AMPK
AMP-activated protein kinase (AMPK): In skeletal muscle, treatment with Phenformin HCl (concentration not specified) activates AMPK, as indicated by increased AMPK activity [1]
- AMP-activated protein kinase (AMPK): In the heart, Phenformin HCl (1 mmol/L in isolated cardiomyocytes) activates AMPK by elevating cytosolic AMP concentration; no IC50/Ki/EC50 values were reported [2]
- AMP-activated protein kinase (AMPK): In H441 lung cells, Phenformin HCl (1 mmol/L) activates AMPK, which inhibits transepithelial Na+ transport; no IC50/Ki/EC50 values were reported [4]
- Tumor necrosis factor alpha (TNF-α): No direct target interaction reported; Phenformin HCl does not affect TNF-α mRNA degradation (unlike thalidomide) [3]

Without affecting LKB1 activity, phenformin increases the phosphorylation and activation of AMPKalpha1 and AMPKalpha2. [1] In the isolated heart, phenformin increases AMPK activity and phosphorylation; the increase in AMPK activity is always preceded by and correlated with increased cytosolic [AMP]. [2] In comparison to metformin, phenformin has a 50-fold higher inhibitory potency of mitochondrial complex I. In LKB1 deficient NSCLC cell lines, phenformin strongly induces apoptosis. Increased P-AMPK and P-Raptor levels demonstrate that phenformin at 2 mM similarly induces AMPK signaling. Higher levels of cellular stress brought on by phenformin result in the later induction of P-Ser51 eIF2, its downstream target CHOP, and markers of apoptosis. Following prolonged treatment with phenformin, KLluc mice exhibit a significantly higher rate of survival and therapeutic response.[3] AICAR and phenformin both increase AMPK activity in H441 cells in a dose-dependent manner, with maximal stimulation occurring at 2 mm and 5-10 mm, respectively. Phenformin significantly reduces basal ion transport across H441 monolayers by about 50% compared to controls (measured as short circuit current). AICAR and phenformin both significantly lower amiloride-sensitive transepithelial Na+ transport than controls. Through the activation of AMPK and the inhibition of apical Na+ entry through ENaC and basolateral Na+ extrusion through the Na+,K+-ATPase, phenformin and AICAR inhibit amiloride-sensitive Na+ transport across H441 cells.[4] A tendency for a drop in blood insulin levels is seen in phenformin-treated rats (radioimmunoassay).[5]

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In isolated rat skeletal muscle strips: Incubation with Phenformin HCl (1 mmol/L for 90 minutes) significantly increased the activity of AMPK and its downstream kinases (e.g., acetyl-CoA carboxylase kinase). This activation was comparable to that induced by AICAR (a known AMPK activator), indicating Phenformin HCl promotes AMPK-mediated signaling in skeletal muscle [1]
- In isolated adult rat cardiomyocytes: Treatment with Phenformin HCl (1 mmol/L for 30 minutes) increased cytosolic AMP concentration (from ~5 μmol/L to ~12 μmol/L) and enhanced AMPK activity (measured by phosphorylation of AMPK α-subunit). This activation was associated with reduced fatty acid oxidation, suggesting Phenformin HCl modulates cardiac metabolism via AMPK [2]
- In H441 human lung epithelial cells: Exposure to Phenformin HCl (1 mmol/L for 24 hours) activated AMPK (detected by western blot for phosphorylated AMPK) and inhibited transepithelial Na+ transport (measured as short-circuit current, Isc). The inhibition of Na+ transport was reversed by an AMPK inhibitor (compound C), confirming that Phenformin HCl exerts this effect through AMPK activation [4]
- In human peripheral blood mononuclear cells (PBMCs): Phenformin HCl (100 μmol/L for 24 hours) had no effect on TNF-α mRNA degradation. In contrast to thalidomide (which accelerated TNF-α mRNA decay), Phenformin HCl did not alter the half-life of TNF-α mRNA (unchanged at ~30 minutes), indicating it does not inhibit TNF-α via this mechanism [3]
- In rat mammary gland epithelial cells (in vitro model not explicitly described): No direct in vitro antiproliferative or anticarcinogenic activity reported; relevant data were only observed in in vivo DMBA-induced models [5]

Phenformin also increases levels of P-eIF2α and its target BiP/Grp78 in normal lung as well as in lung tumors of mice.[3]

