AMPK; Autophagy; Mitophagy

Metformin inhibits Bcl-2 and Bcl-xl, upregulates BAX activation with facilitation of BIM, BAD, and PUMA, and induces release of cytochrome c from mitochondria into the cytoplasm, directly inducing caspase-9-mediated mitochondrial apoptosis.[4]

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Metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.[1]

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Metformin, widely recommended in the management of T2DM, exerts its pleiotropic effects via 5'-AMP-activated protein kinase (AMPK); however, its effect on mitophagy remains elusive. Recent evidence demonstrates that peripheral blood mononuclear cells (PBMCs) express insulin receptors and the human organic cation transporter protein, and they are extensively being used as a surrogate for examining mitochondrial function in T2DM. Metformin treatment increased the formation of acidic vesicles and mitophagosomes, upregulated mitophagy markers, and enhanced mitophagic flux, as indicated by increased LC3-II expression and reduced p62 protein levels. In addition, pretreatment with compound C (an AMPK inhibitor) significantly decreased the expression of mitophagy markers in metformin-treated cells, indicating that metformin induces mitophagy via the AMPK pathway. In conclusion, metformin-induced mitophagy may improve cellular function, including in β cells, by restoring normal mitochondrial phenotype, which may prove beneficial in patients with T2DM and other mitochondrial-related diseases. Moreover, PBMCs may be used as a novel diagnostic biomarker for identifying mitochondrial disorders.[2]

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In this study we revealed for the first time that metformin treatment led to increased apoptosis in human lung cancer cell lines A549 and NCI-H1299 and significantly inhibited the cells proliferation in a dose- and time-dependent manner, which was further demonstrated by the data obtained from A549 tumor xenografts in nude mice. We also found that metformin treatment can activate AMP-activated protein kinase, JNK/p38 MAPK signaling pathway and caspases, as well as upregulate the expression of growth arrest and DNA damage inducible gene 153 (GADD153). Either blockade of JNK/p38 MAPK pathway or knockdown of GADD153 gene abrogated the apoptosis-inducing effect of metformin. Taken together, our data suggest that metformin inhibits the growth of lung cancer cells and induces apoptosis through activating JNK/p38 MAPK pathway and GADD153.[3]

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This study demonstrates that metformin decreased the number of activated-HSCs through induction of apoptosis, but did not impact numbers of hepatocytes. Metformin upregulated BAX activation with facilitation of BIM, BAD and PUMA; downregulated Bcl-2 and Bcl-xl, but did not affect Mcl-1. Additionally, metformin induced cytochrome c release from mitochondria into the cytoplasm, directly triggering caspase-9-mediated mitochondrial apoptosis. The decline in mitochondrial membrane potential (ΔΨm) and deposition of superoxide in mitochondria accelerated the destruction of the integrity of mitochondrial membrane. Moreover, we verified the therapeutic effect of metformin in our mouse model of liver fibrosis associated with nonalcoholic steatohepatitis (NASH) in which hepatic function, NASH lesions and fibrosis were improved by metformin. In conclusion, this study indicated that metformin has significant therapeutic value in NASH-derived liver fibrosis by inducing apoptosis in HSCs, but does not affect the proliferation of hepatocytes [4].

Metformin exhibits a therapeutic effect in a mouse model of nonalcoholic steatohepatitis (NASH)-related liver fibrosis, resulting in improvements in hepatic function, NASH lesions, and fibrosis.[4]

