Tripeptide

Light-exposed fibroblasts are affected by copper tripeptide (1 nM; 0-96 hours), and their population doubling time is similar to that of controls [1]. In 24 irradiated fibroblast cells, copper tripeptide (1 nM; 0-120 hours) generated considerably more basic fibroblast growth factor than normal controls [1].

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Irradiated fibroblasts survived and replicated in serum-free media. The population-doubling times of normal and irradiated fibroblasts exposed to GHK-Cu were faster than those of nontreated controls. Irradiated fibroblasts treated with GHK-Cu doubled at a rate that approximated that of untreated controls, and produced significantly more basic fibroblast growth factor and vascular endothelial growth factor than untreated controls early after GHK-Cu exposure. Conclusions: Irradiated fibroblasts survive and replicate in serum-free media, establishing this model as ideal for evaluating growth factor production in vitro. Copper tripeptide accelerates the growth of normal and irradiated fibroblasts to the point where treated irradiated fibroblasts approximate the population-doubling time of normal controls. An early increase in basic fibroblast growth factor and vascular endothelial growth factor production by GHK-Cu-treated irradiated fibroblasts may improve wound healing. [1]

Glycyl-L-histidyl-L-lysine/Gly-His-Lys (ip; 1.5, 5, 50, 150, and 450 mg/kg; 10 times) promotes mitotic activity of hepatocytes and dose-dependently decreases immune Reactivity[2]. Glycyl-L-histidyl-L-lysine (ip; 0.5, 5, 50 μg/kg) exerts anxiolytic effects in the elevated plus maze test [3].

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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).[2]

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Intraperitoneal administration of tripeptide Gly-His-Lys to male rats in doses of 0.5, 5, and 50 μg/kg 12 min before the start of the experiment produced an anxiolytic effect in the elevated plus maze test manifested in an increase in the time spent in open arms and shortened time spent in the closed arms. The anxiolytic effect was most pronounced after injection of 0.5 μg/kg peptide and decreased with increasing the dose of the peptide. Replacement of L-lysine with D-lysine in the tripeptide molecule was accompanied by a significant weakening of the neurotropic effects in all studied doses. Attachment of D-alanine to N- or C-terminus of Gly-His-Lys peptide leveled its anxiolytic action in all doses; significant changes in some measures of increased anxiety after administration at 50 μg/kg were found. [3]

