Mitochondrial permeability transition pore (PTP).
Mitochondrial complex I (specifically inhibits reverse electron transfer-associated reactive oxygen species production, without inhibiting forward electron transfer or complex I activity). [2]

Complete prevention of cell death induced by tert-butyl hydroperoxide (tBH) was similarly achieved by preincubation with Imeglimin (10 mM, 4 h or 100 μM, 24 h) [2].

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Imeglimin (10 mM for 4h or 100 µM for 24h preincubation) fully prevented tert-butylhydroperoxide (tBH, 0.5 mM)-induced cell death in human microvascular endothelial cells (HMEC-1) as assessed by annexin V/propidium iodide staining. This protective effect was comparable to that of the reference PTP inhibitor cyclosporine A (CsA, 1 µM).
Imeglimin (100 µM for 24h preincubation) fully prevented hyperglycemia (33 mM glucose for 48h)-induced cell death in HMEC-1 cells.
Imeglimin prevented the cytochrome c release from mitochondria to the cytoplasm induced by either tBH exposure or high glucose in HMEC-1 cells, as observed by immunocytochemistry.
Imeglimin (100 µM for 24h) increased the calcium retention capacity (CRC) in digitonin-permeabilized HMEC-1 cells, indicating delayed Ca2+-induced PTP opening. This effect was observed when mitochondria were energized with either complex I (glutamate/malate) or complex II (succinate) substrates.
Imeglimin (100 µM for 24h) delayed tBH-induced PTP opening in intact HMEC-1 cells, as monitored by calcein/cobalt fluorescence quenching.
Imeglimin (10 mM for 4h or 100 µM for 24h) did not inhibit rotenone-sensitive complex I activity (NADH-ubiquinone oxidoreductase activity) in permeabilized HMEC-1 cells.
Imeglimin (10 mM for 4h) did not affect the basal, oligomycin-inhibited, or dinitrophenol (DNP)-uncoupled oxygen consumption rates in intact HMEC-1 cells. It also did not affect lactate production, lactate/pyruvate ratio (cytosolic redox potential), or ATP/ADP ratio (phosphate potential). In contrast, metformin (10 mM for 30min) inhibited oxygen consumption and altered these parameters.
Imeglimin (100 µM for 24h) did not inhibit H2O2 production in permeabilized HMEC-1 cells when mitochondria were energized with complex I substrates (glutamate/malate) under basal conditions or in the presence of rotenone or antimycin A.
Imeglimin (100 µM for 24h) dramatically decreased H2O2 production in permeabilized HMEC-1 cells when mitochondria were energized with the complex II substrate succinate (alone or with glutamate/malate), a condition where ROS production is primarily due to reverse electron transfer through complex I. This inhibitory effect was similar to that of rotenone. [2]

During the final six weeks of HFHSD feeding, meglimin (200 mg/kg b.i.d. orally) dramatically lowers hyperglycemia and returns normal glucose tolerance [1].

For the measurement of respiratory chain complex I activity, confluent HMEC-1 cells were incubated with or without Imeglimin (10 mM for 4h or 100 µM for 24h). Cells were harvested, permeabilized with a digitonin-containing cold buffer, and centrifuged to remove cytosolic contaminants. The permeabilized cell pellet was subjected to a hypotonic shock in water to break mitochondrial membranes. The reaction was performed in a cuvette by adding Tris buffer containing NADH, followed by the initiation of the reaction with decylubiquinone as the final electron acceptor. The oxidation rate of NADH was measured fluorimetrically. The rotenone-sensitive complex I activity was calculated by subtracting the residual NADH oxidation rate after the addition of rotenone. [2]

Cell Viability Assay [2]
Cell Types: Human Endothelial Cells (HMEC-1)
Tested Concentrations: 100 μM and 10 mM
Incubation Duration: 100 μM 24 hrs (hours), 10 mM 4 hrs (hours)
Experimental Results: Prevents cell death.

