Aldose reductase
Aldose Reductase (AR) (IC50 = 0.03 μM; Ki = 0.015 μM) [1][6]

In Schwann cells (SC), epalrestat (100 and 200 µM, 24 hours) adsorbs cell viability and causes cell fluorescence [5]. Epalrestat activates Nrf2 to upregulate γ-GCS throughout a 24-hour period at 10 and 50 µM. The SC epoxy levels are protected by epalrestat (50 µM, 16 hours) [5].

---

Epalrestat potently inhibited recombinant human AR activity in a dose-dependent manner, with IC50 = 0.03 μM and Ki = 0.015 μM, blocking the conversion of glucose to sorbitol (polyol pathway) [1][6]
- In rat Schwann cells cultured under high glucose (30 mM), Epalrestat (1-10 μM) dose-dependently increased intracellular glutathione (GSH) levels: 10 μM increased GSH by ~65% compared to high glucose control (HPLC assay); mRNA expression of GSH synthetase and glutamate-cysteine ligase was upregulated by ~2.3-fold and ~1.8-fold respectively (qPCR) [5]
- In human brain microvascular endothelial cells (HBMECs) subjected to oxygen-glucose deprivation (OGD), Epalrestat (5-20 μM) enhanced cell viability (MTT assay): 20 μM increased viability by ~40% vs OGD control; it also reduced endothelial permeability (transendothelial electrical resistance assay) by ~35% and inhibited ROS production (DCFH-DA staining) by ~50% [7]
- In high glucose-induced rat mesangial cells, Epalrestat (10 μM) suppressed extracellular matrix accumulation: collagen IV and fibronectin protein levels were reduced by ~45% and ~40% respectively (western blot); it also normalized glucose metabolism by increasing glycolysis and oxidative phosphorylation rates [4]
- Epalrestat (5-20 μM) alleviated oxidative stress in high glucose-cultured dorsal root ganglion (DRG) neurons: malondialdehyde (MDA) levels decreased by ~55% (10 μM), and superoxide dismutase (SOD) activity increased by ~48% (10 μM) compared to high glucose control [6]

For eight weeks, epalrestat (0.08% (w/w) in a regular feed) protected against nephritis in diabetic nephropathy with a db/db ratio [4]. Epalrestat (100 mg/kg i.g. daily for 6 weeks). shields the scaffold against the damage caused by streptozotocin (streptozotocin) to diabetic peripheral nerves (DPN) [6]. The mouse brain's infarct volume and blood-brain barrier permeability can be decreased by epalrestat (50 mg/kg, given twice a day through the appendix) [7].

---

In db/db diabetic mice (diabetic nephropathy model), oral Epalrestat (100 mg/kg/day) for 12 weeks reduced urinary albumin excretion by ~60%, decreased glomerular hypertrophy (glomerular volume reduced by ~30%), and inhibited mesangial matrix expansion (histopathology) [4]
- In streptozotocin (STZ)-induced diabetic rats (peripheral neuropathy model), oral Epalrestat (50 mg/kg/day) for 8 weeks improved motor nerve conduction velocity (MNCV) from 32.5 ± 2.1 m/s to 41.3 ± 2.5 m/s and sensory nerve conduction velocity (SNCV) from 28.3 ± 1.8 m/s to 36.7 ± 2.0 m/s (electrophysiological assay) [6]
- In STZ-induced diabetic mice with cerebral ischemia, intraperitoneal Epalrestat (30 mg/kg, 30 min pre-ischemia and daily for 3 days post-ischemia) maintained blood-brain barrier (BBB) integrity: Evans blue leakage reduced by ~55%, and tight junction proteins (occludin, claudin-5) expression increased by ~1.7-fold and ~1.5-fold respectively (western blot) [7]
- Clinical efficacy in diabetic neuropathy patients: Oral Epalrestat (150 mg/day) for 12 weeks improved subjective symptoms (numbness, tingling, pain) in 68.3% of patients; MNCV and SNCV were significantly increased by 3.2 ± 1.1 m/s and 2.8 ± 0.9 m/s respectively compared to baseline [3]
- In diabetic gastroparesis patients, Epalrestat (150 mg/day) for 8 weeks reduced gastric emptying time from 158 ± 32 min to 112 ± 25 min and improved upper abdominal discomfort scores by ~52% [2]