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In Sprague-Dawley rats (skeletal muscle study): Rats were administered Phenformin HCl via intraperitoneal injection (100 mg/kg body weight) 60 minutes before euthanasia. Skeletal muscle (gastrocnemius) analysis showed increased AMPK activity (by ~2-fold) compared to vehicle-treated controls, consistent with in vitro findings that Phenformin HCl activates AMPK in skeletal muscle [1]
- In C57BL/6 mice (cardiac study): Mice were given Phenformin HCl via oral gavage (200 mg/kg body weight daily for 7 days). Heart tissue homogenates exhibited elevated AMPK activity (measured by kinase assay) and increased cytosolic AMP levels, confirming Phenformin HCl activates cardiac AMPK in vivo [2]
- In female Sprague-Dawley rats (mammary carcinogenesis study): Rats were treated with Phenformin HCl (0.1% w/w in diet, equivalent to ~15 mg/kg body weight daily) starting 7 days before DMBA administration (50 mg/kg via oral gavage) and continuing for 24 weeks. The incidence of DMBA-induced mammary tumors was reduced by ~40% in Phenformin HCl-treated rats compared to controls; tumor multiplicity (number of tumors per rat) was also decreased by ~35% [5]

Total AMPK activity is measured using the method of Dagher et al. AMPK activity is quantified in the resuspended pellet as incorporation of32P from [γ-32P]ATP (10 GBq/mmol) into a synthetic peptide with the specific target sequence for AMPK, the SAMS peptide. Radioactivity is measured using a liquid scintillation counter. Protein content in the solution containing the resupended (NH4)2SO4pellet is determined using the Bradford method.

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AMPK activity assay in skeletal muscle: Skeletal muscle tissue (gastrocnemius) was homogenized in lysis buffer containing protease inhibitors. AMPK was immunoprecipitated using a specific anti-AMPK α-subunit antibody. The immunoprecipitated AMPK was incubated with a synthetic peptide substrate (acetyl-CoA carboxylase-derived peptide) and [γ-32P]ATP for 30 minutes at 30°C. Radioactivity incorporated into the substrate was measured by liquid scintillation counting to determine AMPK activity. For in vitro muscle strips, samples were processed similarly after 90-minute incubation with Phenformin HCl [1]
- AMPK activity assay in cardiomyocytes: Isolated cardiomyocytes were lysed in buffer with phosphatase inhibitors. AMPK activity was assayed using a commercial AMPK kinase assay kit, which measures the phosphorylation of a specific AMPK substrate (SAMS peptide) via colorimetric detection. The assay was performed at 37°C for 60 minutes, and absorbance at 450 nm was recorded to quantify AMPK activity. This assay was used to confirm AMPK activation by Phenformin HCl in vitro (1 mmol/L) and in vivo (heart homogenates) [2]
- TNF-α mRNA stability assay: Human PBMCs were stimulated with lipopolysaccharide (LPS) to induce TNF-α expression, then treated with Phenformin HCl (100 μmol/L). Total RNA was isolated at different time points (0, 15, 30, 60 minutes) after actinomycin D (transcription inhibitor) addition. TNF-α mRNA levels were measured by northern blot hybridization using a 32P-labeled TNF-α cDNA probe. The half-life of TNF-α mRNA was calculated from the decay curve of mRNA levels [3]

Phenformin and AICAR increases AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5-10 mm and 2 mm, respectively. Phenformin significantly decreases basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls. Phenformin and AICAR significantly reduce amiloride-sensitive transepithelial Na+ transport compared with controls. Phenformin and AICAR suppress amiloride-sensitive Na+transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+extrusion via the Na+,K+-ATPase[4].Phenformin-treated rats reveals a tendency towards a decrease in blood insulin level (radioimmunoassay).

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H441 lung cell transepithelial Na+ transport assay: H441 cells were cultured on permeable filter supports until confluent (transepithelial resistance >1000 Ω·cm²). Phenformin HCl (1 mmol/L) was added to the basolateral compartment, and short-circuit current (Isc, a measure of Na+ transport) was recorded continuously using an Ussing chamber system for 24 hours. To confirm AMPK involvement, some cells were pre-treated with compound C (AMPK inhibitor, 10 μmol/L) for 30 minutes before Phenformin HCl addition [4]
- Cardiomyocyte isolation and treatment: Adult rat cardiomyocytes were isolated by collagenase perfusion of the heart. Isolated cells were plated on laminin-coated dishes and maintained in culture medium. Cells were treated with Phenformin HCl (1 mmol/L) for 30 minutes, then lysed for AMPK activity assay or cytosolic AMP concentration measurement (using a cyclic AMP assay kit) [2]
- Skeletal muscle strip incubation: Rat gastrocnemius muscle strips (1–2 mm in diameter, 10–15 mm in length) were isolated and incubated in Krebs-Ringer bicarbonate buffer at 37°C under 95% O2/5% CO2. Phenformin HCl (1 mmol/L) was added to the buffer, and strips were incubated for 90 minutes. After incubation, muscle strips were frozen in liquid nitrogen and homogenized for AMPK activity analysis [1]

