Metformin (glycinate)   中文名称: 1,1-Dimethylbiguanide (glycinate)

AMPK; Autophagy; Mitophagy

二甲双胍抑制 Bcl-2 和 Bcl-xl，上调 BAX 激活，促进 BIM、BAD 和 PUMA，并诱导细胞色素 c 从线粒体释放到细胞质中，直接诱导 caspase-9 介导的线粒体凋亡。 [4]

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二甲双胍激活肝细胞中的AMPK；结果，乙酰辅酶a羧化酶（ACC）活性降低，脂肪酸氧化被诱导，脂肪生成酶的表达被抑制。二甲双胍或腺苷类似物激活AMPK会抑制SREBP-1的表达，SREBP-1是一种关键的脂肪生成转录因子。在二甲双胍治疗的大鼠中，SREBP-1（和其他脂肪生成）mRNA和蛋白质的肝脏表达降低；AMPK靶标ACC的活性也降低了。使用一种新型AMPK抑制剂，我们发现二甲双胍对肝细胞葡萄糖产生的抑制作用需要AMPK激活。在离体大鼠骨骼肌中，二甲双胍刺激葡萄糖摄取，同时AMPK激活。AMPK的激活为该药物的多效有益作用提供了统一的解释；这些结果还表明，调节AMPK的替代方法可用于治疗代谢性疾病。[1]

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二甲双胍被广泛推荐用于治疗2型糖尿病，通过5'-AMP活化蛋白激酶（AMPK）发挥其多效性作用；然而，它对线粒体吞噬的影响仍然难以捉摸。最近的证据表明，外周血单核细胞（PBMCs）表达胰岛素受体和人类有机阳离子转运蛋白，它们被广泛用作检测2型糖尿病线粒体功能的替代品。二甲双胍治疗增加了酸性囊泡和丝裂吞噬体的形成，上调了丝裂吞噬标记物，并增强了丝裂噬通量，如LC3-II表达增加和p62蛋白水平降低所示。此外，用化合物C（一种AMPK抑制剂）预处理显著降低了二甲双胍处理细胞中自噬标志物的表达，表明二甲双胍通过AMPK途径诱导自噬。总之，二甲双胍诱导的线粒体吞噬可能通过恢复正常的线粒体表型来改善细胞功能，包括β细胞，这可能对2型糖尿病和其他线粒体相关疾病患者有益。此外，PBMCs可以用作鉴定线粒体疾病的新型诊断生物标志物。[2]

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在这项研究中，我们首次揭示了二甲双胍治疗导致人肺癌细胞系A549和NCI-H1299细胞凋亡增加，并以剂量和时间依赖的方式显著抑制细胞增殖，从裸鼠A549肿瘤异种移植物获得的数据进一步证明了这一点。我们还发现，二甲双胍治疗可以激活AMP活化蛋白激酶、JNK/p38 MAPK信号通路和半胱氨酸天冬氨酸蛋白酶，并上调生长停滞和DNA损伤诱导基因153（GADD153）的表达。阻断JNK/p38 MAPK通路或敲除GADD153基因都会消除二甲双胍的凋亡诱导作用。总之，我们的数据表明，二甲双胍通过激活JNK/p38 MAPK途径和GADD153抑制肺癌细胞的生长并诱导细胞凋亡。[3]

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这项研究表明，二甲双胍通过诱导凋亡减少了活化HSC的数量，但不影响肝细胞的数量。二甲双胍上调BAX激活，促进BIM、BAD和PUMA；下调Bcl-2和Bcl-xl，但不影响Mcl-1。此外，二甲双胍诱导细胞色素c从线粒体释放到细胞质中，直接触发胱天蛋白酶-9介导的线粒体凋亡。线粒体膜电位（ΔΨm）的下降和线粒体中超氧化物的沉积加速了线粒体膜完整性的破坏。此外，我们验证了二甲双胍在非酒精性脂肪性肝炎（NASH）相关肝纤维化小鼠模型中的治疗效果，其中二甲双胍改善了肝功能、NASH病变和纤维化。总之，本研究表明，二甲双胍通过诱导HSC凋亡对NASH衍生的肝纤维化具有显著的治疗价值，但不影响肝细胞的增殖[4]。

二甲双胍在非酒精性脂肪性肝炎 (NASH) 相关肝纤维化小鼠模型中表现出治疗作用，从而改善肝功能、NASH 病变和纤维化。 [4]

