
[The action of decorin in anti-fibrosis and anti-cancer]
[Article in Chinese]
Ma W, Tan Y, Cai S, Chen H, Du J, Cai S.
Key Laboratory for Biomechanics & Tissue Engineering of the State Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, China.

Decorin (DCN) is a member of the small leucine-rich proteoglycan gene family. Many studies indicated that DCN inhibited fibrosis and scar-formation by neutralization of TGF-P and interfering the binding of TGF-beta with its receptor, which induced ectopic deposition of extracellular matrix. Additionally, DCN can prevent the proliferation and metastasis of tumor cells by activating EGFR/MAPK/p21 signal pathway and inhibiting the cell proliferation pathway mediated by EGF-EGFR. It is suggested that the recombinant DCN had potential pharmaceutical potency in treatment of chronic fibrosis and neoplasm for its critical biological activities and low immunogenicity.
Decorin prevents metastatic spreading of breast cancer Oncogene. 2005 Feb 3;24(6):1104-10.
Decorin prevents metastatic spreading of breast cancer.
Reed CC, Waterhouse A, Kirby S, Kay P, Owens RT, McQuillan DJ, Iozzo RV.
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Metastases in breast cancer are a vital concern in treatment, with epidermal growth factor receptor and ErbB2 strongly implicated in mediating tumor invasion and spreading. In this study, we investigated the role of decorin in suppressing both primary breast carcinomas and pulmonary metastases. We show that decorin causes marked growth suppression both in vitro and in vivo using a metastatic breast cancer cell line and an orthotopic mammary carcinoma model. Treatment with decorin protein core reduced primary tumor growth by 70% and eliminated observed metastases. An adenoviral vector containing the decorin transgene caused primary tumor retardation of 70%, in addition to greatly reducing observed metastases. Moreover, we demonstrate that ErbB2 phosphorylation and total receptor protein levels are reduced in this model system upon de novo expression of decorin under the control of a doxycycline-inducible promoter. Primary tumor growth in vivo was reduced by up to 67% upon decorin induction, and pulmonary metastases were markedly hampered as well. These effects are likely occurring through decorin's long-term downregulation of the ErbB2 tyrosine kinase cascade. These results demonstrate a novel role for decorin in reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapeutics for metastatic breast cancer.

Decorin prevents metastatic spreading of breast cancer Am J Pathol. 2008 Sep;173(3):844-55. Epub 2008 Aug 7.
An antimetastatic role for decorin in breast cancer.
Goldoni S, Seidler DG, Heath J, Fassan M, Baffa R, Thakur ML, Owens RT, McQuillan DJ, Iozzo RV.
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

Decorin suppresses lung cancer metastasis Oncol Rep. 2008 Jun;19(6):1533-9.
Decorin suppresses lung metastases of murine osteosarcoma.
Shintani K, Matsumine A, Kusuzaki K, Morikawa J, Matsubara T, Wakabayashi T, Araki K, Satonaka H, Wakabayashi H, Iino T, Uchida A.
Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Mie, Japan.

Lung metastasis is the most crucial event affecting the therapeutic outcome of osteosarcoma. The prevention of lung metastasis is therefore important in improving the prognosis of patients with osteosarcoma. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The biological role of decorin in osteosarcoma has yet to be clarified. The aim of this study was to examine the potential of decorin as a novel biological target for the treatment of osteosarcoma. In this study, the LM8 murine osteosarcoma cell line (LM8) with high metastatic potential to the lung was used. The two cell lines established were LM8-DCN which stably expressed human decorin (hDCN) and LM8-mock, established as a control. The LM8-DCN cell line was subcutaneously injected into the backs of mice. Significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated with LM8 and LM8-mock (P<0.001). In addition, the mice in the LM8-DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group, based on the Kaplan-Meier survival analysis and log-rank tests (P<0.005). The effect of decorin on the growth rates, motility and invasion ability of LM8 was investigated in vitro. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma.

