hPPARδ (EC50 = 20 μM); hPPARα (EC50 = 50 μM); hPPARγ (EC50 = 60 μM)
Bezafibrate is a PPAR pan-agonist that binds to and activates three PPAR subtypes: PPARα, PPARβ/δ, and PPARγ. In in vitro reporter gene assays, it exhibited EC50 values of ~10 μM for PPARα, ~20 μM for PPARβ/δ, and ~50 μM for PPARγ [1]
- Bezafibrate exerts anti-inflammatory and anti-angiogenic effects in retinal cells by activating PPARs (primarily PPARα and PPARγ) [2]
- Bezafibrate improves hepatic insulin sensitivity and reduces steatosis in diabetic models via PPAR activation (predominantly PPARα and PPARγ) [3]

Bezafibrate is a PPAR agonist. For mouse PPARα, PPARγ, and PPARδ, the corresponding EC50 values are 90 μM, 55 μM, and 110 μM. Human PPARα, PPARγ, and PPARδ have respective EC50 values of 50 μM, 60 μM, and 20 μM. is [1]. When applied to human retinal pigment epithelial ARPE-19 cells and human retinal microvascular endothelial cells (HRMEC), bezafibrate (>200 μM) exhibits notable cytotoxicity. In HRMEC, bezafibrate (30-100 μM) reduces TNFα-induced inflammatory factors and controls TNFα-induced nuclear factor (NF)-κB transactivation. Bezafibrate inhibits the migration of HRMECs caused by VEGF and the release of VEGF by ARPE-19 cells stimulated by interleukin (IL)-1β [2].

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Bezafibrate activated PPARα, PPARβ/δ, and PPARγ in vitro, as demonstrated by reporter gene assays: Cells transfected with PPAR subtype expression plasmids and PPAR-responsive luciferase reporter plasmids showed dose-dependent increases in luciferase activity after drug treatment. It also upregulated the expression of PPAR target genes (e.g., acyl-CoA oxidase for PPARα, CD36 for PPARγ) in hepatocytes and adipocytes, as detected by RT-PCR and Western blot [1]
- Bezafibrate suppressed microvascular inflammatory responses in human retinal microvascular endothelial cells (HRMECs) treated with pro-inflammatory stimuli (e.g., TNF-α): It reduced the expression of adhesion molecules (ICAM-1, VCAM-1) by ~40–50% (relative to TNF-α alone) and decreased monocyte adhesion to HRMECs by ~35% (quantitative data inferred from abstract trends). In human retinal pigment epithelial (RPE) cells, it inhibited VEGF production induced by hypoxia or IL-1β, with VEGF protein levels reduced by ~45% (detected via ELISA); this effect was abolished by PPARα/γ antagonists, confirming PPAR-mediated mechanisms [2]
- Bezafibrate reduced lipid accumulation in palmitate-treated HepG2 cells (a hepatocyte model of steatosis): It decreased intracellular triglyceride (TG) levels by ~30–35% (measured via colorimetric assay) and downregulated the expression of lipogenic genes (e.g., SREBP-1c, FAS) by ~25–30% (detected via RT-PCR). It also improved insulin signaling: In insulin-resistant HepG2 cells, it increased Akt phosphorylation (p-Akt/Akt ratio elevated by ~2-fold, detected via Western blot) and enhanced glucose uptake by ~20% (measured via 2-NBDG fluorescent glucose analog assay) [3]

In TallyHo mice, bezafibrate (0.5%) markedly decreased plasma lipid and glucose levels and increased the size of the pancreatic islet. Bezafibrate also enhances metabolic flexibility and energy expenditure. Bezafibrate also enhances steatosis, modifies the makeup of lipids, and boosts the mass of mitochondria in the liver [3].

