Tripeptide

Effects of glycyl-L-histidyl-L-lysine, its copper complex, and yeast/copper fermentate on normal human dermal fibroblast NHDF cell line's release of pro-inflammatory IL-6 [1]. Glucose-L-histidyl-L-lysine and its copper complex inhibit fibroblasts' TNF-α-dependent IL-6 release [1].

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Cosmeceuticals represent a marriage between cosmetics and pharmaceuticals. There are numerous cosmeceutically active products which can be broadly classified into the following categories: antioxidants, oligopeptides, growth factors and pigment lightning agents. Much attention has been focused on the tripeptides such as Gly-His-Lys (GHK) and Gly-Gly-His (GGH) and their copper complexes, which have a high activity and good skin tolerance. Recent data suggested their physiological role in process of wound healing, tissue repair and skin inflammation. The mechanism of anti-inflammatory properties of these peptides is not clear. The aim of the study was evaluation of influence of two peptides GGH. GHK and their copper complexes and saccharomyces/copper ferment (Oligolides Copper) on secretion of pro-inflammatory IL-6 in normal human dermal fibroblasts NHDF cell line. IL-6 was evaluated using the ELISA kit. GGH, GHK, CuCl2 and their copper complexes decreased TNF-alpha-dependent IL-6 secretion in fibroblasts. IL-6 is crucial for normal wound healing, skin inflammation and UVB-induced erythema. Because of the anti-inflammatory properties, the copper-peptides could be used on the skin surface instead of corticosteroids or non-steroidal anti-inflammatory drugs, which have more side effects. Our observations provide some new information about the role of these tripeptides in skin inflammation. [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]

IL-6 assay [1]
TNF-α stimulated IL-6 secretion by normal human dermal fibroblasts was evaluated using the enzyme-linked immunosorbent assay kit. Normal human dermal fibroblasts were cultured in 96-well plates for 7-10 days. Cells were grown to the 90-95% confluence. Twenty four hours before initiation of the proper experiment, the medium was replaced with a medium without FBS containing 200 µg/mL bovine serum albumin (BSA). 1 nM or 1 µM solutions of GGH, GGH-Cu, Gly-His-Lys/GHK or GHK-Cu and 1 or 10% solutions of OligolidesÆ Copper were added into each well. Medium contained 100 ng/mL TNF-α. IL-6 concentration was evaluated after 72 h. The absorbance was measured at λ = 450 nm in a microplate reader [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
Prezatide both free and in complex with copper can pass through the stratum corneum. Its absorption is pH dependent with the highest absorption occurring at physiological pH.

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Metabolism / Metabolites
Prezatide is broken down to histidyl-lysine which is likely further degraded to other metabolites of proteolysis.

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Biological Half-Life
Prezatide is rapidly eliminated within minutes.

[1]. 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.

[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.

Gly-His-Lys is a tripeptide composed of glycine, L-histidine and L-lysine residues joined in sequence. It has a role as a metabolite, a chelator, a vulnerary and a hepatoprotective agent.
Prezatide is a tripeptide consisting of glycine, histidine, and lysine which readily forms a complex with copper ions. Prezatide is used in cosmetic products for the 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 being investigated.

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Drug Indication
Commonly used in cosmetic products for the skin and hair.
FDA Label

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Mechanism of Action
Prezatide in complex with copper increases the synthesis and deposition of type I collagen and glycosaminoglycan. It also increases the expression of matrix metalloproteinase-2 as well as tissue inhibitor of matrix metalloproteinases-1 and -2, suggesting that it plays a role in the modulation of tissue remodeling. It is thought that prezatide's antioxidant activity is due to its ability to supply copper for superoxide dismutase and its anti inflammatory ability due to the blockage the of iron (Fe2+) release during injury. Prezatide also increases angiogenesis to injury sites. The precise mechanisms of these effects are unknown. It is also unknown whether prezatide's effects are due to the action of the tripeptide itself or its ability to localize and transport copper. Prezatide is known to be bound by heparin and heparin sulfate

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Pharmacodynamics
Prezatide in complex with copper improve skin elasticity, density, and firmness, reduces fine lines and wrinkles, reduces photodamage, increases keratinocyte proliferation. Prezatide also displays anti-oxidant and angiogenic effects and appears to modulate tissue remodeling in injury.

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There are no many publications on the topic of influence of the peptides like Gly-His-Lys/GHK or GGH on cytokines secretion at the moment. Fields et al. wrote about palmitoyl tetrapeptide-7, which is an active ingredient of any cosmetics. It is able to down-regulate IL-6 in vitro in both resting and inflamed cells. Marketing materials related to RiginTM indicate that this reduction in IL-6 can produce increased skin firmness, smoothness, and elasticity. One mM GHK significantly inhibited IL-6 expression in SZ95 sebocytes. In the past, several laboratories demonstrated local and systemic anti-inflammatory activity of copper compounds, particularly after subcutaneous administration. Copper-peptides could be used on the skin surface instead of corticosteroids or nonsteroidal anti-inflammatory drugs, which have more side effects. [1]

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Replacement of L-lysine with D-lysine was performed to study the role of this amino acid in the tripeptide molecule. L-lysine is known to affect functioning of the nervous system, in particular, due to modulation of serotonin release in the central nucleus of the amygdala and norepinephrine release in the ventromedial hypothalamus. Signifi cant weakening of the studied effects of the neurotropic peptide after modifi cation demonstrates the important role of lysine in functional activity of the molecule. The purpose of modifi cation of tripeptide using D-alanine was to increase its resistance to destructive proteases and, as a consequence, the expected enhancement of the effect. However, changes in the molecule structure leveled the anxiolytic effect or its inversion (increased anxiety). The latter observation also indirectly confi rms the participation of the tripeptide in the development of fear and anxiety. Altered reception of modifi ed molecules can be one of the mechanisms responsible for the obtained results; this issue however requires further study [3].

C14H24N6O4

340.3782

340.185

C, 49.40; H, 7.11; N, 24.69; O, 18.80

49557-75-7

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

73587

Gly-His-Lys

GHK; H-GHK-OH

White to off-white solid powder

1.3±0.1 g/cm3

831.0±65.0 °C at 760 mmHg

456.4±34.3 °C

0.0±3.2 mmHg at 25°C

1.583

-2.24

6

7

11

24

434

2

MVORZMQFXBLMHM-QWRGUYRKSA-N

InChI=1S/C14H24N6O4/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)/t10-,11-/m0/s1

(2S)-6-amino-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-(1H-imidazol-5-yl)propanoyl]amino]hexanoic acid

glycyl-l-histidyl-l-lysine; Prezatide; L-Lysine, glycyl-L-histidyl-; Tripeptide-1; Liver cell growth factor; KOLLAREN; ORISTAR GHK; ...; 49557-75-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

H2O : ~100 mg/mL (~293.79 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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