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Administration of Gly-His-Lys peptide in all specifi ed doses has a marked effect on the examined behavioral responses of rats (Table 1). The maximum effect was observed at a dose of 0.5 μg/kg: the time spent in open arms increased by 136% (p<0.01), the number of entries into open arms, by 208% (p<0.01), and the time spent on the central platform, by 109% (p<0.05). Increasing the peptide dose to 5 μg/kg was not accompanied by enhancement of the anxiolytic action, and the majority of the studied parameters were similar to those recorded in the previous group. Further increase in the injected dose of Gly-HisLys to 50 μg/kg attenuated these effects and appearance of signifi cant differences between studied parameters in the experimental groups. Thus, the time spent in open arms did not substantially differ from the control values and was signifi cantly lower than at lower and medium doses (by 45 and 39% respectively at p<0.05). Moreover, the time spent in closed arms and the number of entries into closed arms in this group were lower than after administration of the peptide in a dose of 0.5 μg/kg: by 28% (p<0.05) and 37% (p<0.05), respectively. Only the peptide dose of 50 μg/kg signifi cantly increased the number of entries into closed arms (by 45%; p<0.05) in comparison with the control. This phenomenon can be explained by the increase in motor activity of rats associated with increasing the dose of the peptide. The results of the study of neurotropic effects of Gly-His-Lys prompt us to study behavioral effects of its modifi cations. Replacement of L-lysine with D-lysine led to a signifi cant reduction in behavioral activity of rats and practically leveled the anxiolytic action of the peptide (Table 2). Only the time spent on the central platform after peptide injection in a dose of 0.5 μg/kg (by 134%; p<0.01) and 50 μg/kg (by 56%; p<0.05) and the number of entries into closed arms at the lesser dose (by 59%; p<0.05) signifi cantly surpassed the control values. Attachment of D-alanine to N-terminus of the Gly-His-Lys molecule did not signifi cantly affect the studied behavioral parameters of the peptide in doses of 0.5 and 5 μg/kg. Administration of the peptide in the maximum dose (50 μg/kg) signifi cantly reduced the time spent in open arms (by 66%; p<0.05) and on central platform (by 48%; p<0.05); the time spent in closed arms increased by 31% (p<0.05). These behavioral shifts indicated anxiety in rats. After attachment of D-alanine to C-terminus of Gly-His-Lys molecule, the neurotropic effects of the tripeptide were considerably leveled as in previous modifi cation, and their individual manifestations had the opposite nature. In particular, the number of entries into open arms was reduced after injection of the peptide in doses of 5 μg/kg (by 65%; p<0.05) and 50 μg/kg (by 72%; p<0.05) as well as the time spent on the central platform after injection of the highest dose (by 42%; p<0.05). These behavioral changes, similar to those observed in case of N-terminal localization of D-alanine indicate increased anxiety in rats. Thus, an anxiolytic effect of Gly-His-Lys peptide was observed after intraperitoneal administration in doses ranging from 0.5 to 50 μg/kg, the lowest dose being most effective. Maximum activity of the peptide used in a low dose typical of regulatory peptides might be achieved via triggering the cascade amplifi cation mechanisms of intracellular formation of a large number of second messenger molecules, function of super-affi nity receptors, and existence of acceptor molecules capable of accumulation of circulating signaling molecules. The data on the anxiolytic effects of Gly-His-Lys peptide one more time conform the concept of multifunctional nature of the effects of regulatory peptides. [3]

Experiments were performed with cells in their first or second passage. At the time of experimentation, the fibroblasts were washed with phosphate-buffered saline solution, and 0.05% trypsin was used to release the confluent cells from the flask wall. Trypsin soybean inhibitor (GIBCO) in a 1:1 ratio inactivated the trypsin. Cell culture viability was determined by means of trypan-blue dye exclusion, and cells counts were performed in duplicate using a hemocytometer and phase-contrast microscopy. Cells were then seeded at a density of 5 × 103 (normal) and 3 × 103 (irradiated) cells/well in each well of a sterile 96-well plate using a commercially available serum-free medium. This medium has been shown to sustain dermal fibroblast growth to at least 7 days with greater than 90% viability.
At 0 hours, GHK-Cu solution (1 × 10−9 M) in the serum-free medium was added to the treatment groups, and an equalvolume of plain serum-free medium was added to the untreated control groups. Untreated cells from each cell line were used for controls. Cell counts were performed using a cell proliferation assay system with reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) 24, 48, 72, and 96 hours after initiation for growth curve generation. The WST-1 assay is a colorimetric assay used in the quantification of cell proliferation and cell viability based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. It is a nonradioactive alternative to the tritium-thymidine incorporation assay. Assays were read using an automated plate reader. Optical densities were analyzed with commercially available software. Cell counts were determined by comparison with a standard curve derived from known cell quantities calculated for each cell type and medium.
At each 24-hour interval, cell-free supernatant was collected from the testing wells in triplicate. Samples were stored at −80°C in microcentrifuge tubes for later growth factor assays. Expression of bFGF, TGF-β1, and VEGF was evaluated for each group by means of a solid-phase enzyme-linked immunosorbent assay at 24-hour intervals. We calculated cell population-doubling times (PDT) from logarithmic best-fit curves.[1]