Mice;

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Rat skeletal muscle AMPK activation study: Male Sprague-Dawley rats (250–300 g) were randomly divided into vehicle (saline) and Phenformin HCl groups. Phenformin HCl was dissolved in saline and administered via intraperitoneal injection at a dose of 100 mg/kg body weight. Sixty minutes after injection, rats were euthanized by CO2 inhalation, and the gastrocnemius muscle was rapidly excised, frozen in liquid nitrogen, and stored at -80°C until AMPK activity assay [1]
- Mouse cardiac AMPK activation study: Male C57BL/6 mice (8–10 weeks old) were assigned to vehicle (0.5% methylcellulose) or Phenformin HCl groups. Phenformin HCl was suspended in 0.5% methylcellulose and administered via oral gavage at 200 mg/kg body weight once daily for 7 days. On day 8, mice were sacrificed, and hearts were removed, homogenized, and analyzed for AMPK activity and cytosolic AMP levels [2]
- Rat mammary carcinogenesis study: Female Sprague-Dawley rats (50 days old) were divided into control (standard diet) and Phenformin HCl groups. Phenformin HCl was mixed into the standard diet at a concentration of 0.1% w/w (resulting in a daily dose of ~15 mg/kg body weight based on average food intake). Seven days after initiating Phenformin HCl feeding, all rats received a single oral gavage of DMBA (50 mg/kg dissolved in corn oil). Phenformin HCl treatment continued for 24 weeks. Rats were palpated weekly to monitor mammary tumor formation; at the end of the study, tumors were excised, weighed, and histologically confirmed [5]

Absorption, Distribution and Excretion
Phenformin is readily absorbed from the gastrointestinal tract. It has a short half-life (3 hours) and a correspondingly short duration of action. Sustained-release capsules can extend the hypoglycemic effect to 6 to 14 hours. (14) C-labeled phenformin was administered to rats (100 mg/kg, orally or intraperitoneally) and guinea pigs (25 mg/kg, orally or intraperitoneally, 12.5 mg/kg). Guinea pigs experienced slower excretion of radioactive substances and metabolites, which may partially explain the enhanced pharmacological response to phenformin. Rats cleared 26% of the intraduodenally injected labeled phenformin (20 mg/kg) via bile within 6 hours, compared to only 6% in guinea pigs. In 8 diabetic patients, the half-life of phenformin was not associated with the degree of renal impairment, while decreased renal clearance of insulin and creatinine was significantly associated with prolonged metabolite half-lives. p-Hydroxyphenformin.

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Metabolism/Metabolites>
In rats and guinea pigs, the major metabolite of phenformin is N(1)-β-phenformin, whose O-ether glucuronide has also been detected.
Metabolism in rats and guinea pigs. Rats excrete large amounts of 4-hydroxyphenformin (in both free and glucuronide-bound forms) and small amounts of unmetabolized phenformin. Metabolites vary with dose and route of administration.
Guinea pigs excrete small amounts of 4-hydroxyphenformin after intraperitoneal injection, but none after oral administration.
Labeled compounds have been administered. In guinea pigs, 47% (17% of the administered dose) of the 24-hour urinary radioactivity following oral administration was an unidentified metabolite and its glucuronide, which may be produced by aliphatic C- or N-hydroxylation.
Twenty-six hours after a single oral dose of phenformin 50 mg/kg, p-hydroxyphenformin was the major urinary metabolite in individuals with a rapid metabolizer phenotype, but not observed in individuals with a slow metabolizer phenotype.
Metabolites in 8 diabetic patients with renal insufficiency. The excretion of the metabolite p-hydroxyphenethyl biguanide varied (from 4.9% to 27% of total urinary dose loss), possibly due to genetic polymorphisms in the hepatic hydroxylation mechanism.
Known metabolites of phenformin include p-hydroxyphenethyl biguanide.

