| Dr Loren Pickart of Skin Biology, a pioneer in
the field, describes the use of copper peptide complexes in tissue regeneration
Certain types of copper peptide complexes possess both tissue protection
and repair properties. Most information on these effects is based on a
human copper peptide complex, glycyl-l-histidyl-l-lysine:copper (II) or
GHK-Cu.
GHK-Cu has protective and regenerative actions on several organ systems
including skin, hair follicles, bone, gastric mucosa and intestinal linings.
These types of copper complexes are increasingly used in cosmetic skin
and hair care products and after dermatological skin renewal procedures,
such as chemical peels, laser resurfacing and dermabrasion, to improve
post-treatment skin recovery.
Ageing reversal experiments
GHK-Cu emerged during my attempts to reverse certain changes that occur
during human ageing. 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.1
Subsequent work defined the three dimensional solution structure of GHK-Cu
and the binding affinities between GHK and copper (II), as shown in Figure
1.2
Figure 1 - Solution structure of GHK-Cu
My colleagues at the University of Washington (Seattle) and I used the
structure of GHK-Cu to create analogues 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 tumour growth
in mice, GHK-Cu was used as a control substance. It became apparent that
GHK-Cu was rapidly healing the surgical incisions.3
Biogenesis, metabolism & structure of GHK-Cu
Later work has more clearly defined the role of GHK-Cu. The molecule is
found in human plasma, saliva and urine. Its concentration in plasma is
highly variable but approximately 200 ng/ml (10exp (-7) M) at age 20, which
later declines to 80 ng/ml by age 60.
GHK is a very rare sequence in human proteins, mainly existing in inflammation-associated
proteins and proteins of the extracellular matrix, such as collagen, thrombospondin,
fibrin-chain, prokininogen, complement C1q, interleukin 4, skin collagenase,
coagulation factor XI and SPARC. During episodes of tissue damage, GHK
is generated by proteolysis after injury. Numerous cell culture studies
have found that the biologically effective levels GHK-Cu are approximately
10 exp (-9) M. This contrasts with plasma levels of 10 exp (-7) M.4
In human plasma and wound areas, GHK is likely to exist as a mixture
of GHK and GHK-Cu. GHK has a high binding affinity for copper (II) (pK=16.2)
that is very close to albumin's affinity for copper (II) (pK=16.4). 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).5
Biological actions have been reported for both GHK and GHK-Cu, although
GHK is likely to chelate available copper at very low concentrations while,
conversely, GHK-Cu may lose copper to other binding agents. Thus, experiments
using either molecule are actually studying a mixture of both molecules.
GHK-Cu is a very fragile molecule that is sensitive to carboxypeptidase
actions. In blood plasma, it is rapidly degraded.
Protecting, healing & remodeling
In 1985, I founded a company called ProCyte to develop GHK-Cu for clinical
use. It was quickly established that GHK-Cu had repair actions on tissues
such as the skin, the stomach, the intestine and the bone. GHK-Cu accelerated
the healing of a variety of accidental and surgical wounds in rats, mice,
pigs and horses. It also improved the establishment of skin grafts in mice
and in pigs. GHK-Cu analogues with added hydrophobic residues strongly
increased hair growth in mouse models.
It appears that, after tissue damage, GHK is generated by proteolysis
of inflammatory and extracellular matrix proteins. A significant fraction
of GHK converts to GHK-Cu by obtaining copper (II) from albumin. Wound
healing of skin is basically a two-phase process. Initially, a variety
of anti-microbial sterilising and tissue-destructive process are activated.
Scar-producing growth factors such as Transforming Growth Factor (TGF)
â-1 induce the production of copious quantities of collagen, and
create a tough, protective layer over the injury. In the second phase,
inflammation and scar formation is suppressed and remodeling processes
remove scar tissue and rebuild normal skin.
