The Effect of Human Peptide GHK Relevant to
Nervous System Function and Cognitive Decline
Brain Sciences (2017)
New Data of the Cosmeceutical and TriPeptide GHK
SOFW Journal (2015)
GHK-Cu May Prevent Oxidative Stress in Skin
by Regulating Copper and Modifying Expression of
Numerous Antioxidant Genes Cosmetics (2015)
GHK Peptide as a Natural Modulator of
Multiple Cellular Pathways in Skin Regeneration (2015)
GHK, the Human Skin Remodeling Peptide Induces Anti-Cancer
Expression and DNA Repair Analytical Oncology (2014)
GHK and DNA: Resetting the Human Genome to Health
BioMed Research International (2014)
Avoid Buying Fake Copper Peptides Dangerous







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       

In more recent years, 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 used currently / 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, newer formulas are non-ionic, but stable. 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.   


Questions or Advice?

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Alternate Email: GHkcopperpeptides@gmail.com

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