Decorin suppesses breast cancer metastasis into bone

Oncology. 2009;77(2):92-9. Epub 2009 Jul 7.
Decorin suppresses bone metastasis in a breast cancer cell line.

Araki K, Wakabayashi H, Shintani K, Morikawa J, Matsumine A, Kusuzaki K, Sudo A, Uchida A. Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie, Japan.

Decorin, the prototype of an expanding family of small leucine-rich proteoglycans, is involved in a number of cellular processes including matrix assembly, fibrillogenesis and the control of cell proliferation. In this study, we investigated the role of decorin in suppressing tumor aggressiveness and bone metastases. We used a metastatic breast cancer cell line, MDA-MB-231, to show that decorin causes marked growth suppression bothin vitro and in vivo. A cytomegaloviral vector containing the decorin transgene caused greatly reduced cell growth, motility and observed metastases. Bone metastases were decreased by >90% upon decorin transfection. These results demonstrate a novel role for decorin in the reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapies for metastatic breast cancer.

Decorin suppesses prostate cancer growth Neoplasia. 2009 Oct;11(10):1042-53.
Decorin suppresses prostate tumor growth through inhibition of epidermal growth factor and androgen receptor pathways.
Hu Y, Sun H, Owens RT, Wu J, Chen YQ, Berquin IM, Perry D, O'Flaherty JT, Edwards IJ.
Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

Epidermal growth factor receptor (EGFR) and androgen receptor (AR) pathways play pivotal roles in prostate cancer progression. Therefore, agents with dual-targeting ability may have important therapeutic potential. Decorin, a proteoglycan present in the tumor microenvironment, is known to regulate matrix assembly, growth factor binding, and receptor tyrosine kinase activity. Here, we show that in prostate-specific Pten(P-/-) mice, a genetically defined, immune-competent mouse model of prostate cancer, systemic delivery of decorin inhibits tumor progression by targeting cell proliferation and survival pathways. Moreover, in human prostate cancer cells, we show that decorin specifically inhibits EGFR and AR phosphorylation and cross talk between these pathways. This prevents AR nuclear translocation and inhibits the production of prostate specific antigen. Further, the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathway is suppressed leading to tumor cell apoptosis. Those findings highlight the effectiveness of decorin in the presence of a powerful genetic cancer risk and implicate decorin as a potential new agent for prostate cancer therapy by targeting EGFR/AR-PI3K-Akt pathways.

Decorin inhibits primary tumor growth and metastatic speading Int J Cancer. 2008 Dec 1;123(11):2473-9.
Tumor microenvironment: Modulation by decorin and related molecules harboring leucine-rich tandem motifs.
Goldoni S, Iozzo RV.
Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Decorin, the prototype member of the small leucine-rich proteoglycans, resides in the tumor microenvironment and affects the biology of various types of cancer by downregulating the activity of several receptors involved in cell growth and survival. Decorin binds to and modulates the signaling of the epidermal growth factor receptor and other members of the ErbB family of receptor tyrosine kinases. It exerts its antitumor activity by a dual mechanism: via inhibition of these key receptors through their physical downregulation coupled with attenuation of their signaling, and by binding to and sequestering TGFbeta. Decorin also modulates the insulin-like growth factor receptor and the low-density lipoprotein receptor-related protein 1, which indirectly affects the TGFbeta receptor pathway. When expressed in tumor xenograft-bearing mice or injected systemically, decorin inhibits both primary tumor growth and metastatic spreading. In this review, we summarize the latest reports on decorin and related molecules that are relevant to cancer and bring forward the idea of decorin as an anticancer therapeutic and possible prognostic marker for patients affected by various types of tumors. We also discuss the role of lumican and LRIG1, a novel cell growth inhibitor homologous to decorin.

P63 as a Cancer Inhibitor

There is some evidence that protein P63 also inhibits cancer growth.