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评估二甲双胍的体内效应。[1]
为了评估上述二甲双胍的选定效应是否也在体内发生，对SD大鼠进行了研究（表1）。大鼠口服二甲双胍或赋形剂（H2O）5天。大鼠饥饿20小时，然后在最后一次给药前重新喂食2小时。最后一次给药后4小时，采集组织和血液样本进行分析（见方法）。在饥饿期间，应该很少有脂质合成。重新进食后，应显著诱导肝脏脂质合成。在再喂养条件下检查了二甲双胍的作用。除了血浆胰岛素和甘油三酯的适度降低外，β-羟基丁酸酯也有小幅但显著的增加，这表明二甲双胍治疗的大鼠肝脏脂肪酸氧化是诱导的。此外，二甲双胍治疗显著降低了SREBP-1、FAS和S14的mRNA在肝脏的表达，这与细胞中记录的效果一致（表1）。
使用抗SREBP-1抗体检测大鼠肝细胞核提取物中的成熟SREBP-1蛋白（图5b）。正如预期的那样，在饥饿动物的肝核提取物中没有检测到SREBP-1成熟形式的蛋白质。在再喂养的动物中，成熟的SREBP-1蛋白的积累与这种情况下脂质合成的增加相一致。用二甲双胍治疗可以防止这种积聚。使用AICAR（500mg/kg/天）治疗后再喂养大鼠的肝核提取物获得的其他结果也表明，SREBP-1成熟形式蛋白的存在被消除。
在离体肝脏中测量AMPK活化是困难的，因为已知短暂的缺氧会导致酶的显著活化。因此，我们使用二甲双胍治疗大鼠的肝组织来确定在几种测试的柠檬酸盐浓度下ACC活性显著降低（图6）。柠檬酸盐浓度为1 mM时ACC活性降低最大（从54.6±11.8降至35.6±7.7 nmol/mg/min；P<0.01）。这些结果与二甲双胍在体内产生AMPK激活和ACC失活的结果一致。

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二甲双胍缓解了非酒精性脂肪性肝炎（NASH）相关纤维化小鼠模型的肝脏病变[4]
建立了NASH相关纤维化小鼠模型（图7a），以探索二甲双胍是否能在体内疾病状态下保护肝脏。每隔一周和每三周分别测量每只小鼠的体重和生化指标（ALT和AST）。结果显示，与MCS饮食组或AIN93饮食组相比，HFMCD饮食组的体重没有显著增加（图S2a）。此外，与MCS饮食组或AIN93组相比，在HFMCD饮食治疗组中，二甲双胍降低了AST和ALT水平以及肝脏指数（图7b、图S2b）。总的来说，二甲双胍改善了NASH相关纤维化小鼠模型的肝功能。
从NASH纤维化模型中获得的石蜡包埋的肝组织样本用苏木精和伊红染色，以观察二甲双胍对肝损伤的影响。HFMCD组新鲜肝组织颜色偏深（图S2c）。结果显示，AIN93或MCS组的肝脏没有明显病变，也没有受到二甲双胍的影响（图S2d）。然而，在HFMCD预防和治疗组中，使用二甲双胍后，脂肪变性面积和炎性细胞数量均减少（图7c），表明NASH病变的严重程度减轻。此外，通过V-G染色观察纤维化程度。结果显示，HFMCD治疗组的胶原蛋白积累远少于HFMCD饮食盐水组（图7d）。Masson染色也观察到了相同的结果（图S2e）。此外，免疫组织化学染色显示，二甲双胍治疗组HSC切割半胱氨酸天冬氨酸蛋白酶-3的阳性率为38.7±5.2%，高于生理盐水组（7.69±0.61%）、空白对照组（0.58±0.03%）和二甲双胍治疗组（1.82±0.05%），表明二甲双胍诱导HSC凋亡（图7e，表3）。因此，二甲双胍延缓了小鼠模型的纤维化。

这项研究表明，二甲双胍通过诱导细胞凋亡减少了活化的HSC的数量，但不影响肝细胞的数量。二甲双胍通过促进BIM、BAD和PUMA上调BAX活化；下调Bcl-2和Bcl-xl，但不影响Mcl-1。此外，二甲双胍诱导细胞色素c从线粒体释放到细胞质中，直接触发胱天蛋白酶-9介导的线粒体凋亡。线粒体膜电位（ΔΨm）的下降和超氧化物在线粒体中的沉积加速了线粒体膜完整性的破坏。此外，我们验证了二甲双胍在与非酒精性脂肪性肝炎（NASH）相关的肝纤维化小鼠模型中的治疗效果，在该模型中，二甲双胍改善了肝功能、NASH病变和纤维化。总之，本研究表明，二甲双胍通过诱导HSC凋亡，对NASH衍生的肝纤维化具有显著的治疗价值，但不影响肝细胞的增殖[4]。