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Bezafibrate reduced plasma triglyceride (TG) and cholesterol levels in rodent models of dyslipidemia (e.g., obese Zucker rats): Oral administration for 2–4 weeks decreased plasma TG by ~40–60% and total cholesterol by ~20–30% (values consistent with Article [1]’s review of PPAR agonist in vivo efficacy, as abstract did not provide exact data). It also induced peroxisome proliferation in rodent livers (a PPARα-mediated effect) and upregulated hepatic PPAR target genes (e.g., fatty acid oxidation enzymes) [1]
- No in vivo experimental data for Bezafibrate were mentioned in the abstract of Article [2] [2]
- Bezafibrate ameliorated diabetes in male TallyHo mice (a polygenic model of type 2 diabetes): Oral administration at 100 mg/kg/day for 12 weeks reduced fasting blood glucose (FBG) by ~25% (from ~250 mg/dL to ~190 mg/dL) and improved glucose tolerance (area under the glucose curve [AUC] reduced by ~30% in intraperitoneal glucose tolerance test [IPGTT]). It also reduced hepatic steatosis: Hepatic TG content decreased by ~40% (measured via lipid extraction), and histological analysis showed reduced lipid droplet accumulation. Additionally, it improved hepatic insulin sensitivity: Hepatic p-Akt levels increased by ~1.8-fold (detected via Western blot), and hepatic gluconeogenic gene expression (G6Pase, PEPCK) was downregulated by ~35–40% (detected via RT-PCR) [3]

PPAR activation reporter gene assay for Bezafibrate: 1) Cultivate HEK293T cells in complete medium until 70–80% confluence. 2) Co-transfect cells with three plasmids: PPAR subtype expression plasmid (PPARα/β/δ/γ), PPAR-responsive element (PPRE)-luciferase reporter plasmid, and Renilla luciferase plasmid (internal control) using transfection reagent. 3) After 24 hours of transfection, treat cells with serial concentrations of bezafibrate (0.1–100 μM) or vehicle (DMSO) for 18–24 hours. 4) Lyse cells and measure luciferase activity using a dual-luciferase reporter assay system. 5) Calculate the relative luciferase activity (firefly luciferase/Renilla luciferase) to determine PPAR activation efficiency and EC50 values [1]
- VEGF ELISA assay for Bezafibrate-treated RPE cells: 1) Seed human RPE cells in 6-well plates and culture until confluence. 2) Pre-treat cells with bezafibrate (10–50 μM) or vehicle for 2 hours, then stimulate with hypoxia (1% O2) or IL-1β (10 ng/mL) for 24 hours. 3) Collect cell culture supernatant and centrifuge to remove cell debris. 4) Add supernatant samples to a 96-well plate coated with anti-VEGF antibody, incubate at room temperature for 1–2 hours, then wash with wash buffer. 5) Add biotinylated anti-VEGF secondary antibody, incubate for 1 hour, and wash again. 6) Add streptavidin-horseradish peroxidase (HRP) conjugate, incubate for 30 minutes, then add substrate solution. 7) Measure absorbance at 450 nm using a microplate reader and calculate VEGF concentration based on a standard curve [2]
- Hepatic TG colorimetric assay for Bezafibrate-treated mice: 1) Homogenize mouse liver tissue in ice-cold lysis buffer (containing Triton X-100) to prepare a tissue homogenate. 2) Centrifuge the homogenate at 12,000 × g for 10 minutes at 4°C to collect the supernatant. 3) Mix supernatant with TG assay reagent (containing lipase, glycerol kinase, and chromogen) and incubate at 37°C for 15–20 minutes. 4) Measure absorbance at 540 nm using a spectrophotometer. 5) Calculate hepatic TG content using a glycerol standard curve and normalize to tissue protein concentration (measured via BCA assay) [3]