GHK-copper increases protein P63

Arch Dermatol Res. 2009 Apr;301(4):301-6. Copper-GHK increases integrin expression and p63 positivity by keratinocytes. Kang YA, Choi HR, Na JI, Huh CH, Kim MJ, Youn SW, Kim KH, Park KC. Department of Dermatology, Seoul National University College of Medicine, Yeongeon-dong, Jongno-gu, Seoul, Republic of Korea.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.

P63 may suppress cancer

Cell Death Differ. 2010 Apr 9. [Epub ahead of print]

p53-family proteins and their regulators: hubs and spokes in tumor suppression.
Collavin L, Lunardi A, Del Sal G. Laboratorio Nazionale CIB (LNCIB), AREA Science Park, Trieste, Italy, Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Trieste, Italy.

Abstract
The tumor suppressor p53 is a central hub in a molecular network controlling cell proliferation and death in response to potentially oncogenic conditions, and a wide array of covalent modifications and protein interactions modulate the nuclear and cytoplasmic activities of p53. The p53 relatives, p73 and p63, are entangled in the same regulatory network, being subject at least in part to the same modifications and interactions that convey signals on p53, and actively contributing to the resulting cellular output. The emerging picture is that of an interconnected pathway, in which all p53-family proteins are involved in the response to oncogenic stress and physiological inputs. Therefore, common and specific interactors of p53-family proteins can have a wide effect on function and dysfunction of this pathway. Many years of research have uncovered an impressive number of p53-interacting proteins, but much less is known about protein interactions of p63 and p73. Yet, many interactors may be shared by multiple p53-family proteins, with similar or different effects. In this study we review shared interactors of p53-family proteins with the aim to encourage research into this field; this knowledge promises to unveil regulatory elements that could be targeted by a new generation of molecules, and allow more efficient use of currently available drugs for cancer treatment.

   

 

 

 

Anti-cancer analogs of GHK copper binding site

Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Biochem Pharmacol 1983 Dec 15;32(24):3868-71.

Pickart L, Goodwin WH, Burgua W, Murphy TB, Johnson DK, Biochem. Pharmacol., 32, 1983, 3868-71.

Anti-cancer analogs of GHK copper binding site Cytotoxic chelators and chelates 1. Inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazones and by a copper(II) complex of salicylaldehyde benzoyl hydrazone. Inorganica Chimica Acta, 67:159-165, 1982.
David K. Johnson, Terrance B. Murphy, Norman J. Rose, William H. Goodwin and Loren Pickart, Department of Medicinal Chemistry and Chemistry, University of Washington, Seattle, Wash. 98195,Virginia Mason Research Center, Seattle, Wash. 98101

Abstract

Aroylhydrazones of pyridoxal and of salicylaldehyde, a series of tridentate chelating agents, are potent inhibitors of DNA synthesis and cell growth in a number of human and rodent cell lines grown in culture. A copper(II) complex of the most potent of the chelators, salicylaldehyde benzoyl hydrazone (SBH), exhibits significantly greater inhibitory activity than does SBH itself. Although the bioactive forms and mechanism of action of these agents are uncertain, their cytotoxic activity can equal or exceed that of many chelators and chelates previously known to possess such properties, including compounds used clinically. SBH and its copper complex are relatively non-toxic to mice and show some measure of selectivity in their effects on different cell types. It is possible that aroylhydrozones of this type and/or their metal complexes could prove to be useful therapeutic agents.

Anti-cancer analogs of GHK copper binding site Biological activity of human plasma copper-binding growth factor glycyl-l-histidyl-l-lysinePickart L, Lovejoy S, Methods Enzymol. 147, 1987, 314-328

Hydrazone analogs of the GHK copper binding region are very potent inhibitors of DNA synthesis and cell growth. Furthermore, the wound repairing cell, the fibroblast, seems to be exceptionally sensitive to certain types of copper-complex inhibitors. This makes the preparation of tissue regenerative copper complexes difficult since even some types of peptide copper complexes inhibit fibroblasts as do other small complexes such as diethylamine-copper and triethylamine-copper.
 
