Copper tripeptide (GHK-Cu)   中文名称: [N2-(N-甘氨酰-L-组氨酰)-L-赖氨酸]铜

Tripeptide

暴露在光下的成纤维细胞受到GHK-Cu/铜三肽（1 nM；0-96 小时）的影响，其群体倍增时间与对照组相似[1]。在 24 个受辐射的成纤维细胞中，GHK-Cu/铜三肽（1 nM；0-120 小时）产生的碱性成纤维细胞生长因子比正常对照要多得多 [1]。

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辐照后的成纤维细胞在无血清培养基中存活并复制。暴露于GHK-Cu的正常和辐照成纤维细胞的种群倍增速度比未处理的对照组快。GHK-Cu处理的成纤维细胞的生长速率与未处理的对照组接近，并且在GHK-Cu暴露后早期产生的碱性成纤维细胞生长因子和血管内皮生长因子明显多于未处理的对照组。 结论：受辐照的成纤维细胞在无血清培养基中存活和复制，建立了体外评价生长因子生成的理想模型。铜三肽加速正常和受辐照的成纤维细胞的生长，使受辐照的成纤维细胞接近正常对照的群体倍增时间。GHK-Cu-处理的成纤维细胞早期增加碱性成纤维细胞生长因子和血管内皮生长因子的产生可能促进伤口愈合。［1］

Glycyl-L-histidyl-L-lysine（腹腔注射；1.5、5、50、150 和 450 mg/kg；10 次）可促进肝细胞的有丝分裂活性，并以剂量依赖性方式降低免疫反应性[2]。甘氨酰-L-组氨酰-L-赖氨酸（腹腔注射；0.5、5、50 μg/kg）在高架十字迷宫测试中发挥抗焦虑作用[3]。

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腹腔注射三肽Gly-His-Lys，剂量分别为1.5、5、50、150和450 mg/kg，可刺激肝细胞的有丝分裂活性，并剂量依赖性地抑制免疫反应性（产生抗体的细胞数量和延迟型超敏反应）。[2]

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在实验开始前12分钟，以0.5、5、50 μg/kg的剂量，腹腔注射三肽Gly-His-Lys，雄性大鼠在升高+迷宫试验中具有抗焦虑作用，表现为张开臂时间增加，闭合臂时间缩短。注射0.5 μg/kg多肽后抗焦虑作用最明显，随剂量的增加而减弱。在所有研究剂量中，用三肽分子中的d -赖氨酸代替l -赖氨酸都伴随着嗜神经效应的显著减弱。在所有剂量下，d -丙氨酸与Gly-His-Lys肽的N端或c端连接可使其抗焦虑作用趋于平衡；在给药剂量为50 μg/kg后，一些增加焦虑的指标发生了显著变化。[3]

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在所有指定剂量下给予Gly-His-Lys肽对大鼠所检查的行为反应有显著影响（表1）。以0.5 μg/kg剂量组效果最佳，小鼠张开臂时间增加136% (p<0.01)，进入张开臂次数增加208% (p<0.01)，中央平台停留时间增加109% （p<0.05）。将多肽剂量增加至5 μg/kg时，抗焦虑作用并未增强，大部分研究参数与前一组相似。进一步增加gly - hisys的注射剂量至50 μg/kg后，这些作用减弱，各实验组间各研究参数之间出现显著差异。因此，张开双臂的时间与控制值没有显著差异，显著低于低剂量和中剂量组（分别减少45%和39%，p<0.05）。此外，与0.5 μg/kg剂量肽组相比，该组小鼠的闭臂时间和进入闭臂次数分别减少28% （p<0.05）和37% （p<0.05）。只有肽剂量为50 μg/kg时，小鼠进入闭锁臂的数量显著增加(增加45%；P <0.05)。这种现象可以通过增加肽剂量引起的大鼠运动活动的增加来解释。Gly-His-Lys的嗜神经效应研究结果促使我们研究其修饰的行为效应。用d -赖氨酸替代l -赖氨酸可显著降低大鼠的行为活动，并使肽的抗焦虑作用基本持平（表2）。仅注射0.5 μg/kg多肽后在中央平台停留的时间(增加134%；P <0.01)和50 μg/kg(降低56%；P <0.05)，较小剂量时进入闭合臂的次数(59%；P <0.05)显著高于对照组。在0.5和5 μg/kg剂量下，d -丙氨酸附着在Gly-His-Lys分子的n端对肽的研究行为参数没有显著影响。以最大剂量（50 μg/kg）给予肽可显着减少张开臂的时间(66%；P <0.05)和中心平台(48%；p < 0.05);闭臂时间增加31% （p<0.05）。这些行为变化表明了大鼠的焦虑。d -丙氨酸附着在Gly-His-Lys分子的c端后，三肽的嗜神经作用与之前修饰时相当均匀，其个体表现具有相反的性质。特别是，注射5 μg/kg剂量的肽后，进入张开臂的动物数量减少了65%；P <0.05)和50 μg/kg(降低72%；P <0.05)，以及注射最高剂量后在中心平台停留的时间(增加42%；p < 0.05)。这些行为变化，类似于在d -丙氨酸n端定位的情况下观察到的，表明大鼠焦虑增加。由此可见，Gly-His-Lys肽在0.5 ~ 50 μg/kg的剂量范围内腹腔给药后具有抗焦虑作用，且以最低剂量最有效。在低剂量的典型调节肽中使用的肽的最大活性可能通过触发细胞内形成大量第二信使分子的级联扩增机制、超亲和受体的功能以及能够积累循环信号分子的受体分子的存在来实现。这些关于Gly-His-Lys肽抗焦虑作用的数据再次印证了调控肽作用的多功能化概念。[3]