We used Gly-His-Lys peptide (experimental series I) and its modifi ed analogs Gly-His-D-Lys, D-Ala-GlyHis-Lys, and Gly-His-Lys-D-Ala (experimental series II) synthesized in the Research Institute for Chemistry, Saint Petersburg State University. The peptides were dissolved in saline and administered intraperitoneally 12 min before the experiment in doses of 0.5, 5, and 50 μg/kg. Controls in both series received equivalent volumes of saline (1 ml/kg body weight). Anxiolytic effects of the peptides were studied using the elevated plus maze (EPM) test. The maze consisted of four perpendicular arms (two opposite open arms without the walls and two closed arms with walls of 30 cm height) measured 50 cm long by 14 cm wide and was elevated by 50 cm above the fl oor. At the beginning of the experiment, the rat was placed in the center of the maze with its head directed toward an open arm; the time spent in the open and closed arms and central area and the number of entries into the open and closed arms were recorded over 5 min. Anxiolytic effects of peptides were evaluated by the increase in the number of entries into the open arms and the time spent there. [3]

Absorption, Distribution and Excretion
Prezapeptide (both free and copper-chelated forms) can penetrate the stratum corneum. Its absorption is affected by pH, with the highest absorption rate at physiological pH. Metabolism/Metabolites Prezapeptide is broken down into histidine lysine, which may be further degraded into other proteolytic metabolites. Biological Half-Life Prezapeptide is rapidly eliminated within minutes.

[1]. Effects of copper tripeptide on the growth and expression of growth factors by normal and irradiated fibroblasts. Arch Facial Plast Surg. 2005 Jan-Feb;7(1):27-31.

[2]. Tripeptide Gly-His-Lys is a hepatotropic immunosuppressor. Bull Exp Biol Med. 2002 Jun;133(6):586-7.

[3]. Anxiolytic effects of Gly-His-Lys peptide and its analogs. Bull Exp Biol Med. 2015 Apr;158(6):726-8.

[4]. Effect of Gly-Gly-His, Gly-His-Lys and their copper complexes on TNF-alpha-dependent IL-6 secretion in normal human dermal fibroblasts. Acta Pol Pharm. 2012 Nov-Dec;69(6):1303-6.

Prezatide is a tripeptide composed of glycine, histidine, and lysine that readily forms a complex with copper ions. Prezatide is used in skin and hair care cosmetics. It is known to aid wound healing, and its potential applications in chronic obstructive pulmonary disease and metastatic colon cancer are currently under investigation. Pharmaceutical Indications It is commonly used in skin and hair care cosmetics. Mechanism of Action After forming a complex with copper, prezatide increases the synthesis and deposition of type I collagen and glycosaminoglycans. It also increases the expression of matrix metalloproteinase-2 and tissue inhibitors of matrix metalloproteinases-1 and -2, suggesting a role in the regulation of tissue remodeling. The antioxidant activity of prezatide is thought to be related to its ability to provide copper to superoxide dismutase, while its anti-inflammatory activity is attributed to its inhibition of iron ion (Fe2+) release during injury. Prezatide also promotes angiogenesis at the site of injury. The specific mechanisms of these effects are not yet fully understood. Whether the effects of prezapeptide are due to the tripeptide itself or its ability to target and transport copper remains unclear. Prezapeptide is known to bind to heparin and heparin sulfate.

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Pharmacodynamics
The copper-based complex of prezapeptide improves skin elasticity, density, and firmness, reduces fine lines and wrinkles, alleviates photodamage, and promotes keratinocyte proliferation. Prezapeptide also has antioxidant and angiogenic effects and appears to modulate post-injury tissue remodeling.

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Objective: To evaluate the effects of the copper tripeptide (GHK-Cu) on the growth of normal and irradiated fibroblasts and the autocrine production of basic fibroblast growth factor, transforming growth factor β1, and vascular endothelial growth factor in a serum-free in vitro environment.
Methods: Primary human dermal fibroblast lines were isolated from intraoperative specimens of patients receiving radiotherapy for head and neck tumors. Normal and irradiated fibroblasts were cultured in serum-free and growth factor-free media. The treatment group was exposed to GHK-Cu (1 x 10⁻⁹ mol/L). We measured cell counts and the production of basic fibroblast growth factor, transforming growth factor β1, and vascular endothelial growth factor. [1]