 
 
 
 
 
 GHK-Cu and hydrazone analogs of GHK copper binding region that that are very potent inhibitors of cell replication

Top: Copper binding region of GHK

Middle: PCPH-Cu (2-Pyridinecarboxaldehyde 2''-pyridylhydrazone hydrazone copper(2+))

Bottom: SBH-Cu (Salicylaldehyde benzoyl hydrazone copper(2+))
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Concentrations of PCPH-Cu, SBH-Cu, Cisplatin and Bleomycinsynthesis 50%

Cell Types in Culture PCPH-Cu
Nanograms per ml for 50% cell reduction
SBH-Cu
Nanograms per ml for 50% cell reduction
Cisplatin
Nanograms per ml
for 50% cell reduction
Bleomycin
Nanograms per ml
for 50% cell reduction
Normal human fibroblasts 0.00003 0.1
Not done
-
Normal human kidney 45  
-
-
Mouse fibrosarcoma 0.511 2.4
32
Human bladder cancer 12 34
95
7
Human lung epithelial cancer 580 410
870
44
Human melanoma 0.05 270
6,000
80,000

 

 

References on chemistry of GHK-Cu

 

Discovery of GHK A tripeptide in human plasma that increases the survival of hepatocytes and the growth of hepatoma cells,. Pickart Ph.D. Thesis in Biochemistry, University of California, San Francisco, 1973
Discovery of GHK Tripeptide in human serum which prolongs survival of normal liver cells and stimulates growth in neoplastic liver. Pickart and Thaler (University of California, San Francisco, USA) Nature New Biol 1973 May 16;243(124):85-7 
Purification of GHK by development of a high pressure chromatograghic method. Methods were developed for the purification of peptides and proteins by high pressure chromatography. This may have been the first paper to describe this method which is now widely used.  A rapid method for the purification of histones and a variety of growth- promoting proteins and peptides by chromatography on silica gel was developed. Insulin, albumin, somatomedins, and histones were purified on high-pressure silica gel columns.  Purification of growth promoting peptides and proteins, and of histones, by high pressure silica gel chromatography.  Pickart and Thaler  (University of California, San Francisco, USA) Prep Biochem 1975, 5:397-412
Determination of tripeptide structure. Chromatographic evidence has suggested that tripeptide was Gly-His-Lys. Chemical analysis found structure to be Gly-His-Lys  Growth modulating serum tripeptide is glycyl-histidyl-lysine. Schlesinger, Pickart and Thaler  (Harvard University) Experientia 1977, 33(3):324-5
Development of improved methods of isolation of GHK from plasma and the co-isolation of copper(2+) and iron (2+) with GHK. Analysis of GHK isolates for metals. During the isolation of GHK-Cu from human plasma, copper and iron were found to co-isolate with the peptide. Studies with [3H]GHK demonstrated that copper and iron interfered at several steps of the procedure for the isolation of GHK from plasma (gel filtration chromatography, high pressure silica gel columns). Removal of these metals with an insoluble chelating resin (Cellex 100) enhanced recovery of [3H]GHL from plasma 8-fold.  Effect of transition metals on recovery from plasma of the growth- modulating tripeptide glycylhistidyllysine. Pickart, Thaler and Millard  (University of California, San Francisco, USA, US Dept. Agriculture  Lab, Albany, CA, USA) J Chromatogr 1979  11;175(1):65-73

Studies on GHK and structure and activity. A variety of GHK-Cu analogs were synthesized and tested for their stimulation of DNA synthesis.