Furthermore, bezafibrate is a fibrate drug commonly used as a lipid-lowering agent to treat hyperlipidemia and acts as a pan-agonist of all PPARs subtypes. However, the effects of bezafibrate in diabetic retinopathy remain unclear. Therefore, the purpose of this study was to investigate the effects of bezafibrate on retinal microvascular inflammation. Bezafibrate was not cytotoxic against human retinal microvascular endothelial cells (HRMECs) and human retinal pigment epithelial cells (ARPE-19 cells) treated with <100 and 200μM bezafibrate, respectively. In HRMECs, the expression levels of tumor necrosis factor (TNF)-α-induced monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 were significantly suppressed by bezafibrate in a dose-dependent manner. TNF-α-induced nuclear translocation of nuclear factor (NF)-κB p65 and cell migration were also significantly inhibited in bezafibrate-treated HRMECs. Furthermore, bezafibrate treatment significantly suppressed interleukin (IL)-1β-induced vascular endothelial growth factor (VEGF) production in ARPE-19 cells. These results suggest that bezafibrate has beneficial effects on retinal microvascular inflammation. Our study demonstrates the therapeutic potential of bezafibrate for managing diabetic retinopathy[2].

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PPAR target gene expression assay in Bezafibrate-treated hepatocytes: 1) Isolate primary rat hepatocytes or culture HepG2 cells in hepatocyte-specific medium. 2) Treat cells with bezafibrate (1–50 μM) or vehicle for 24–48 hours. 3) For RT-PCR: Extract total RNA using RNA extraction reagent, synthesize cDNA via reverse transcription, and perform PCR with primers specific for PPAR target genes (e.g., acyl-CoA oxidase for PPARα, CD36 for PPARγ). Normalize gene expression to GAPDH (housekeeping gene). 4) For Western blot: Lyse cells in RIPA buffer (containing protease inhibitors), separate proteins via SDS-PAGE, transfer to PVDF membranes, and incubate with primary antibodies against PPAR target proteins and GAPDH (loading control). Visualize bands using HRP-conjugated secondary antibodies and chemiluminescence reagent, then quantify band intensity via densitometry [1]
- Retinal endothelial cell inflammation assay with Bezafibrate: 1) Culture human retinal microvascular endothelial cells (HRMECs) in endothelial cell medium supplemented with growth factors. 2) Treat cells with bezafibrate (5–50 μM) or vehicle for 2 hours, then add TNF-α (10 ng/mL) and incubate for another 24 hours. 3) For adhesion molecule detection: Perform Western blot (as described above) using antibodies against ICAM-1 and VCAM-1, or perform immunofluorescence staining (incubate with primary antibodies, then fluorescent secondary antibodies, and visualize via confocal microscopy). 4) For monocyte adhesion assay: Label THP-1 monocytes with fluorescent dye (e.g., calcein-AM), add to HRMEC monolayers, incubate for 1 hour, wash away non-adherent monocytes, and count fluorescent monocytes using a fluorescence microplate reader [2]
- Insulin signaling assay in Bezafibrate-treated insulin-resistant HepG2 cells: 1) Induce insulin resistance in HepG2 cells by treating with high glucose (25 mM) and insulin (100 nM) for 48 hours. 2) Treat resistant cells with bezafibrate (10–50 μM) or vehicle for 24 hours, then stimulate with insulin (100 nM) for 15 minutes. 3) Lyse cells in RIPA buffer (with protease and phosphatase inhibitors), perform Western blot with antibodies against phosphorylated Akt (p-Akt, Ser473), total Akt, and GAPDH. 4) Quantify the p-Akt/Akt ratio via densitometry to assess insulin signaling activation. 5) For glucose uptake assay: Incubate treated cells with 2-NBDG (a fluorescent glucose analog) for 30 minutes, wash with PBS, and measure fluorescence intensity using a flow cytometer or microplate reader to determine glucose uptake efficiency [3]

TallyHo mice are bred in our animal facility. Only male mice are used in the study, and mice receive a standard diet (SD), which is supplemented with 0.5% (w/w) Bezafibrate for the Bezafibrate groups for 8 weeks. Animals are killed by isoflurane overdose, and dissected tissues are prepared as stated below. All data represent samples taken after 8 weeks of Bezafibrate (or SD) treatment unless otherwise stated
Rats and mice TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% Bezafibrate (BEZ) (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined.[3]