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The results showed some interesting findings. First, we demonstrated that irradiated fibroblasts could survive and proliferate in serum-free medium. To our knowledge, our laboratory is the first to record this phenomenon using irradiated human fibroblasts. Our laboratory has previously demonstrated that normal fibroblasts, fetal fibroblasts, and keloid fibroblasts can survive and proliferate in a serum-free environment. Serum-free cell culture is essential for measuring changes in the growth factor environment and is now a viable model for future studies of irradiated fibroblasts. Second, our data identified differences in baseline growth factor production between normal and irradiated fibroblasts by direct comparison with the model. Except at 72 hours, normal fibroblasts produced significantly more bFGF than irradiated fibroblasts at all time points. At 24 hours, normal fibroblasts produced significantly more TGF-β1 than irradiated fibroblasts. Finally, compared to irradiated fibroblasts, normal fibroblasts showed significantly increased VEGF production at both 24 and 48 hours. It is reasonable to assume that these differences play a significant role in the clinical significance of these differences in wound healing properties. Third, the data indicate that the GHK-Cu-modulated environment is associated with changes in the growth factor environment. Irradiated fibroblasts treated with GHK-Cu produced significantly more bFGF than the control group at both 24 and 72 hours. In fact, irradiated fibroblasts treated with GHK-Cu produced significantly more bFGF than the normal control group at 24 hours. Furthermore, irradiated fibroblasts treated with GHK-Cu also produced significantly more VEGF than the normal control group at 24 hours. Given the known benefits of the early presence of these growth factors in the wound healing process, this finding is significant. Finally, the data show that the use of GHK-Cu-modulated environment is associated with a significant enhancement of fibroblast photodynamic therapy (PDT). This was confirmed in both normal and irradiated cell lines. A notable finding was that the population growth of irradiated fibroblasts treated with GHK-Cu was similar to that of the normal control group. Its clinical significance is unclear. However, given the important role of fibroblasts in wound healing, it can be hypothesized that an increase in the number of fibroblasts in the irradiated wound bed will lead to an overall improvement in wound healing. Gly-His-Lys is a tripeptide composed of glycine, L-histidine, and L-lysine residues linked in sequence. It has the functions of a metabolite, chelating agent, wound healing agent, and hepatoprotective agent. Prezatide is a tripeptide composed of glycine, histidine, and lysine that readily forms complexes with copper ions. Prezatide is used in cosmetics for skin and hair. It is known to aid wound healing, and its potential applications in chronic obstructive pulmonary disease and metastatic colon cancer are currently under investigation. Commonly used in cosmetics for skin and hair.
FDA Label
Mechanism of Action
The complex formed by prezapeptide and copper increases the synthesis and deposition of type I collagen and glycosaminoglycans. It also increases the expression of matrix metalloproteinase-2 and tissue inhibitors of matrix metalloproteinase-1 and -2, suggesting a role in the regulation of tissue remodeling. The antioxidant activity of prezapeptide is thought to stem from its ability to provide copper to superoxide dismutase, while its anti-inflammatory effect is attributed to its ability to block the release of iron ions (Fe2+) during injury. Prezapeptide also promotes angiogenesis at the site of injury. The specific mechanisms of these effects are not yet clear. Whether the action of prezapeptide is due to the tripeptide itself or its ability to localize and transport copper is also currently unclear. Prezapeptide is known to bind to heparin and heparin sulfate.
Pharmacodynamics
After forming a complex with copper, prezapeptide improves skin elasticity, density, and firmness, reduces fine lines and wrinkles, alleviates photodamage, and promotes keratinocyte proliferation.
Prezatide also has antioxidant and angiogenic effects and appears to regulate tissue remodeling after injury.