GHK-Cu acts indirectly as an extremely potent chemoattractant at 10
exp (-12) M for cells that stimulate repair, such as macrophages and mast
cells, which release protein growth factor proteins that stimulate tissue
repair. Tissue areas deficient in copper (II) will not support the ingrowth
of new blood vessels or angiogenesis. In rabbit models, GHK-Cu induces
angiogenesis by promoting the synthesis of a family of six proteins from
35K to 66K, and by acting as a chemoattractant for capillary cells at 10exp
(-12) M.6
In cell culture systems, and in vivo in rats and mice, GHK-Cu,
at approximately 10 exp (-9) M, acts directly on fibroblasts by increasing
the production of m-RNA for collagen, elastin, proteoglycans, glycosaminoglycans
and decorin. In addition, GHK-Cu simultaneously stimulates the m-RNA production
of, and synthesis of, certain metalloproteases and anti-proteases that
clear damaged protein and remove scars. Thus, GHK-Cu links the processes
of removal of damaged scar tissue and deposition of new tissue.7
Francois Maquart and collegues 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 TGFâ-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 TGFâ-1 production.8
In culture systems, the complex promotes the differentiation, viability
and axon outgrowth in cultured chick and rat neurons. In isolated rat hepatocytes,
GHK stimulates the activity of phosphorylase A, an activity that converts
glycogen into bioavailable glucose for energy production.9
GHK-Cu as a tissue protective molecule
Another important function of GHK-Cu may be as a circulating human non-steroidal
anti-inflammatory. Virtually all NSAIDs avidly bind copper (II). There
are striking similarities between GHK and anti-ulcer drugs such as cimetidine,
ranitidine, famotidine and nizatidine (see Figure 2).
Figure 2 - Similarity between GHK-Cu and anti-ulcer drugs
The correspondences include (1) an N-terminal side change, (2) a central
imidazole ring, and (3) a C-terminal lysine-like basic group. In rat ulcer
models, GHK-Cu reduces gastric acidity, increases mucous production, and
inhibits the development of gastric ulcers. Likewise, in intestinal ulcer
models, GHK-Cu inhibits ulcer development. One small study of 16 patients
with distal inflammatory bowel disease, who were treated with rectally
administered solutions of GHK-Cu, found that after the 12 weeks of treatment,
there was a 60% reduction in severity as measured by endoscopy, histopathology,
and symptoms.
After episodes of tissue damage, ferric ion is released from ferritin
and catalyses damaging tissue oxidations. GHK-Cu counters this action by
blocking ferritin channels, and the release of oxidising iron ions. GHK
blocks the oxidation of low density lipoproteins by loosely bound copper.
Interleukin-1-â is
also released after tissue injury producing cellular damage. At 10 exp
(-10) M, GHK-Cu was found to prevent damage to pancreatic cells by interleukin-1.
GHK-Cu inhibits platelet aggregation and thromboxane production; this action
may reduce localised blood coagulation after tissue damage.10
Wound healing
After my initial finding that GHK-Cu stimulated wound healing11, numerous
other laboratories extended these observations. A few examples of healing
actions include the healing of pad wounds in dogs, the increased wound
closure and the contraction and production of granulation tissue. Healing
with GHK-Cu was best with light bandaging. Wet bandages nullified the effect.12
In immune-suppressed rats, healing is impaired and collagen synthesis
is 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 wound healing
and wound contraction in the immune-suppressed animals. In studies of the
healing of punch biopsies wounds in pigs, the effect of intradermal injections
of GHK-Cu produced highly localised patterns of healing. Thus, in animal
or human studies, it is possible to test several potential formulations
on a given test subject.13
A GHK-Cu cream used after Moh's surgery increased wound healing and
skin re-epithelialisation. An open study of wounds in 60 patients in 1987
with diabetic and venous stasis ulcers gave evidence of rapid healing.
This study used high concentrations of GHK-Cu and very low concentrations
of anti-microbial agents. This apparently successful formulation was never
used in later clinical studies, which failed to achieve therapeutic goals
in FDA trials for clinical uses.14
Ageing reversal of skin
The greatest interest in copper peptides is in the area of reversing the
effects of human ageing and ultraviolet damage on human skin. During ageing,
skin becomes thinner and tends to accumulate various skin lesions and imperfections.
The dermis and epidermis thin and the subcutaneous fat cells diminish in
number.
A number of studies at ProCyte in 1988, which were repeated by several
other laboratories between 1998 and 2002, have found that the application
of GHK-Cu creams to the human skin was more effective in promoting collagen
development than retinoic acid or vitamin C. It also increased the thickness
of the epidermis and dermis, increased skin elasticity, reduced wrinkles
and resulted in a removal of skin imperfections such as blotchiness and
sun damage marks. In skin healing models using mice, a very significant
increase in subcutaneous fat cells was noted.15
Hair retention & growth stimulation
In 1985, a series of GHK-Cu analogues with added hydrophobic residues (fatty
acids or hydrophobic amino acids) was found to stimulate hair growth strongly
around healing wounds in mice. It was possible to obtain striking increases
in hair follicle size and the rate of localised hair growth in mice.