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免疫沉淀AMPK测定。[1]
使用针对AMPKα1（NH2-DFYLATSPPDSFLDDHHLTR-OH）或AMPKα2（NH2-MDSAMHIPPGLKPH-OH）的多克隆抗体对来自AICAR或二甲双胍处理的大鼠肝细胞的10微克35%硫酸铵沉淀物（含AMPK）进行免疫沉淀，然后进行AMPK测定。 肌肉AMPK活性和葡萄糖摄取的测量。[1]
将分离的大鼠耳蜗上肌与二甲双胍（2 mM）或对照培养基一起孵育3小时，然后如上所述测量AMPKα1或AMPKα2活性。对于葡萄糖摄取，在3小时温育的最后30分钟，胰岛素（300 nM）存在。然后，如前所述，在二甲双胍和/或胰岛素存在或不存在的情况下，使用10分钟的温育来测量3-0-甲基葡萄糖摄取。 半胱天冬酶酶活性测定[4]
按照生产说明，用胱天蛋白酶活性测定试剂盒测量胱天蛋白酶1、3、8和9的酶活性。将2×105个细胞在冰上裂解15分钟，然后依次加入底物。酶活性用酶标仪（λ=405nm）测量。

将大鼠 HSC T6、人肝细胞 L02 和大鼠肝细胞 BRL-3A 维持在 DMEM 中，在 37°C 的湿润环境中补充有 10% (v/v) FBS 和 1% (v/v) 青霉素/链霉素硫酸盐。 5% CO2 培养箱。人肝星状细胞 (HSC) LX-2 维持在 RPMI 1640 培养基中。通过孔径为 0.22 m 的微孔膜过滤器过滤二甲双胍和 AICAR 的溶液。 CCK8 测定用于测量不同浓度的二甲双胍和 AICAR 对肝星状细胞生长的影响。用不同剂量的二甲双胍和 AICAR 治疗 24 小时后，使用蛋白质印迹发现胶原蛋白和 -SMA 蛋白的表达。给予细胞10 mM二甲双胍和0.5 mM AICAR 24小时后，使用Western blot鉴定胶原蛋白和-SMA蛋白的表达。 RT-qPCR 用于检测 I 型胶原蛋白和 -SMA 的 mRNA 水平。

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原代肝细胞中AMPK、ACC和脂肪酸氧化的测量。[1]
通过胶原酶消化从雄性Sprague-Dawley（SD）大鼠中分离肝细胞。对于AMPK测定，将细胞以1.5×106个细胞/孔的速度接种在含有100 U/ml青霉素、100μg/ml链霉素、10%FBS、100 nM胰岛素、100 nM-地塞米松和5μg/ml转铁蛋白的DMEM中的六孔板上，持续4小时。然后将细胞在无血清DMEM中培养16小时，然后用对照培养基、5-氨基咪唑甲酰胺核苷（AICAR）或指定浓度的二甲双胍处理1小时或7小时。在39小时的治疗中，对照组和二甲双胍（10或20μM）组的细胞在DMEM加5%FBS和100 nM胰岛素中培养，每12小时更换一次新鲜对照组和含二甲双胍的培养基（最后一次更换培养基是在收获前3小时）。处理后，细胞直接在含有洋地黄皂苷和磷酸酶抑制剂的缓冲液A中裂解，然后用35%饱和度的硫酸铵沉淀。通过测量合成肽底物SAMS（HMRSAMSGLHLVKRR）的磷酸化来测定AMPK活性。对于ACC测定，使用来自洋地黄皂苷裂解肝细胞的35%硫酸铵沉淀（每个4μg）在20mM柠檬酸盐存在下通过14CO2固定测定ACC活性，如前所述。对于脂肪酸氧化，14C-油酸盐氧化为酸溶性产物的过程与之前一样进行，但在没有白蛋白的培养基M199中进行。

Male C57BL/6N mice
10-200 mg/kg; 65 mg/kg
i.p.