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Researchers replaced L-lysine with D-lysine to study the role of this amino acid in the tripeptide molecule. L-lysine is known to affect the function of the nervous system, particularly by regulating serotonin release in the central amygdala and norepinephrine release in the ventromedial hypothalamus. After modification, the effects of the neurotrophic peptides studied were significantly reduced, suggesting that lysine plays an important role in the functional activity of the molecule. The purpose of modifying the tripeptide with D-alanine was to enhance its resistance to destructive proteases, thereby enhancing its intended effect. However, the change in molecular structure weakened or even reversed the anti-anxiety effect (increased anxiety). The latter observation also indirectly confirmed that the tripeptide was involved in the generation of fear and anxiety. Receptor changes in the modified molecule may be one of the mechanisms leading to the above results; however, this issue needs further investigation [3].

C14H22CUN6O4

401.91

402.107

89030-95-5

72957-37-0 (monoacetate); 130120-57-9 (copper acetate salt/solvate); 89030-95-5 (copper salt)

71587328

Gly-His-Lys + Cu²⁺

GHK-Cu

Light blue to blue solid powder

1.045

5

7

10

25

428

2

C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)[O-])NC(=O)CN.[Cu+2]

NZWIFMYRRCMYMN-ACMTZBLWSA-M

InChI=1S/C14H24N6O4.Cu/c15-4-2-1-3-10(14(23)24)20-13(22)11(19-12(21)6-16)5-9-7-17-8-18-9;/h7-8,10-11H,1-6,15-16H2,(H,17,18)(H,19,21)(H,20,22)(H,23,24);/q;+2/p-1/t10-,11-;/m0./s1

copper;(2S)-6-amino-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(1H-imidazol-5-yl)propanoyl]amino]hexanoate

Prezatide copper; GHK copper; 89030-95-5; CG-copper peptide; Copper tripeptide-1; Oristar Cu-GHK; UNII-6BJQ43T1I9; 6BJQ43T1I9;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~124.41 mM)

GHK-Cu Formulation Tips

Avoid strong oxidizing ingredients.

 

Avoid ingredients that form complexes with Cu ions. For example:

 

Carnosine has a structure similar to GHK, so it may compete for copper ions and turn the solution purple.

 

Similarly, EDTA may chelate copper ions from GHK-Cu and turn the solution green.

 

GHK-Cu is water-soluble (1g GHK-Cu can easily dissolve in 20ml water). To prevent decomposition and color change due to excessively low or high pH:

 

Adjust the pH to near neutral.

 

Add all other ingredients (including preservatives) except GHK-Cu first.

 

Add GHK-Cu in the final step.

 

The process should be carried out below 40°C.

 

Troubleshooting color changes: To identify the problematic ingredient (often the optimal one):

 

Mix GHK-Cu with each individual ingredient and observe the reaction.

 

Consider removing or replacing any ingredient that causes a color change.

 

Usage & Dosage Recommendations

Based on conventional applications and efficacy studies, the suitable concentration range for GHK-Cu is typically between 500 ppm and 5,000 ppm.

 

To achieve a concentration of 2,000 ppm in the final product:

 

Add 0.2% GHK-Cu (Copper Tripeptide-1) powder, or

 

Add 10% of a GHK-Cu (Copper Tripeptide-1) 20,000 ppm solution.

 

Incompatibility List (Avoid Combining With)

Avoid mixing with Retinoids and Retinoid-like drugs (e.g., HPR).

 

Avoid mixing with Chelating Agents, such as:

 

Disodium EDTA

 

Capryloyl Hydroxamic Acid

 

Carnosine

 

Avoid mixing with Acids, such as:

 

Lactic Acid

 

Salicylic Acid

 

Fruit Acids (AHAs)

 

Mandelic Acid

 

Lactobionic Acid

 

Azelaic Acid

 

Avoid mixing with Vitamin C & Derivatives, Potassium Methoxysalicylate, Tranexamic Acid, Glabridin, Arbutin.

 

Avoid mixing with Sodium Polyglutamate (PGA Sodium), Carbomer, and other anionic polymers.

 

Avoid mixing with Niacinamide.

 

Avoid mixing with Color-changing and colored substances, such as:

 

Serine

 

Metabisulfite

 

Salicylic Acid

 (Please use freshly prepared in vivo formulations for optimal results.)