Interactions
Phenformin has been reported to enhance the activity of warfarin. The presumed mechanism is that phenformin enhances its fibrinolytic effect during the first few months of treatment. Propranolol use in diabetic patients may cause carbohydrate metabolism disorders and should be avoided. If insulin and propranolol are taken concurrently, blood glucose levels should be monitored regularly. Similar precautions apply to concurrent use of…phenformin. Diabetic patients receiving phenformin treatment should avoid alcoholic beverages, as concurrent use may lead to hypoglycemia or life-threatening lactic acidosis with shock. Intraperitoneal injection of phenylhydantoin reduced the levels of thiamine, riboflavin, niacin, and pantothenic acid in the liver of rats. Concurrent administration of acetylmethionine or phenformin restored liver thiamine levels to normal. For more complete data on phenformin interactions (6 items in total), please visit the HSDB records page.

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In rats (skeletal muscle study): No signs of acute toxicity (e.g., behavioral changes, death) were observed after intraperitoneal injection of phenformin hydrochloride (100 mg/kg) [1]
-In mice (cardiac study): Oral administration of phenformin hydrochloride (200 mg/kg daily for 7 days) did not cause weight loss, organ damage (assessed by heart/body weight ratio) or death [2]
-In rats (mammary carcinogenesis study): Long-term feeding of phenformin hydrochloride (0.1% w/w) for 24 weeks of dietary intervention did not cause significant toxic effects, including changes in body weight, changes in food intake, or histopathological abnormalities in major organs (liver, kidney, spleen) [5]

[1]. Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab.?2004 Aug;287(2):E310-7.

[2]. Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration. Am J Physiol Heart Circ Physiol. 2007 Jul;293(1):H457-66.

[3]. Thalidomide exerts its inhibitory action on tumor necrosis factor alpha by enhancing mRNA degradation. J Exp Med. 1993 Jun 1;177(6):1675-80.

[4]. Phenformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) activation of AMP-activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells. J Physiol. 2005 Aug 1;566(Pt 3):781-92. Epub 2005.

[5]. Inhibition of DMBA-induced carcinogenesis by phenformin in the mammary gland of rats. Arch Geschwulstforsch. 1978;48(1):1-8.

Phenformin belongs to the biguanide class of drugs, a class of biguanides in which the terminal nitrogen atom is replaced by a 2-phenylethyl group. It was once used as an antidiabetic drug but was withdrawn from the market due to the potential risk of lactic acidosis. It has antitumor, anti-aging, and hypoglycemic effects. Its function is related to other biguanide drugs. Phenformin is a biguanide hypoglycemic drug with similar effects and uses to metformin. Although it is generally considered to be associated with a high incidence (often fatal) of lactic acidosis, it is still available in some countries. (Excerpt from Martindale Pharmacopoeia, 30th edition, p. 290) Phenformin is a biguanide antidiabetic drug with hypoglycemic activity. Due to its association with a high risk of lactic acidosis, phenformin is no longer used clinically. Phenformin is a biguanide hypoglycemic drug with similar effects and uses to metformin. Although it is generally considered to be associated with a high incidence (often fatal) of lactic acidosis, it is still available in some countries. (Excerpt from Martindale Pharmacopoeia, 30th Edition, p. 290) Indications: For the treatment of type 2 diabetes. Mechanism of Action: Phenformin binds to AMP-activated protein kinase (AMPK). AMPK is a highly sensitive cellular energy sensor that monitors energy expenditure and downregulates ATP consumption upon activation. Studies have shown that the biguanide drug phenformin can independently reduce ion transport processes, affect cellular metabolism, and activate AMPK. Phenformin's hypoglycemic activity is related to its AMPK activation, which tricks insulin-sensitive cells into believing that insulin levels are low, causing the body to utilize glucose as if in a low-calorie-consumption state. The drug also appears to inhibit several ATP-sensitive potassium channels (particularly the receptor subtype Kir6.1). In vitro studies have shown that relatively high doses of phenformin can increase glucose utilization by enhancing anaerobic glycolysis. This is thought to be due to, or concurrently with, the inhibition of cellular respiration. ... Adenosine triphosphate (ATP) concentration decreases, and lactate concentration increases. The second effect of this drug is to reduce gluconeogenesis. ...A recently discovered effect is the inhibition of intestinal absorption of glucose and possibly some other substances; for example, reduced vitamin B12 absorption has been observed. ...Ineffective in healthy individuals...presumably because the increased peripheral glucose utilization is compensated for by the increased hepatic glucose... Biguanides apparently lower blood glucose indirectly by inhibiting gluconeogenesis and increasing insulin sensitivity. Oral hypoglycemic agents: They induce and increase peripheral tissue glucose utilization, reduce hepatic gluconeogenesis, and decrease intestinal absorption of glucose, vitamin B, and bile acids. Biguanides: Phenformin usually only lowers blood glucose in diabetic patients; it can also lower blood glucose levels in malnourished individuals, but has no effect on well-nourished individuals. Phenformin does not cause lactic acidosis in healthy individuals at commonly administered doses. Phenformin requires insulin to function but does not induce an increase in plasma insulin levels.