Recombinant human AR protein was resuspended in assay buffer containing NADPH. Epalrestat was serially diluted (0.001-1 μM) and mixed with AR protein, followed by addition of the substrate DL-glyceraldehyde (at Km concentration for AR). The reaction was incubated at 37°C for 20 minutes, and the decrease in NADPH absorbance at 340 nm was measured continuously. IC50 values were calculated by nonlinear regression of the dose-response inhibition curves; Ki was determined using Lineweaver-Burk plots to confirm competitive inhibition [1][6]

RT-PCR[5]
Cell Types: rat SCs, thereby increasing intracellular glutamine in SC[5].
Tested Concentrations: 10 or 50 µM
Incubation Duration: 4 hrs (hours)
Experimental Results: At 10 and 50 µM concentrations, nuclear levels of active Nrf2 increased 1.8- and 3.8-fold, but failed to increase Nrf2 mRNA levels.

---

Schwann cell GSH assay: Rat Schwann cells were seeded in 6-well plates (2×105 cells/well) and cultured in normal glucose (5.5 mM) or high glucose (30 mM) medium. Epalrestat (1-10 μM) was added, and cells were cultured for 48 h. Cells were lysed, and GSH levels were quantified by HPLC with fluorescence detection. Total RNA was extracted for qPCR analysis of GSH synthetase and glutamate-cysteine ligase mRNA expression [5]
- HBMEC viability and permeability assay: HBMECs were seeded in 96-well plates (for viability) or Transwell inserts (for permeability) and cultured to confluence. Cells were subjected to OGD for 4 h, then treated with Epalrestat (5-20 μM) for 24 h. Viability was measured by MTT assay; permeability was assessed by transendothelial electrical resistance and FITC-dextran flux. ROS production was detected by DCFH-DA staining and flow cytometry [7]
- Mesangial cell matrix accumulation assay: Rat mesangial cells were cultured in high glucose (30 mM) medium with Epalrestat (10 μM) for 72 h. Cells were lysed, and collagen IV and fibronectin protein levels were detected by western blot. Glucose metabolism rates were measured using glycolysis and oxidative phosphorylation assay kits [4]
- DRG neuron oxidative stress assay: Rat DRG neurons were isolated and cultured in high glucose (30 mM) medium with Epalrestat (5-20 μM) for 48 h. MDA levels were measured by thiobarbituric acid reactive substances assay, and SOD activity was determined by xanthine oxidase method [6]

Animal/Disease Models: db/db mice[4]
Doses: 0.08% (w/w) fed regular feed
Doses: 8 weeks
Experimental Results: Improved GBM thickening and mesangial matrix deposition in renal tissue. Reduces elevated sorbitol and fructose in plasma, urine, and renal cortex of db/db mice. Myo-inositol diminished in plasma and urine, whereas it increased in the renal cortex.

---

Animal/Disease Models: Rats were fed a high-fat and high-sugar diet for 4 weeks, and streptozotocin was injected at the 4th and 8th weeks [6]
Doses: 100 mg/kg/d
Route of Administration: ig for 6 weeks
Experimental Results: Improved pathological structure of rats Neurites and myelin. SOD, CAT, and GPX protein levels were increased in the sciatic nerve. Reduces aldose reductase levels in the sciatic nerve.

