Complex I of the mitochondrial respiratory chain (inhibition) [1]
REDD1 protein (activation) [1]
Insulin receptor substrate (IRS) receptors (inhibition) [1]
AMP-activated protein kinase (AMPK) (activation) [1]
mTOR pathway (blocking) [1]

In HPV+ head and neck cancer models, the EC50 of phenformin for inducing cell death was 840 times lower than that of metformin [1].
In cholangiocarcinoma (CCA) cells, phenformin inhibits cell growth. It also induces apoptosis and autophagy in these cells [1].
In ovarian cancer (OC) cell lines (SKOV3, IGROV-1, and Hey), phenformin inhibits cell proliferation and induces G1 cell cycle arrest and apoptosis. An Annexin V assay showed that phenformin treatment induces G0/G1 cell cycle arrest and decreases the S phase in a dose-dependent manner [1].
In melanoma, phenformin strongly reduces the viability, growth, and invasion of melanoma cells by inducing apoptosis in both two-dimensional and three-dimensional (spheroid) models. It also decreases CSC selective markers, an effect not observed with metformin [1].
In glioblastoma stem cells (GSCs), phenformin inhibits self-renewal and induces cell death at higher concentrations. GSCs appear much more sensitive to phenformin than non-stem glioma cells, showing more growth arrest and induction of apoptosis [1]. This effect is mediated by the upregulation of let-7, miR-137, and miR-124 and the downregulation of HMGA2 [1].
Phenformin inhibits the mitochondrial complex I, increasing the cellular AMP (and ADP) to ATP ratio, leading to the overproduction of reactive oxygen species (ROS) [1].
AMPK activation by phenformin leads to the inhibition of mTOR pathways via TSC2 protein activation, resulting in S6K protein inhibition and 4E-BP1 protein activation, which decreases tumor cellular growth [1].
Phenformin blocks the IR/IGF1R transduction and reduces the expression of proteins downstream such as ErbB2, AKT, ERK, and mTOR [1].
Phenformin inhibits CDK4 and D glycine and activates p21 proteins [1].
Phenformin depletes nucleotide triphosphates (NTP), potentially preventing nucleotide synthesis [1].

In breast cancer xenografts in vivo, phenformin demonstrates potential as an antineoplastic agent in both estrogen receptor (ER)-positive MCF-7 and triple-negative MDA-MB-231 models [1].
In cholangiocarcinoma (CCA), phenformin inhibits tumor growth in vivo [1].
In glioma xenografts, phenformin prolongs the survival of mice, especially when combined with dichloroacetate (DCA) [1].
In a syngeneic mouse model using several tumor cell lines, a phenformin/oxamate combination induced a reduced tumor size and increased apoptosis compared to controls [1].
In a mouse model of serous ovarian cancer, phenformin showed anti-tumorigenic effects [1].

Cell Proliferation and Viability Assay: The effect of phenformin on cell proliferation was assessed in various cancer cell lines, including ovarian cancer (SKOV3, IGROV-1, Hey), breast cancer (MCF-7, MDA-MB-231), head and neck cancer, cholangiocarcinoma, and melanoma. Cells were treated with increasing concentrations of phenformin, and cell viability was measured using standard assays (e.g., MTT, CellTiter-Glo) after a specified period (e.g., 48-72 hours). The results demonstrated a dose-dependent inhibition of cell proliferation, with phenformin being significantly more potent than metformin [1].
Cell Cycle Analysis: Ovarian cancer cells (SKOV3, IGROV-1, Hey) were treated with phenformin for a set time. Cells were then fixed, stained with propidium iodide (PI), and analyzed by flow cytometry. The assay revealed that phenformin treatment induced G0/G1 phase cell cycle arrest and decreased the percentage of cells in the S phase in a dose-dependent manner [1].
Apoptosis Assay: Apoptosis induction by phenformin was evaluated using an Annexin V-FITC/PI staining kit followed by flow cytometry. Cancer cells (e.g., ovarian, melanoma) treated with phenformin showed a significant increase in the percentage of Annexin V-positive cells, indicating apoptotic cell death [1].
Western Blot Analysis: To investigate the mechanism of action, whole-cell lysates from phenformin-treated and control cells were subjected to Western blot analysis using specific antibodies. Key findings include: phenformin treatment led to increased phosphorylation of AMPK and its substrate ACC, indicating AMPK activation. It also resulted in decreased phosphorylation of S6K and 4E-BP1, demonstrating mTOR pathway inhibition. Furthermore, phenformin reduced the expression of proteins like ErbB2, AKT, ERK, and CDK4, while increasing p21 levels [1].
Cancer Stem Cell (CSC) Assay: The effect of phenformin on CSCs was assessed by measuring the expression of CSC selective markers (e.g., ALDH activity) or by using side population (SP) analysis in lung cancer and sphere formation assays in glioblastoma and melanoma. Phenformin treatment decreased CSC markers and inhibited self-renewal in these models [1].

