Tripeptide

甘氨酰-L-组氨酰-L-赖氨酸、其铜复合物和酵母/铜发酵物对正常人真皮成纤维细胞 NHDF 细胞系释放促炎性 IL-6 的影响 [1]。葡萄糖-L-组氨酰-L-赖氨酸及其铜复合物抑制成纤维细胞的 TNF-α 依赖性 IL-6 释放 [1]。

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药妆品是化妆品和药品的结合。有许多药妆活性产品可大致分为以下几类：抗氧化剂、寡肽、生长因子和色素闪电剂。Gly-His-Lys (GHK)和Gly-Gly-His （GGH）等三肽及其铜配合物具有较高的活性和良好的皮肤耐受性。最近的研究表明，它们在伤口愈合、组织修复和皮肤炎症过程中具有生理作用。这些肽的抗炎作用机制尚不清楚。本研究的目的是评价两种多肽GGH的影响。GHK及其铜复合物和酵母菌/铜发酵（低聚铜）对正常人真皮成纤维细胞NHDF分泌促炎IL-6的影响。采用ELISA试剂盒检测IL-6。GGH、GHK、CuCl2及其铜复合物可降低成纤维细胞中tnf - α依赖性IL-6的分泌。IL-6对正常伤口愈合、皮肤炎症和uvb诱导的红斑至关重要。由于铜肽具有抗炎的特性，可以用于皮肤表面，而不是使用副作用较大的皮质类固醇或非甾体类抗炎药物。我们的观察结果为这些三肽在皮肤炎症中的作用提供了一些新的信息。［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]

IL-6测定[1]
采用酶联免疫吸附测定试剂盒评价TNF-α刺激正常人真皮成纤维细胞分泌IL-6的情况。正常人真皮成纤维细胞在96孔板中培养7-10天。细胞生长到90-95%的合度。在适当实验开始前24小时，将培养基替换为含有200µg/mL牛血清白蛋白（BSA）的不含牛血清白蛋白的培养基。每孔加入1 nM或1µM的GGH、ghh - cu、Gly-His-Lys/GHK /GHK或GHK- cu溶液和1或10%的OligolidesÆ Copper溶液。培养基含100 ng/mL TNF-α。72h后测定IL-6浓度。在λ = 450 nm处用微孔板阅读器[1]测定吸光度。

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

176.22

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

注意： 本产品在运输和储存过程中需避光。

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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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。