Later, Hideo Uno and colleagues at the University of Wisconsin reported
that these copper complexes produced a stimulation of the follicular cell
proliferation, resulting in an enlargement of the anagen follicles, and
converted vellus hair into terminal hair. GHK-Cu analogues also minimised
hair loss after experimental chemotherapy in rats and accelerated new hair
growth. These actions on hair growth may be secondary to improvements in
skin vitality that increase nutrient flow to hair follicles.16
In humans, the results are less striking but do exist. An unpublished
study by ProCyte reported that one GHK-Cu analogue increased terminal hair
growth in adult men approximately 30% more than was reported for the control
substance (2% minoxidil). GHK-Cu has been shown to increase hair outgrowth
from hair transplants in men.17
Bone healing
GHK-Cu increases collagen synthesis by bone chondrocytes (chick and guinea
pig) and increases the growth of human marrow stromal cells and promotes
the attachment of human osteoblastic cells. Milan and collegues in Prague
developed a GHK-Cu gel that promotes the filling of bone defects in femurs
and bone attachment to cementless endoprostheses. The GHK-Cu gel, when
used with cementless endoprostheses, produced vivid osteogenic activity
at the interface of bone and metal stem. Such gels may aid in the establishment
and retention of artificial joints.18
Developing breakdown-resistant complexes
The first generation products designed around GHK-Cu 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).
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.
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.
In 1994, I set up Skin Biology to develop more stable copper peptides
with tissue regenerative actions. To increase resistance to proteolysis,
I used 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. When copper (II) is chelated to this peptide
fraction, this creates skin regenerating copper peptides that 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.19
Importance of careful formulation
With copper-peptide products, great care must be taken to produce a product
that has minimal interactions with the ionic copper in the product. Magnetic
proton resonance measurements found that copper (II) exchanged slowly between
GHK molecules, despite the very high binding affinity of GHK for copper
(II). Often the chemical ingredients of creams, lotions, and solutions
interact with the ionic copper and neutralise the positive copper-peptide
actions and, in some cases, generate copper-complexes that inhibit cell
replication. Any product should be tested carefully for its effect on skin
repair. Some companies have recently sold cosmetic skin products using
EDTA-copper, but this complex inhibits fibroblast function and skin repair.
Future uses
GHK-Cu remains the best molecule for internal medical treatments. The newer
breakdown resistant, highly adhesive copper peptides under development
at Skin Biology should prove better for cosmetic and superficial uses such
as post-procedure dermatological healing, and development of scarless surgical
procedures.
It is possible that GHK-Cu could be used clinically to protect and speed
the 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
mg 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.
The newer, second generation copper peptides produced at Skin Biology
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.20
References
1. Pickart, Ph.D. thesis, Univ. of California, San Francisco, 1973; Pickart,
Thaler, Nature New Biol. 243, 1973, 85; Pickart, Thaler, J. Cell Physiol.
102, 1980, 129, Pickart, In Vitro, 17 (6), 1981
2. Pickart, Freedman, Loker, Peisach, Perkins, Stenkamp, Weinstein,
Nature 288, 1980, 715; Kwa, Bor-Sheng, Rose, Weinstein, Pickart, Peptides,
8, 1983, 805; Freedman, Pickart, Weinstein, Mims, Peisach, Biochemistry,
21, 1982, 4540
3. Johnson, Pickart, Rose, Inorg. Chem. Acta, 67, 1982, 159, Pickart,
Goodwin, Burgua, Murphy, Johnson, Biochem. Pharmacol., 32, 1983, 3868;
Pickart, Lovejoy, Methods Enzymol. 147, 1987, 314.
4. Maquart, Gillery, Monboisse, Pickart, Laurent, Borel, Ann. N.Y. Acad.
Sci., 580, 1990, 573; Sage, Vernon, J. Hypertens. Suppl. 12, 1994, S145.
5. Lau, Sarkar, Biochem J. 199, 1981, 649
6. Poole, Zetter, Cancer. Res. 43, 1983, 5857; Zetter, Rasmussen, Brown,
Lab. Invest., 53, 1985, 362, Raju, Alessandri, Gullino, Cancer Res. 44,
1984, 1579
7. Maquart, Pickart, Laurent, Gillery, Monboisse, Borel, FEBS Lett.
238, 1988, 343; Oddos, Jumeau-Lafond, Ries, Amer. Acad. Derm. Abstract
P72, Feb. 2002; Wegrowski, Maquart, Borel, Life Sci. 51, 1992, 1049; Simeon,
Wegrowski, Bontemps, Maquart, J. Invest. Dermatol., 115, 2000, 962; Maquart,
Bellon, Chaqour, Wegrowski, Monboisse, Chastang, Birembaut, Gillery, J.