实验在细胞的第一或第二代进行。实验时，用磷酸盐缓冲的生理盐水洗涤成纤维细胞，用0.05%胰蛋白酶将融合细胞从烧瓶壁上释放出来。胰酶大豆抑制剂（GIBCO）以1:1的比例灭活胰蛋白酶。通过台盼蓝染色排除法测定细胞培养活力，并使用血细胞计和相差显微镜进行重复细胞计数。然后使用市售无血清培养基，以5 × 103（正常）和3 × 103（辐照）细胞/孔的密度在无菌96孔板的每孔中接种细胞。该培养基已被证明能维持真皮成纤维细胞生长至少7天，存活率高于90%。
在0小时时，在治疗组中加入无血清培养基中的GHK-Cu溶液（1 × 10−9 M），在未处理的对照组中加入等体积的无血清普通培养基。将每个细胞系中未经处理的细胞作为对照。细胞计数使用细胞增殖试验系统，在启动24、48、72和96小时后使用试剂4-[3-（4-碘苯基）-2-（4-硝基苯基）- 2h -5-四氮唑]-1,3-苯二磺酸盐（WST-1）进行细胞计数，以生成生长曲线。WST-1测定法是一种定量测定细胞增殖和细胞活力的比色法，基于线粒体脱氢酶在活细胞中切割四氮唑盐WST-1。它是一种非放射性的替代氚胸腺嘧啶掺入试验。使用自动车牌阅读器读取化验结果。用市售软件分析光密度。细胞计数通过与标准曲线进行比较来确定，标准曲线是根据每种细胞类型和培养基计算的已知细胞数量得出的。
每隔24小时，从测试井中收集三份无细胞上清液。样品保存在- 80°C的微离心管中，用于随后的生长因子测定。采用固相酶联免疫吸附法每隔24小时检测各组bFGF、TGF-β1和VEGF的表达。我们从对数最佳拟合曲线中计算细胞群体加倍时间（PDT）。［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]. 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 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.

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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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Objective: To evaluate the effects of copper tripeptide (GHK-Cu) on the growth and autocrine production of basic fibroblast growth factor, transforming growth factor beta1, and vascular endothelial growth factor by normal and irradiated fibroblasts in a serum-free in vitro environment.
Methods: Primary human dermal fibroblast cell lines were established after explantation from intraoperative specimens obtained from patients who had undergone radiation therapy for head and neck cancer. Normal and irradiated fibroblasts were propagated in serum- and growth factor-free media. Treatment groups were exposed to GHK-Cu (1 x 10(-9) mol/L). We measured cell counts and production of basic fibroblast growth factor, transforming growth factor beta1, and vascular endothelial growth factor.[1]

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Several interesting findings are demonstrated. First, survival and growth of irradiated fibroblasts was demonstrated within the serum-free media. To our knowledge, our laboratory is the first to document this phenomenon using irradiated human fibroblasts. Our laboratory has already demonstrated survival and growth of normal, fetal, and keloid fibroblasts in this serum-free environment. Serum-free cell culture is essential when measuring changes in the growth factor milieu and is now a viable model for future studies involving irradiated fibroblasts.
Second, the data established differences in the baseline production of growth factors between normal and irradiated fibroblasts in a head-to-head model. Production of bFGF by normal fibroblasts was significantly increased when compared with that of irradiated fibroblasts at all but 1 time point (72 hours). Production of TGF-β1 by normal fibroblasts was significantly increased when compared with that of irradiated fibroblasts at the 24-hour mark. Finally, production of VEGF by normal fibroblasts was significantly increased when compared with that of irradiated fibroblasts at 24 and 48 hours. It is reasonable to suppose that these differences play an influential role in the differing wound-healing properties of these wounds clinically.
Third, the data show that modulation of the environment with GHK-Cu is associated with changes in the growth factor milieu. The GHK-Cu–treated irradiated fibroblasts showed significantly greater production of bFGF than controls at 24 and 72 hours. In fact, GHK-Cu–treated irradiated fibroblasts produced significantly more bFGF than normal controls at the 24-hour interval. Furthermore, GHK-Cu–treated irradiated fibroblasts produced significantly more VEGF than normal controls at the 24-hour interval. This finding is of importance given the known benefit of an early presence of these growth factors in the healing wound.
Finally, the data show that modulation of the environment with GHK-Cu is associated with a dramatic increase in fibroblast PDT. This was demonstrated in the normal and irradiated cell lines. One striking finding is that population growth in GHK-Cu–treated irradiated fibroblasts assumed that of normal controls. The clinical implications of this are not yet known. However, given the important role of fibroblasts in wound healing, one might hypothesize that more fibroblasts in an irradiated wound bed would lead to a generalized improvement in wound healing.

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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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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].

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

186.03

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

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

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。