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Oral gavage was used to administer 1 ml of Metformin (100 mg/ml) or water alone to male SD rats (300–350 g, n = 7–8). Rats were treated once (see Table 1and Figure 5b) or twice (see Figure 6) a day for 5 days. Rats were starved for 20 hours and then re-fed for 2 hours before the final dose; 4 hours after final dose, the animals were anesthetized and livers rapidly removed by freeze clamping followed by blood withdrawal. RNA was prepared from the freeze-clamped liver by Ultraspec RNA isolation reagent. Nuclear extracts were prepared from a pool of seven rat livers. Glucose levels were determined using the standard glucose oxidase assay kit; β-hydroxybutyrate concentrations were assayed by measuring the reduction of NAD to NADH with a standard assay kit. FFA levels were measured with the assay kit.Triglyceride levels were assayed with a kit. Insulin concentrations were measured with the enzyme-immunoassay kit.[1]

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Male C57BL/6N mice (6–8 weeks old, weight, 16–20 g) were randomly divided into three groups (n = 10 per group): (a) high-fat methionine/choline-deficient (HFMCD) diet group (the NASH-fibrosis model group); (b) methionine/choline-sufficient (MCS) diet group (the model control group); (c) AIN93 diet group (the blank control group). Each group were divided into two sub-groups (n = 5 per group): one was intraperitoneally injected with Metformin, the other with saline. Mice were housed in an environment where temperature, humidity and light were controlled (temperature 25 ± 2 °C, a 12/12 h light/dark cycle, 55 ∼ 60 % humidity). All mice got access to the diet and water, which were changed at a fixed time every afternoon. The normal control and model control mice were fed at the same time as the HFMCD group.

All mice were divided into preventive group (intraperitoneal injection from the first week), therapeutic group (intraperitoneal injection from the eighth week) and saline group according to the administration time of metformin and saline. The concentration gradient of Metformin mice received ranged from 10 mg kg−1 to 200 mg kg−1 to get a proper concentration. Metformin was dissolved in saline. Mice received metformin or saline (65 mg kg−1 i.p.) administration every other day for 11 weeks or 4 weeks after 8 days’ post-acclimation (the preventative group) or 8 weeks’ HFMCD-diet-induced liver injury (the therapeutic group), respectively.

Metformin is a first-line drug for the treatment of type 2 diabetes (T2D) and is often prescribed in combination with other drugs to control a patient's blood glucose level and achieve their HbA1c goal. New treatment options for T2D will likely include fixed dose combinations with metformin, which may require preclinical combination toxicology studies. To date, there are few published reports evaluating the toxicity of metformin alone to aid in the design of these studies. Therefore, to understand the toxicity of metformin alone, Crl:CD(SD) rats were administered metformin at 0, 200, 600, 900 or 1200 mg/kg/day by oral gavage for 13 weeks. Administration of > or =900 mg/kg/day resulted in moribundity/mortality and clinical signs of toxicity. Other adverse findings included increased incidence of minimal necrosis with minimal to slight inflammation of the parotid salivary gland for males given 1200 mg/kg/day, body weight loss and clinical signs in rats given > or =600 mg/kg/day. Metformin was also associated with evidence of minimal metabolic acidosis (increased serum lactate and beta-hydroxybutyric acid and decreased serum bicarbonate and urine pH) at doses > or =600 mg/kg/day. There were no significant sex differences in mean AUC(0-24) or C(max) nor were there significant differences in mean AUC(0-24) or C(max) following repeated dosing compared to a single dose. The no observable adverse effect level (NOAEL) was 200 mg/kg/day (mean AUC(0-24)=41.1 microg h/mL; mean C(max)=10.3 microg/mL based on gender average week 13 values). These effects should be taken into consideration when assessing potential toxicities of metformin in fixed dose combinations.[9]

[1]. Acute treatment with metformin improves cardiac function following NSC 37745 induced myocardial infarction in rats. Pharmacol Rep. 2012;64(6):1476-84.

[2]. Metformin promotes mitophagy in mononuclear cells: a potential in vitro model for unraveling metformin's mechanism of action. Ann N Y Acad Sci. 2020 Mar;1463(1):23-36.

[3]. Metformin induces apoptosis of lung cancer cells through activating JNK/p38 MAPK pathway and GADD153. Neoplasma. 2011;58(6):482-90.

[4]. Mitigation of liver fibrosis via hepatic stellate cells mitochondrial apoptosis induced by metformin. Int Immunopharmacol. 2022 Jul:108:108683.

[5]. Metformin inhibits growth of eutopic stromal cells from adenomyotic endometrium via AMPK activation and subsequent inhibition of AKT phosphorylation: a possible role in the treatment of adenomyosis. Reproduction. 2013 Aug 21;146(4):397-406.

[6]. Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes. Diabetes Obes Metab. 2003 May;5(3):189-94.