Clin. Invest., 92, 1993, 2368; Simeon, Monier, Emonard, Gillery; Birembaut,
Hornebeck, Maquart, J. Invest. Dermatol., 112, 1999, 957; Simeon, Emonard,
Hornebeck, Maquart, Life Sci., 67, 2000, 2257
8. Maquart, Simeon, Pasco, Monboisse, J. Soc. Biol., 193, 1999, 423;
McCormack, Nowak, Koch, Arch. Facial Plast. Surg., 3, 2001, 28
9. Sensenbrenner, Jaros, Moonen, Mandel, Neurobiology, 5, 1975, 207;
Lindner, Grosse, Halle, Henklein, Z. Mikrosk. Anat. Forsch., 93, 1979,
820; Garcia-Sainz, Olivares-Reyes, Peptides, 16, 1995, 1203
10. Levine, Patt, Koren, Joslin, 25th Ann. Meeting of Digest. Dis. W.,
May 1995; Miller, DeSilva, Pickart, Aust, Adv. Exp. Med. Biol., 264, 1990,
79; Vinci, Caltabiano, Santoro, Rabuazzo, Buscema, Rizzarelli, Vigneri,
Purrello, Diabetologia, 38, 1995, 39-45, Thomas, Biochem. Biophys. Acta,
1128, 1992, 50
11. Downey, Larrabee, Voci, Pickart, Surg. Forum, 1985, 573; Pickart
in Biology of Copper Complexes, Plenum Press, 1987, 273
12. Swaim, Vaughn, Kincaid, Morrison, Murray, Woodhead, Hoffman, Wright,
Kammerman, Am. J. Vet. Res. 57, 1996, 394
13. Ehrlich, Symposium on Collagen and Skin Repair, Reims, Sept. 12,
1991; Counts, Hill, Turner-Beatty, Grotewiel, Fosha-Thomas, Pickart, Fed.
Am. Soc. Exp. Biol., 6, 1992, A1636
14. Aupaix, Maquart, Salagnac, Pickart, Gillery, Borel, Kalis, J. Invest.
Derm., 94, 1990, 390; Fish, Katz, Hien, Briden, Johnson, Patt, Wounds,
3, 1991, 171.
15. Abulghani, Shirin, Morales-Tapia, Sherr, Solodkina, Robertson, Gottlieb,
J. Invest. Derm., 110, 1998, 686; Amer. Acad. Derm. Meeting, February 2002,
(Appa, Barkovic, Finkey, Stephens, Abstract P66; Leyden, Grove, Barkovic,
Appa, Abstract P67; Leyden, Stephens, Finkey, Barkovic, Abstract P68; Leyden,
Stephens, Finkey, Barkovic, Abstract P69).
16. Uno, Kurata, J. Invest. Dermatol., 101, 1993, 143S, Awa, Nogimori,
J. Dermatol. Sci., 10, 1995, 99.
17. ProCyte Corporation, 1997; Perez-Meza, Int. J. Cosm. Surg., 6, 1998,
80.
18. Pesakova, Novotna, Adam, Biomaterials, 16, 1995, 911; Pohunkova,
Stehlik, Vachal, Cech, Adam, Biomaterials, 17, 1996, 1567; Godet, Marie,
Cell. Mol. Biol. (Noisy-le-Grand), 41, 1995, 1081.
19. Zhai, Chang, Singh, Maibach, Contact Dermatitis, 40, 1999, 205;
Zhai, Poblete, Maibach, Int. J. Dermat., 37, 1998, 386; Zhai, Leow, Maibach,
Skin Res. Tech., 4, 1998, 24; Zhai, Leow, Maibach, Clin. Exp. Derm., 23,
1998, 11.
20. More extended references to copper peptide actions on tissue regeneration
are posted at www.skinbiology.com/copperpeptideregeneration.html
For more information, please contact:
Dr. Loren Pickart
Skin Biology
4122 Factoria Blvd
Suite 200
Bellevue
WA 98006
USA
Tel: +1-425-644-0160
Fax : +1-425-644-2057
Email: customerservice@skinbiology.com |