Breast Cancer Xenograft Model:** Female athymic nude mice were orthotopically implanted with either MCF-7 (ER-positive) or MDA-MB-231 (triple-negative) human breast cancer cells. When tumors became established, mice were randomized to receive either vehicle control or phenformin. Phenformin was administered at a specific dose and regimen (details not specified in the text). Tumor growth was monitored by caliper measurements, and the results showed that phenformin significantly inhibited tumor growth in both models compared to the control group [1].
* **Melanoma Xenograft Model:** Mice bearing melanoma xenografts (details not specified) were treated with phenformin alone, a BRAF inhibitor (e.g., PLX4720) alone, or a combination of both. Phenformin was administered via oral gavage (PO) at a specified dose (e.g., a range of doses) and frequency (e.g., once or twice daily). Tumor size and mouse survival were monitored. The combination therapy showed enhanced anti-tumor efficacy compared to either single agent alone [1].
* **Glioblastoma Xenograft Model:** Nude mice were intracranially implanted with glioblastoma stem cells (GSCs). Mice were then treated with phenformin, temozolomide (TMZ), or a combination of both. Phenformin was administered via oral gavage. The combination treatment synergistically induced GSC death and prolonged the survival of mice [1].

---

Breast Cancer Xenograft Model: Female athymic nude mice were orthotopically implanted with either MCF-7 (ER-positive) or MDA-MB-231 (triple-negative) human breast cancer cells. When tumors became established, mice were randomized to receive either vehicle control or phenformin. Phenformin was administered at a specific dose and regimen (details not specified in the text). Tumor growth was monitored by caliper measurements, and the results showed that phenformin significantly inhibited tumor growth in both models compared to the control group [1].
Melanoma Xenograft Model: Mice bearing melanoma xenografts (details not specified) were treated with phenformin alone, a BRAF inhibitor (e.g., PLX4720) alone, or a combination of both. Phenformin was administered via oral gavage (PO) at a specified dose (e.g., a range of doses) and frequency (e.g., once or twice daily). Tumor size and mouse survival were monitored. The combination therapy showed enhanced anti-tumor efficacy compared to either single agent alone [1].
Glioblastoma Xenograft Model: Nude mice were intracranially implanted with glioblastoma stem cells (GSCs). Mice were then treated with phenformin, temozolomide (TMZ), or a combination of both. Phenformin was administered via oral gavage. The combination treatment synergistically induced GSC death and prolonged the survival of mice [1].

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.

---

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 (free and glucuronide-bound) 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.

---

Phenformin is more lipophilic than metformin, which gives it a high affinity for mitochondria membranes [1].
Due to its lipophilic nature, phenformin can pass through the cellular membrane freely and does not require organic cation transporters (OCTs) for cellular uptake, unlike metformin [1]. This allows for a higher concentration of phenformin inside tumor cells and makes it effective in tumors with no OCT overexpression [1].
Phenformin has greater tissue bioavailability compared to metformin [1].

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.

---

Phenformin was withdrawn from the market in the late 1970s due to its high risk of causing fatal lactic acidosis [1].
The lipophilic structure of phenformin gives it a high affinity for mitochondria membranes, leading to interference with oxidative phosphorylation and causing lactic acidosis [1].
Phenformin is nearly 50 times more potent than metformin but is also 20 times more likely to cause lactic acidosis [1].

[1]. Phenformin as an Anticancer Agent: Challenges and Prospects. Int J Mol Sci. 2019 Jul 5;20(13):3316.

[2]. A review of phenformin, metformin, and imeglimin. Drug Dev Res. 2020 Jun;81(4):390-401.

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.

---

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.

---

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.

---

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 II diabetes who do not have liver, kidney, or cardiovascular disease and cannot control their blood sugar through diet.

---

Phenformin is an oral biguanide drug originally used to treat type 2 diabetes mellitus [1].
Phenformin acts as an antitumor agent not only by inducing hypoglycemia via AMPK activation but also as a blocker of the mTOR regulatory complex [1].
The addition of phenformin eliminates resistance to antiangiogenic tyrosine kinase inhibitors (TKI) [1].
Phenformin is being investigated as an anticancer agent, especially in combination with other therapies. A Phase I clinical trial (NCT03026517) at the Memorial Sloan Kettering Cancer Center is testing the safety of phenformin in combination with standard chemotherapy (dabrafenib plus trametinib) in patients with BRAF-mutated melanoma [1].
Phenformin shows potential in targeting Cancer Stem Cells (CSCs) due to its ability to inhibit oxidative phosphorylation (OXPHOS), which is often upregulated in CSCs [1].
Proposed combination therapies with phenformin include: with oxamate/dichloroacetate (DCA) to induce metabolic catastrophe; with BRAF inhibitors (e.g., PLX4720, vemurafenib) to overcome resistance in melanoma; with ERK inhibitors (e.g., SCH772984) for NF1-mutant cancer; and with temozolomide (TMZ) for glioblastoma treatment [1].

C10H15N5

205.27

205.132

114-86-3

Phenformin hydrochloride;834-28-6

8249

White to off-white solid powder

1.2±0.1 g/cm3

332.2±35.0 °C at 760 mmHg

280-282°C

154.7±25.9 °C

0.0±0.7 mmHg at 25°C

1.620

-0.6

3

1

4

15

236

0

ICFJFFQQTFMIBG-UHFFFAOYSA-N

InChI=1S/C10H15N5/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)

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

BRN 1977317 Azucaps DebeonePhenformin Insoral

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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