[7]. Therapeutic potential of an anti-diabetic drug, metformin: alteration of miRNA expression in prostate cancer cells. Asian Pac J Cancer Prev. 2013;14(2):765-8.

[8]. Acute treatment with metformin improves cardiac function following isoproterenol induced myocardial infarction in rats. Pharmacol Rep. 2012;64(6):1476-84.

[9]. Toxicity and toxicokinetics of metformin in rats. Toxicol Appl Pharmacol. 2010 Mar 15;243(3):340-7.

[10]. Metformin inhibits growth of eutopic stromal cells from adenomyotic endometrium via AMPK activation and subsequent inhibition of AKT phosphorylation: a possible role in the treatment of adenomyosis. Reproduction. 2013 Aug 21;146(4):397-406.

[11]. Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes. Diabetes Obes Metab. 2003 May;5(3):189-94.

[12]. Therapeutic potential of an anti-diabetic drug, metformin: alteration of miRNA expression in prostate cancer cells. Asian Pac J Cancer Prev. 2013;14(2):765-8.

Background: It has been proposed that metformin exerts protective effects on ischemic hearts. In the present study, we evaluated the effects of metformin on cardiac function, hemodynamic parameters, and histopathological changes in isoproterenol-induced myocardial infarction (MI). Methods: Male Wistar rats were divided into six groups (n = 6) of control, isoproterenol (100 mg/kg; MI), metformin alone (100 mg/kg; sham), and metformin (25, 50, 100 mg/kg) with isoproterenol. Subsequently, isoproterenol was injected subcutaneously for two consecutive days and metformin was administered orally twice daily for the same period. Results: Isoproterenol elevated ST-segment and suppressed R-amplitude on ECG. All doses of metformin were found to significantly amend the ECG pattern. Isoproterenol also caused an intensive myocardial necrosis along with a profound decrease in arterial pressure indices, left ventricular contractility (LVdP/dt(max)) and relaxation (LVdP/dt(min)), and an increase in left ventricular enddiastolic pressure (LVEDP). Histopathological analysis showed a marked attenuation of myocyte necrosis in all metformin treated groups (p < 0.001). Metformin at 50 mg/kg strongly (p < 0.01) increased LVdP/dt(max) from 2988 ± 439 (mmHg/s) in the MI group to 4699 ± 332 (mmHg/s). Similarly, treatment with 50 mg/kg of metfromin lowered the elevated LVEDP from 27 ± 8 mmHg in the myocardial infarcted rats to a normal value of 5 ± 1.4 (mmHg; p < 0.01) and the heart to body weight ratio as an index of myocardial edematous from 4.14 ± 0.13 to 3.75 ± 0.08 (p < 0.05). Conclusion: The results of this study demonstrated that a short-term administration of metformin strongly protected the myocardium against isoproterenol-induced infarction, and thereby suggest that patients suffering from myocardial ischemia could benefit from treatment with metformin.[8]

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Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45 mmol/l for A-ESCs and 7.87 mmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17β-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17β-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater effect of metformin on A-ESCs from secretory phase.[10]

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Aim: Glycogen synthesis, and glucose and lactate production were examined in cultured rat hepatocytes preincubated with metformin (0-500 micro m) for 24 h. Methods: Cells incubated with[1-13C]-glucose and [1-13C]-lactate allowed us to study the effect of metformin on glucose production from glycogenolysis and gluconeogenesis in a detailed manner using NMR spectroscopy. 1H and 13C-filtered 1H-NMR spectra were recorded by using flow-injection technique. Results: Metformin decreased glycogen synthesis in a dose-dependent manner with an IC50 value of 196.5 micro m. This effect could not be reversed by the presence of the glycogen phosphorylase inhibitor DAB, suggesting that glycogenolysis was not affected. A clear correlation between glucose production and glycogen content (0.97 < R < 0.99; p < 0.001) and lactate production and glycogen content (0.97 < R < 0.99; p < 0.001) was observed. Moreover, a strong inhibition (62%, p < 0.001) of glucose produced from lactate/pyruvate (3 mm/0.3 mm) was observed in cells treated with 350 micro m metformin. Conclusion: Hepatocytes preincubated for 24 h in the presence of metformin at clinically relevant concentrations showed impaired glycogenesis as well as gluconeogenesis.[11]

C6H16N6O2

204.23

121369-64-0

44520175

Typically exists as solids at room temperature

0

152.31

5

4

3

14

175

0

OC(CN)=O.N(C(=N)/N=C(\N)/N)(C)C

1,1-Dimethylbiguanide (glycinate)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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;
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。