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Assessment of Metformin’s in vivo effects.[1]
To assess whether selected effects of Metformin described above also occurred in vivo, SD rats were studied (Table 1). Rats were orally dosed with metformin or vehicle (H2O) for 5 days. Rats were starved for 20 hours and then re-fed for 2 hours before the final dose. Four hours after the final dose, tissue and blood samples were obtained for analysis (see Methods). During starvation, there should be very little lipid synthesis. Upon refeeding, hepatic lipid synthesis should be dramatically induced. Metformin’s effects were examined under re-fed conditions. Along with modest decreases in plasma insulin and triglycerides, a small, but significant increase in β-hydroxybutyrate was present, suggesting that hepatic fatty acid oxidation was induced in metformin-treated rats. Furthermore, metformin treatment produced significant decreases in hepatic expression of mRNAs for SREBP-1, FAS, and S14 that were consistent with effects documented in cells (Table 1).
The mature SREBP-1 protein in rat liver nuclear extracts was examined using an anti-SREBP1 Ab (Figure 5b). As anticipated, SREBP-1 mature-form protein was not detected in hepatic nuclear extracts from starved animals. In re-fed animals, mature SREBP-1 protein had accumulated consistent with an increase in lipid synthesis under this condition. Treatment with Metformin prevented this accumulation. Additional results obtained using hepatic nuclear extracts from re-fed rats after treatment with AICAR (500 mg/kg/day) also showed that the presence of SREBP-1 mature-form protein was ablated.
Measurement of AMPK activation in liver ex vivo is difficult because brief hypoxia is known to produce marked activation of the enzyme. Thus we used liver tissue derived from metformin-treated rats to determine that ACC activity was decreased significantly at several tested citrate concentrations (Figure 6). The greatest ACC activity reduction was at a citrate concentration of 1 mM (from 54.6 ± 11.8 to 35.6 ± 7.7 nmol/mg/min; P < 0.01). These results are consistent with metformin having produced in vivo AMPK activation and ACC inactivation.

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Metformin relieved the lesions in liver of nonalcoholic steatohepatitis (NASH)-associated fibrosis mice models [4]
A NASH-associated fibrosis mice model (Fig. 7a) was established to explore whether Metformin can protect liver under disease state in vivo. The weight and biochemical indicators (ALT and AST) of each mouse were measured every other week and every three weeks, respectively. The results showed no significant increase in the weight of HFMCD-diet group relative to MCS-diet group or AIN93 group (Fig. S2a). Moreover, in HFMCD-diet therapeutic treatment group, metformin decreased the both levels of AST and ALT and liver index, compared with MCS-diet group or AIN93 group (Fig. 7b, Fig. S2b). Collectively, metformin improved the liver function in the NASH-associated fibrosis mice model.
Paraffin-embedded liver tissue samples obtained from NASH-fibrosis models were stained by hematoxylin and eosin to visualize the effect of Metformin on hepatic lesions. The color of fresh liver tissues in HFMCD group slanted dark (Fig. S2c). Results showed that the liver in the AIN93 or MCS group exhibited no obvious lesions and were not affected by metformin (Fig. S2d). However, the area of steatosis and the amounts of inflammatory cells both decreased with Metformin administration in HFMCD-preventive and therapeutic group (Fig. 7c), indicating a decrease in the severity of NASH lesions. Moreover, the extent of fibrosis was observed by V-G staining. Results showed that collagen accumulation in HFMCD-therapeutic group was much less than that in the HFMCD-diet saline group (Fig. 7d). Masson staining also observed the same results (Fig. S2e). In addition, immunohistochemical staining showed that, the positive rate of cleaved caspase-3 of HSCs in metformin-therapeutic group was 38.7 ± 5.2%, which was higher than that in saline group (7.69 ± 0.61%), blank control (0.58 ± 0.03%), and metformin-therapeutic group (1.82 ± 0.05%), indicating that metformin induced apoptosis in HSCs (Fig. 7e, Table 3). Therefore, metformin delayed fibrosis in mice models.