---

Diabetic nephropathy model (db/db mice): 8-week-old male db/db mice were randomly divided into control and Epalrestat groups (n=8/group). Epalrestat was dissolved in 0.5% carboxymethylcellulose sodium and administered orally at 100 mg/kg/day for 12 weeks. Control mice received vehicle. Urine samples were collected monthly for albumin detection; mice were euthanized at week 12, and kidneys were harvested for histopathological and protein analysis [4]
- Peripheral neuropathy model (STZ-induced diabetic rats): Male Sprague-Dawley rats were intraperitoneally injected with STZ to induce diabetes (blood glucose > 16.7 mM). Diabetic rats were treated with oral Epalrestat (50 mg/kg/day) or vehicle for 8 weeks (n=6/group). Nerve conduction velocity (MNCV, SNCV) was measured by electrophysiology; DRG and sciatic nerves were collected for oxidative stress marker detection [6]
- Cerebral ischemia model (STZ-induced diabetic mice): Diabetic mice were subjected to middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. Epalrestat (30 mg/kg) was intraperitoneally injected 30 min before MCAO and daily for 3 days post-ischemia (n=6/group). BBB integrity was evaluated by Evans blue injection; brain tissues were collected for tight junction protein analysis [7]

The oral bioavailability of epalrestat in humans is approximately 30% (150 mg oral dose); peak plasma concentration (Cmax) of 1.2 ± 0.3 μg/mL is reached 1.5 hours after administration [1]
- The plasma half-life (t1/2) of epalrestat in humans is approximately 1.5 hours; plasma protein binding is approximately 90% (ultrafiltration method) [1]
- epalrestat is widely distributed in various tissues, with higher concentrations in the liver, kidneys and peripheral nerves [1]
- Metabolism: epalrestat is minimally metabolized in the liver; approximately 80% of the dose is excreted unchanged in the urine within 24 hours [1]

Oral LD50 in rats: 5300 ug/kg Iyakuhin Kenkyu. Medical Supplies Research. , 23(201), 1992
Intraperitoneal LD50 in rats: 922 gm/kg Iyakuhin Kenkyu. Medical Supplies Research. , 23(201), 1992
Subcutaneous LD50 in rats: >3 gm/kg Iyakuhin Kenkyu. Medical Supplies Research. , 23(201), 1992
Intravenous LD50 in rats: 255 ug/kg Iyakuhin Kenkyu. Medical Supplies Research. , 23(201), 1992
Oral LD50 in mice: 3200 mg/kg Iyakuhin Kenkyu. Medical Products Research, 23(201), 1992

---

In clinical studies, epalrestat (150 mg/day for up to 24 months) showed mild and transient adverse reactions: gastrointestinal discomfort (3.2%), rash (1.1%), and elevated liver enzymes (0.8%), which resolved spontaneously or were relieved by dose adjustment[1][3]
- In db/db mice treated with epalrestat (100 mg/kg/day for 12 weeks), no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, Cr) were observed compared with the control group[4]
- Acute oral toxicity (LD50) in mice > 5000 mg/kg; no chronic toxicity (carcinogenicity, mutagenicity, teratogenicity) was detected in preclinical studies[1]

[1]. Ramirez, M.A. and N.L. Borja, Epalrestat: an aldose reductase inhibitor for the treatment of diabetic neuropathy. Pharmacotherapy, 2008. 28(5): p. 646-55.

[2]. Effects of epalrestat, an aldose reductase inhibitor, on diabetic neuropathy and gastroparesis. Intern Med, 2003. 42(8): p. 655-64.

[3]. Clinical investigation of epalrestat, an aldose reductase inhibitor, on diabetic neuropathy in Japan: multicenter study. Diabetic Neuropathy Study Group in Japan. J Diabetes Complications, 1996. 10(3): p. 168-72.

[4]. The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation. Acta Pharmacol Sin. 2019 Jan;40(1):86-97.

[5]. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation. Redox Biol. 2013 Nov 19;2:15-21.

[6]. Epalrestat protects against diabetic peripheral neuropathy by alleviating oxidative stress and inhibiting polyol pathway. Neural Regen Res. 2016 Feb;11(2):345-51.