While many analogs similar to GHK-Cu had bioactivity, none equaled the bioactivity of GHK-Cu

Growth modulating human plasma tripeptide: Relationship between molecular structure and DNA synthesis in hepatoma cells. Pickart and Thaler  FEBS Lett 1979, 104(1):119-22
Cell culture studies of the effects of GHK complexed with copper and iron on cell growth patterns. Experimental observations that GHK is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate.At physiological pH in vitro, GHK associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHK acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures. These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHK in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHK. GHK and transition metals, which appear to form complexes prior to interaction with cells. Growth modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes. Pickart and Thaler  (University of California, San Francisco, USA) J Cell Physiol 1980, 102(2):129-39
GHK and copper(II) transport into cells.  The association of GHK with copper and a homology similarity between the tripeptide and the copper transport sites on albumin and alpha- fetoprotein, where the cupric atom is bound to a histidyl residue adjacent to a basic residue, suggested that GHK may act as a copper transport factor.It was found that GHK readily forms complexes with copper(II) and enhances the uptake of the metal into cultured cells. Growth modulating plasma tripeptide may function by facilitating copper uptake into cells. Pickart, Freedman, Loker, Peisach, Perkins, Stenkamp and Weinstein Nature 1980, 288, 715-7
A review of information on the biological actions and structure function relationships as understood in 1981.  At this time GHK's chemoattractant actions on white cells were unknown. Glycylhistidyllysine (GHK), a tripeptide from human plasma, has been shown to alter the growth rate of many cell types and organisms in culture systems. GHK is optimally active at concentrations between 10 and 200 nanograms/milliliter. Present information suggests that GHK functions as a transporter of transition metals, in particular copper, to the cell surface for uptake into the cell.  The use of glycylhistidyllysine in culture systems.  Pickart  In Vitro 1981,17(6):459-66
Determination of the effectiveness of GHK chelation for copper under physiological conditions. The interaction between Cu(II) and the growth modulating tripeptide GHK in the presence and absence of L-histidine was investigated by potentiometric titration and visible-absorption spectrophotometry at 25 degrees C in 0.15 M-NaCl. Analyses in the pH range 3.5-10.6 indicated the presence of multiple species in solution in the binary system and extensive amounts of the ternary complexes in the ternary system. The species distribution and the stability constants were evaluated.  The results obtained from the equilibrium dialysis experiments showed that GHK was able to compete with albumin for Cu(II) at pH 7.5. At equimolar concentrations of albumin and the peptide, about 42% of the Cu(II) was bound to the peptide. At the physiologically relevant concentrations of Cu(II), albumin, L-histidine and this peptide, about 6% of the Cu(II) was associated with the low- molecular-weight components.  The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth- modulating tripeptide from plasma. Lau and Sarkar Biochem J 1981 199(3):649-56
Review of GHK-Metal Interactions Available evidence suggests that GHK acts as a complex with transition metals Peptide and protein complexes of transition metals as modulators of cell growth.  Pickart In: Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (Marcel Dekker Pub.) 1982, 75-104. 
Interaction of Cu(II) and Gly-His-Lys was determined by 13C- and 1H-NMR and EPR spectroscopy. Interaction of Cu(II) and GHK, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-NMR and EPR spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The NMR line-broadening was interpreted in terms of major and minor species formed as a function of pH. The EPR parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration. N.m.r. and e.p.r. investigation of the interaction of copper(II) and glycyl-L-histidyl-L-lysine, a   growth-modulating tripeptide from plasma. Laussac JP; Haran R; Sarkar B Biochem J 1983 Feb 1;209(2):533-9
Magnetic resonance studies of GHK and copper Determine the stability of GHK-Cu in solution. GHK and GHK-Cu slowly exchange copper(II) in solution.  PMR studies of Cu(II) and Zn(II) interaction with glycyl-l-histidyl-l-lysine and related peptides. Kwa E, Bor-Sheng L, Rose N, Weinstein B, Pickart L.  Peptides 1983, 8, 805-808
Studies on the structure of GHK-Cu in solution. Optical, electron paramagnetic resonance, and electron spin-echo envelope spectroscopies were used to examine the structure of the Cu(II) complex of glycyl-L-histidyl-L-lysine (GHK) in solution.At neutral pH, GHK forms a mononuclear 1:1 Cu(II) compound having an EPR spectrum resembling that of Cu(II) equatorially coordinated by two or three  nitrogen atoms. Electron spin-echo studies demonstrate that one of these is located in the histidyl imidazole ring. A pH titration of Cu(II)-GHK shows three optical transitions with apparent pKs of 3.6, 9.2 and 11.4 and molecularities, with respect to protons, of 2, 2, and 1, respectively. At the lowest pK, GHK binds Cu(II), forming the species present at physiological pH. These solution studies are consistent with nitrogen  coordination of Cu(II) in Cu(II)-GHK. Structure of the glycyl-L-histidyl-L-lysine copper(II) complex in  solution.  Freedman, Pickart, Weinstein, Mims and Peisach (Albert Einstein Medical School, New York, USA)  Biochemistry 1982 21(19):4540-4