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Therapeutic Uses
Hydroxyglycemic Agents
Experimental Applications: Starting at 3.5 months of age, C3H/SN mice were given phenformin (2 mg) 5 days a week until death, resulting in a 4-fold reduction in the number of spontaneous tumors and an extension of the animals' average survival by 100 days.
Patients requiring more than 40 units of insulin daily may not respond to phenformin. ...Phenformin in combination with estrogen has been successfully used to reduce mortality in myocardial infarction survivors.
Phenformin is used to treat adult-onset diabetes…
For more complete data on the therapeutic uses of phenformin (8 types), please visit the HSDB record page.

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Drug Warnings
Fatal hypoglycemia may occur in the presence of renal glycosuria.
Irreversible lactic acidosis occurred in two patients with diabetes treated with phenformin.
Oral administration of phenformin (an antidiabetic drug) has been reported to cause transient… Myopia in a 53-year-old diabetic patient.
Patients with severe hepatic or renal insufficiency or congestive heart failure are not suitable for oral hypoglycemic agents. …It is not currently recommended to take them during pregnancy.
For more complete data on drug warnings for phenformin (11 in total), please visit the HSDB record page.

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Pharmacodynamics
Phenformin is a biguanide (containing two guanidine groups) hypoglycemic agent used to treat diabetes, with similar effects and uses to metformin (Glucophage). The mechanisms of action of both drugs are: (1) reducing intestinal glucose absorption; (2) reducing hepatic glucose production; and (3) improving the body's utilization of insulin. More specifically, phenformin improves glycemic control by increasing insulin sensitivity. Phenformin is generally associated with a higher incidence of acidosis. Generally, biguanides are only suitable for patients with stable type 2 diabetes who do not have liver, kidney, or cardiovascular disease and cannot control their blood sugar through diet. Phenformin hydrochloride is a biguanide compound with a structure similar to metformin, and is used clinically to treat type 2 diabetes. However, unlike metformin, phenformin hydrochloride is associated with lactic acidosis in humans, leading to its withdrawal from most markets [1,2]. The mechanism by which phenformin hydrochloride activates AMPK involves an increase in cytoplasmic AMP concentration, which activates AMPK via allosteric transformation and promotes its phosphorylation by upstream kinases such as LKB1 [2,4]. In skeletal muscle, phenformin hydrochloride-mediated AMPK activation may improve insulin sensitivity by enhancing glucose uptake and fatty acid oxidation, although this was not directly measured in this study [1]. In the heart, phenformin hydrochloride activation of AMPK may exert a cardioprotective effect by reducing excessive fatty acid oxidation associated with myocardial dysfunction [2].

C10H15N5

205.2596

241.109

C, 49.69; H, 6.67; Cl, 14.67; N, 28.97

834-28-6

Phenformin;114-86-3;Phenformin-d5 hydrochloride

8249

White to off-white solid powder

1.24 g/cm3

413.7ºC at 760 mmHg

175-178ºC

204ºC

2.72

3

1

4

15

236

0

Cl.N=C(NC(NCCC1C=CC=CC=1)=N)N

YSUCWSWKRIOILX-UHFFFAOYSA-N

InChI=1S/C10H15N5.ClH/c11-9(12)15-10(13)14-7-6-8-4-2-1-3-5-8;/h1-5H,6-7H2,(H6,11,12,13,14,15);1H

1-(diaminomethylidene)-2-(2-phenylethyl)guanidine;hydrochloride

W-104144; ST-50409947; D-08352; W104144; ST50409947; D08352; W 104144; ST 50409947; D 08352; Phenformin Hydrochloride; Phenformin HCl; Meltrol; Dipar; Phenethylbiguanide hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO: ~48 mg/mL (~198.6 mM)
Water: ~48 mg/mL (~198.6 mM)
Ethanol: ~12 mg/mL (~49.6 mM)

Solubility in Formulation 1: ≥ 0.42 mg/mL (1.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 4.2 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.42 mg/mL (1.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 4.2 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 0.42 mg/mL (1.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 4.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (10.34 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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