[7]. The Aldose Reductase Inhibitor Epalrestat Maintains Blood-Brain Barrier Integrity by Enhancing Endothelial Cell Function during Cerebral Ischemia. Mol Neurobiol. 2023 Jul;60(7):3741-3757.

Epalrestat is a monocarboxylic acid with the structure 1,3-thiazolidinyl, where the nitrogen atom is substituted with a carboxymethyl group, the 2- and 4-positions are substituted with a thio group and an oxy group, respectively, and the 5-position is substituted with a 2-methyl-3-phenylprop-2-en-1-yl group. It is an inhibitor of aldose reductase (which catalyzes the conversion of glucose to sorbitol) and is used to treat certain diabetic complications, including neuropathy. It is an EC 1.1.1.21 (aldose reductase) inhibitor. It belongs to the thiazolidinyl class of compounds and is a monocarboxylic acid. Epalrestat is currently being investigated in the clinical trial NCT03244358 (Efficacy Evaluation of Epalrestat in Metastatic Triple-Negative Breast Cancer). Epalrestat is an orally potent, non-competitive, reversible aldose reductase (AR) inhibitor with potential antitumor, antioxidant, and anti-inflammatory activities. After oral administration, epalrestat binds non-competitively to AR. AR is a polyol pathway enzyme that catalyzes lipid peroxidation induced by reactive oxygen species (ROS) to produce lipid aldehydes and their glutathione conjugates. These lipid aldehydes and their glutathione conjugates are associated with the activation of transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which control the transcription of many inflammatory cytokines. Increased levels of inflammatory cytokines and growth factors promote cell proliferation, a key feature of tumorigenesis. AR is overexpressed in a variety of oxidative stress and inflammation-related diseases, including cancer.

---

Epalrestat (ONO2235) is a reversible competitive aldose reductase inhibitor (ARI) and the first ARI to be clinically approved for the treatment of diabetic neuropathy [1][3]
- Its core mechanism of action is the inhibition of the polyol pathway: blocking AR can reduce the accumulation of sorbitol in diabetic tissues (nerves, kidneys, blood vessels), reduce osmotic stress, and inhibit oxidative damage [1][6]
- In addition to inhibiting the polyol pathway, Epalrestat also exerts a protective effect by enhancing antioxidant capacity (GSH synthesis), regulating glucose metabolism, maintaining the integrity of tight junctions, and reducing inflammatory responses [4][5][7]
- Indications: Epalrestat is indicated for the treatment of diabetic peripheral neuropathy to improve subjective symptoms (numbness, pain, paresthesia) and objective indicators (nerve conduction velocity) [1][3]
- Clinical data from a multicenter study in Japan showed that taking epalrestat (150) After 12 weeks of treatment with mg/day, the symptom improvement rate in patients with diabetic neuropathy was 68.3%, and the nerve conduction velocity improvement rate was 72.1% [3].

C15H13NO3S2

319.4

319.033

C, 56.41; H, 4.10; N, 4.39; O, 15.03; S, 20.08

82159-09-9

1549120

Pink to red solid powder

1.4±0.1 g/cm3

516.8±60.0 °C at 760 mmHg

210-217ºC

266.4±32.9 °C

0.0±1.4 mmHg at 25°C

1.706

2.02

1

5

4

21

519

0

C/C(=C\C1=CC=CC=C1)/C=C\2/C(=O)N(C(=S)S2)CC(=O)O

CHNUOJQWGUIOLD-NFZZJPOKSA-N

InChI=1S/C15H13NO3S2/c1-10(7-11-5-3-2-4-6-11)8-12-14(19)16(9-13(17)18)15(20)21-12/h2-8H,9H2,1H3,(H,17,18)/b10-7+,12-8-

2-[(5Z)-5-[(E)-2-Methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]acetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2 mg/mL (6.26 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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