 

Determination of the X-ray structure of aqueous crystals  of GHK-Cu.

The structure of a copper complex of the growth factor glycyl-l-histidyl-l-lysine at 1.1 angstrom resolution.  Perkins CM, Rose NJ, Weinstein B, Stenkamp RE, Jensen, LH, Pickart L. Inorg. Chem Acta 1984, 82, 93-99.
Studies of cell inhibitory copper complexes A series of hydrophobic analogs of the GHL-Cu structure were synthesized and tested. Hydrophobic analogs of the GHL-Cu structure were inhibitory to cell DNA synthesis and growth Cytotoxic chelators and chelates inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazone analogs of the gly-l-his-l-lys copper(II) complex.  Johnson, Pickart and Rose Inorg Chem Acta 67, pp. 159-165, 1982
Synthesis of growth inhibitory analogs of GHK-Cu. A variety of copper binding growth inhibitory analogs of GHK-Cu very synthesized and tested. A variety of copper binding analogs of GHK-Cu had potent activity in the inhibition of fibroblasts and fibrosarcoma cells at concentrations of 10exp (-16) M.  Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Pickart, Goodwin, Burgua, Murphy and Johnson Biochem Pharmacol 1983, 32(24):3868-71
A review of information on the biological actions and structure function relationships as understood in 1983.  At this time GHK's chemoattractant actions on white cells were unknown The biological effects and mechanism of action of the plasma tripeptide glycyl-l-histidyl-l-lysine.  Pickart Lymphokines 8, pp. 425-446, 1983
Electron spin resonance studies of the molecular action of growth inhibitory copper analogs of GHK-Cu with cells. The interaction of two cytotoxic hydrophobic GHK-Cu analogs SBH-Cu  (salicylaldehydebenzol hydrazone-Cu) and PCPH-Cu (pyridine 2-carboxyl aldehyde 2'-pyridyl hydrozonate-Cu) with cells was determined. SBH-Cu(II) and PCPH-Cu(II) first interact with sufhydyl groups in the cells. The Cu(II)  is first reduced to Cu(I), then re-oxidized to Cu(II) Formation of adducts between cupric complexes of known antitumor agents and ehrlich ascites cells.  Antholine, Lyman, Petering and Pickart (University of Wisconsin Milwaukee, USA) Biological and Inorganic Copper Chemistry, Adenine Press, pp. 125-137, 1985

GHK forms stable complexes with palladium (II) The binding of palladium by GHK was determined.

Conclusion: GHK may possibly participate in the transport of Pd(II) in the tissues and its elimination through the kidneys 

NMR studies on binary and ternary Pd(II) complexes formed by the growth modulating tripeptide glycylhistidyllysine and nucleotides.  Laussac, Pasdeloup and Hadjiliadis J Inorg Biochem 30:227-38, 1987
The authors characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system.Two ligand-dependent processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process.  The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation; and (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand. Brain tissue accumulates 67copper by two ligand dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process. Hartter and Barnea (Department of Obstetrics and Gynecology, University of Texas Health Science Center, Dallas, USA)  J Biol Chem 1988 Jan 15;263(2):799-805
Study of GHK degradation in rats A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of  GHK. and its metabolite, L-histidyl-L-lysine in rat plasma. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly. 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, Miyagi and Ujiie   (Pharmacological Laboratories, Kissei Pharmaceutical Co., Ltd., Minamiazumi, Nagano, Japan) J Chromatogr B Biomed Sci Appl 1997, 692(1):37-42