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For cell death quantification, HMEC-1 cells preincubated with or without Imeglimin (10 mM for 4h or 100 µM for 24h) or CsA (1 µM for 30min) were washed and then exposed to 0.5 mM tBH in serum-free medium for 45 minutes. After washing, cells were incubated in complete medium for 24 hours. Alternatively, cells were exposed to 5.5 mM (control) or 33 mM glucose for 48 hours in the presence or absence of the compounds. Cell death was evaluated by double staining with annexin V-fluorophore conjugate and propidium iodide, followed by flow cytometric analysis.
For cytochrome c immunostaining, cells treated under the above stress conditions with or without Imeglimin were fixed, permeabilized, and blocked. They were then incubated with a primary monoclonal anti-cytochrome c antibody, followed by a fluorescent secondary antibody. Imaging was performed by confocal microscopy.
For PTP opening assessment in permeabilized cells, HMEC-1 cells were permeabilized with digitonin in a sucrose-based medium containing calcium-sensitive fluorescent dye (Calcium Green 5N) and either complex I or II substrates. Calcium pulses were added sequentially, and the extra-mitochondrial calcium concentration was monitored fluorimetrically until a rapid increase indicated PTP opening. The cumulative calcium added before PTP opening represented the calcium retention capacity (CRC).
For PTP opening assessment in intact cells, HMEC-1 cells grown on coverslips were loaded with calcein-AM and cobalt chloride. Calcein fluorescence localized in mitochondria (protected from cobalt quenching) was monitored by confocal microscopy. PTP opening was indicated by the quenching of mitochondrial calcein fluorescence due to cobalt entry, triggered here by the addition of tBH.
For mitochondrial H2O2 production measurement, HMEC-1 cells preincubated with or without Imeglimin (100 µM for 24h) were digitonin-permeabilized. They were incubated in a KCl-based medium containing Amplex Red and horseradish peroxidase. H2O2 production was initiated by adding respiratory substrates (glutamate/malate, succinate, or both). The rate of H2O2 formation was measured fluorimetrically. Inhibitors (rotenone, antimycin A) were added sequentially to assess their effects. [2]

Animal/Disease Models: Male C57BL/6JOlaHsd mice (4 weeks old) [1]
Doses: 200 mg/kg
Route of Administration: po (oral gavage); bid; 6 weeks
Experimental Results: A slight decrease in body weight and food intake was observed, and There is some diarrhea, but only during the first few days of treatment.

The report points out that imagramine requires a long incubation time (24 hours at a concentration of 100 µM) to exert its effect on cells, which may indicate that it accumulates slowly in cells or has an indirect mechanism of action. [2]

[1]. Imeglimin normalizes glucose tolerance and insulin sensitivity and improves mitochondrialfunction in liver of a high-fat, high-sucrose diet mice model. Diabetes. 2015 Jun;64(6):2254-64.

[2]. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration. Cell Death Discov. 2016 Jan 18;2:15072.

Imegleem is the first novel oral hypoglycemic agent (a glimepiride). It has completed a phase IIb clinical trial and is effective in reducing glycated hemoglobin (HbA1c) levels. Imegleem acts on the liver, muscles, and pancreas, and its mechanism of action is thought to be related to mitochondria and the reduction of oxidative stress. Studies have shown that imagleem is a novel mitochondrial permeability transition pore (PTP) inhibitor. It prevents the opening of the mitochondrial permeability transition pore (PTP) and subsequent cell death without inhibiting mitochondrial respiration or cellular energy status. Imegleem specifically inhibits the production of reactive oxygen species (ROS) driven by reverse electron transport of mitochondrial complex I, a property that is distinct from classic complex I inhibitors such as rotenone. These findings suggest that imagleem may have potential benefits in preventing diabetes-related vascular complications (vascular disease) due to its protective effect on endothelial cells. [2]

C6H14CLN5

191.6619

191.093

775351-61-6

Imeglimin;775351-65-0

54763513

White to off-white solid powder

3

2

1

12

205

1

Cl[H].N(C([H])([H])[H])(C([H])([H])[H])C1=N[C@]([H])(C([H])([H])[H])N=C(N([H])[H])N1[H]

UXHLCYMTNMEXKZ-PGMHMLKASA-N

InChI=1S/C6H13N5.ClH/c1-4-8-5(7)10-6(9-4)11(2)3;/h4H,1-3H3,(H3,7,8,9,10);1H/t4-;/m1./s1

(4R)-6-N,6-N,4-trimethyl-1,4-dihydro-1,3,5-triazine-2,6-diamine;hydrochloride

Imeglimin HCl EMD387008 hydrochlorideImeglimin hydrochloride EMD387008 HCl

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, 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)

H2O : ≥ 50 mg/mL (~260.88 mM)
DMSO : ~25 mg/mL (~130.44 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.85 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 20.8 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: ≥ 2.08 mg/mL (10.85 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 20.8 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: ≥ 2.08 mg/mL (10.85 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (521.76 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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