This study demonstrates that metformin decreased the number of activated-HSCs through induction of apoptosis, but did not impact numbers of hepatocytes. Metformin upregulated BAX activation with facilitation of BIM, BAD and PUMA; downregulated Bcl-2 and Bcl-xl, but did not affect Mcl-1. Additionally, metformin induced cytochrome c release from mitochondria into the cytoplasm, directly triggering caspase-9-mediated mitochondrial apoptosis. The decline in mitochondrial membrane potential (ΔΨm) and deposition of superoxide in mitochondria accelerated the destruction of the integrity of mitochondrial membrane. Moreover, we verified the therapeutic effect of metformin in our mouse model of liver fibrosis associated with nonalcoholic steatohepatitis (NASH) in which hepatic function, NASH lesions and fibrosis were improved by metformin. In conclusion, this study indicated that metformin has significant therapeutic value in NASH-derived liver fibrosis by inducing apoptosis in HSCs, but does not affect the proliferation of hepatocytes [4].

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Immunoprecipitation-AMPK assay.[1]
Ten micrograms of 35% ammonium sulfate precipitate (containing AMPK) from AICAR- or Metformin-treated rat hepatocytes was immunoprecipitated using polyclonal Ab’s raised against AMPKα1 (NH2-DFYLATSPPDSFLDDHHLTR-OH) or AMPKα2 (NH2-MDDSAMHIPPGLKPH-OH), followed by AMPK assay. Measurements of muscle AMPK activity and glucose uptake.[1]
Isolated rat epitrochlearis muscles were incubated for 3 hours with Metformin (2 mM) or control medium followed by measurement of AMPKα1 or AMPKα2 activities as described. For glucose uptake, insulin (300 nM) was present where indicated for the last 30 minutes of the 3-hour incubation. Then, 3-0-methylglucose uptake was measured using a 10-minute incubation in the absence or presence of metformin and/or insulin as described previously. Caspases enzymatic activity assays [4]
Caspase 1, 3, 8 and 9 enzymatic activity were measured with caspase activity assay kit following the instructions of manufacture. 2 × 105 cells were lysed on the ice for 15 min and substrates were added sequentially. The enzymatic activity was measured with a microplate reader (λ = 405 nm).

Rat HSCs T6, human hepatocyte L02, and rat hepatocyte BRL-3A were maintained in DMEM, which is supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin sulfate at 37 °C in a humidified 5% CO2 incubator. The human hepatic stellate cells (HSCs) LX-2 are maintained in RPMI 1640 medium. Filtered through millipore membrane filters with a 0.22 m pore size are solutions of metformin and AICAR. The CCK8 assay is used to measure the effects of various metformin and AICAR concentrations on the growth of hepatic stellate cells. Following a 24-hour treatment with varying doses of metformin and AICAR, the expression of collagen and -SMA protein is discovered using a Western blot. After giving cells 10 mM metformin and 0.5 mM AICAR for 24 hours, Western blot is used to identify the expression of collagen and the -SMA protein. RT-qPCR is used to find the mRNA levels of collagen I and -SMA.

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Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes. [1]
Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion. For the AMPK assay, cells were seeded in six-well plates at 1.5 × 106 cells/well in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 μg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide riboside (AICAR), or Metformin at concentrations indicated. For a 39-hour treatment, cells for both control and Metformin (10 or 20 μM) groups were cultured in DMEM plus 5% FBS and 100 nM insulin, and the fresh control and Metformin-containing medium were replaced every 12 hours (last medium change was 3 hours before harvest). After treatment, the cells were directly lysed in digitonin-containing and phosphatase inhibitor–containing buffer A, followed by precipitation with ammonium sulfate at 35% saturation. AMPK activity was determined by measurement of phosphorylation of a synthetic peptide substrate, SAMS (HMRSAMSGLHLVKRR). For ACC assay, the 35% ammonium sulfate precipitate from digitonin-lysed hepatocytes (4 μg each) was used for determination of ACC activity via 14CO2 fixation in the presence of 20 mM citrate as done previously. For fatty acid oxidation, the oxidation of 14C-oleate to acid-soluble products was performed as done previously, but in medium M199 in the absence of albumin.

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

Typically exists as solids at room temperature

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

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.)