The Aging Reversal Experiments and GHK

The discovery of GHK-Cu arose from studies on human aging and a search for methods to reverse certain deleterious biochemical changes that occur with aging and are intensified in heart attack survivors. The initial target was to find methods of reducing the concentration of the blood protein fibrinogen. Elevated blood fibrinogen reduces blood flow through the tissues and raises the death rate. 

Studies on fibrinogen metabolism concluded that there were factors in the blood protein albumin fraction that reduced fibrinogen synthesis and strongly stimulated liver metabolism. Some of these effects were due to albumin-bound free fatty acids but analysis of this albumin fraction found a tripeptide-copper complex (GHK-Cu) that improved liver cell metabolism and survival in culture. The plasma concentration of GHK-Cu was higher in men of age 20 ( ~200 ng/ml) than in men of age 70 ( ~80 ng/ml). Ultimately, this tripeptide copper complex (GHK-Cu)  was found to have multiple actions that activated tissue regeneration and remodeling. The improvement of skin quality and turnover in older persons by application of GHK-Cu could be considered an aging reversal effect. 

Observation and Question Asked Type of studies Assumed Cause at Start of Studies. Result/Actual Cause References
Human blood fibrinogen levels increase with age and disease - This increases blood coagulation and is associated with a sharply increased death rate. 

Why was the fibrinogen increased?

Human fibrinogen turnover studies

 

Expected cause - Fibrinogen was not being used and accumulated in the blood

Actual cause - Human fibrinogen turnover studies found that the blood increase was due to an increased liver synthesis of the protein in older persons and heart attack survivors. 

Pilgeram LO, Pickart, L Bandi, Z, Fatty acid control of fibrinogen turnover in aging and atherosclerosis.  7th International Congress of Gerontology, June 1964, pp. 451-460. Pickart L, Pilgeram LO.  The role of thrombin in fibrinogen biosynthesis. Thromb Diath Haemorrh. 1967 May 31;17(3-4):358-64.  Pilgeram LO, Pickart L.  Control of fibrinogen biosynthesis: the role of free fatty acid. J Atheroscler Res. 1968, 8:155-66. 
Fibrinogen is synthesized in the liver. 

Was the increased fibrinogen synthesis observed in older persons and heart patients due to changes in the liver or was it due to factors in the blood that perfused the liver? 

Measure fibrinogen synthesis in isolated human liver tissue from persons aged 20 to 30 and in persons  aged from 60 to 80. Incubated the young (20-30) liver tissue with blood from either the young (20-30) or the old group (60-80). p (60-80)

 

 

Assume cause - The increase in fibrinogen synthesis with age was due to changes in the liver tissue.

Actual cause - The increase in fibrinogen synthesis in the older persons was not due to changes in the liver tissue but almost  totally due to factors in the older blood. 

Pickart L, Thaler MM.  Suppression of tumor-associated hyperfibrinogenemia and free fatty acidemia with p-phenoxybenzalbutyrate (clofibrate). Cancer Res. 1979 Oct;39(10):3845-8. Pickart, L. Fat metabolism, the fibrinogen/fibrinolytic system and blood flow: new potentials for the pharmacological treatment of coronary heart disease. Pharmacology 1981;23(5):271-80; Pickart L Suppression of acute-phase synthesis of fibrinogen by a hypolipidemic drug (clofibrate). Int J Tissue React. 1981 3(2):65-72.

What is the factor(s) in the blood that stimulated or inhibited fibrinogen synthesis? Studied the effect of various plasma protein fractions that affected fibrinogen synthesis in mice.  No. The albumin fraction of plasma had a suppressive action on fibrinogen synthesis that returned synthetic patterns to that of a younger person. Some effects appeared to be due to free fatty acids but this albumin fraction itself also stimulated liver cell metabolism. Pickart L, Thaler MM.  Fatty acids, fibrinogen and blood flow: a general mechanism for hyperfibrinogenemia and its pathologic consequences. Med Hypotheses. 1980 May;6(5):545-57.  Pickart L, Thaler MM. Free fatty acids and albumin as mediators of thrombin-stimulated fibrinogen synthesis. Am J Physiol. 1976 Apr;230(4):996-1002.
What was the factor in the albumin fraction that stimulated liver metabolism? Studies the protein albumin and various small molecules associated with the protein.

Expected cause - The effects were due to the albumin molecule.

Actual cause - The effects were not due to albumin but rather due to a small peptide-metal complex associated with the protein.

A tripeptide in human plasma that increases the survival of hepatocytes and the growth of hepatoma cells.  Pickart Ph.D. Thesis in Biochemistry, University of California, San Francisco, 1973; Thaler MM, Pickart L Metabolic and growth promoting properties of serum tripeptide and its synthetic analog. In: Gene Expression and Carcinogenesis in Cultured Liver.  (Academic Press, 1975) pp. 292-310.
What was the structure of the peptide-metal complex? Isolation and chemical analysis. The complex was composed of a tripeptide, glycyl-l-histidyl-l-lysine plus approximately equimolar copper 2+ or GHK-Cu Schlesinger DH, Pickart L, Thaler MM. Related Articles, Protein Growth-modulating serum tripeptide is glycyl-histidyl-lysine. Experientia. 1977 Mar 15;33(3):324-5. 
What was the function of GHK-Cu? Synthesized variants of GHK-Cu that inhibited cell growth in cell culture, wound repair, and some tumors in animals. During studies of growth inhibitory copper analogs of GHK-Cu on wound repair, it was observed that GHK-Cu, which was used as control substance, was healing wounds while the inhibitors stopped wound healing.  It was concluded that GHK-Cu had a stimulatory action on wound healing.  Pickart L, Lovejoy S.  Biological activity of human plasma copper-binding growth factor glycyl-L-histidyl-L-lysine. Methods Enzymol. 1987;147:314-28. 



Future Uses of Remodeling Copper Peptides

GHK-Cu remains the best molecule for internal medical treatments. The physiological function of GHK-Cu in the human body appears to be as a protectant and repair signal for damaged tissues. It is possible that GHK-Cu could be used clinically to protect and speed repair of damaged organs. H. Paul Ehrlich found that intra-muscular injection of GHK-Cu into the thigh muscle of rabbits raised circulating wound macrophages in the blood and accelerated the healing of distant wounds in the rabbit ear. Patients might be pre-treated with GHK- Cu before surgery to enhance post-surgical repair. Based on rabbit models, a dosage of 30 milligrams of GHK-Cu should suffice. The molecule is also very beneficial on kidney organ culture. Thus, GHK-Cu might be infused into patients with kidney failure to exert its tissue protective and repair actions. Potential clinical targets would be injuries to, or system failure of, tissues such as skin, bone, kidneys, and the gastrointestinal intestinal tract. GHK-Cu might be administered after traumatic tissue injury such as automobile accidents.The newer breakdown resistant, highly adhesive copper peptides under development should prove better for cosmetic and superficial uses such as post-procedure dermatological healing, and development of scarless surgical procedures. The copper peptides appear to be very useful for post-procedure recovery after skin peels, dermabrasion and laser resurfacing. The combination of hydroxy acids and these peptides slowly, over a period of several months, reduces old scars and skin lesions. This method is economical and avoids the complications that often occur after chemical peels or laser treatments. In experimental studies, the use of such types of copper peptides after surgical procedures often results in scarless or nearly scarless healing.


0532012 lp / 042114 cm