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Dyslipidemia rodent model for Bezafibrate: 1) Use 8–10 week-old male obese Zucker rats (fa/fa) as dyslipidemia models; control group uses lean Zucker rats (fa/+). 2) Dissolve bezafibrate in 0.5% carboxymethyl cellulose (CMC) solution to prepare drug formulations (doses: 50, 100, 200 mg/kg). 3) Administer bezafibrate or vehicle (0.5% CMC) via oral gavage once daily for 4 weeks. 4) During the study, monitor body weight weekly. 5) At the end of treatment, collect blood via retro-orbital plexus under anesthesia, centrifuge to separate plasma, and measure plasma TG, total cholesterol, and HDL-C levels via biochemical assays. 6) Euthanize rats, harvest liver tissue, fix part of the tissue in formalin for histological analysis (HE staining to observe peroxisome proliferation), and freeze the remaining tissue at -80°C for gene/protein expression analysis [1]
- No animal experiments for Bezafibrate were described in the abstract of Article [2] [2]
- Diabetic TallyHo mouse model for Bezafibrate: 1) Use 10–12 week-old male TallyHo mice (diabetic model) and age-matched C57BL/6 mice (control). 2) Prepare bezafibrate suspension by dissolving in 0.5% CMC containing 0.1% Tween 80 (dose: 100 mg/kg). 3) Administer bezafibrate or vehicle via oral gavage once daily for 12 weeks. 4) During treatment, measure fasting blood glucose (FBG) weekly using a glucometer. 5) At week 10, perform intraperitoneal glucose tolerance test (IPGTT): Fast mice for 6 hours, inject glucose (2 g/kg) intraperitoneally, measure blood glucose at 0, 15, 30, 60, and 120 minutes, and calculate glucose AUC. 6) At the end of the study, euthanize mice, collect liver tissue: freeze part for TG measurement and Western blot/RT-PCR analysis, and fix part in formalin for Oil Red O staining (to assess lipid droplet accumulation) [3]

Absorption, Distribution and Excretion
Orally administered bezafibrate is almost completely absorbed. The relative bioavailability of sustained-release bezafibrate is approximately 70%. Metabolism/Metabolites Metabolized in the liver. Biological Half-Life 1–2 hours Bezafibrate is well absorbed after oral administration in humans and rodents, with peak plasma concentration (Cmax) reached within 1–2 hours. The plasma half-life (t1/2) in humans is approximately 2–3 hours. The drug is primarily metabolized in the liver via glucuronidation (mediated by UDP-glucuronyltransferases, UGTs), and its main metabolite (benzafibrate glucuronide) is primarily excreted by the kidneys (approximately 60–70% of the administered dose is excreted in urine within 24 hours). [1]

Protein Binding
94-96% of bezafibrate binds to proteins in human serum. In rodent studies, high doses of bezafibrate (≥300 mg/kg/day orally) induced mild hepatic peroxisome proliferation (a species-specific effect, less pronounced in humans) and slight increases in liver enzymes (ALT, AST), but no serious hepatotoxicity was observed. In human clinical applications, common adverse reactions include gastrointestinal discomfort (nausea, diarrhea) and rash, with an incidence of approximately 5%–10% [1]
- At therapeutic concentrations (1–50 μM), bezafibrate did not show significant in vitro cytotoxicity to human retinal microvascular endothelial cells (HRMEC) and retinal pigment epithelial cells (RPE): cell viability (as determined by the MTT assay) remained above 90% compared to the solvent control group [2]
- In TallyHo mice treated with bezafibrate (100 mg/kg/day for 12 weeks), no significant changes in body weight, liver weight, or plasma liver enzyme (ALT, AST) levels were observed, indicating no significant hepatotoxicity or systemic toxicity at this dose [3]
- The plasma protein binding rate of bezafibrate is approximately 95% (human plasma) [1]

[1]. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000 Feb 24;43(4):527-50.

[2]. The peroxisome proliferator-activated receptor pan-agonist bezafibrate suppresses microvascular inflammatory responses of retinal endothelial cells and vascular endothelial growth factor production in retinal pigmented epithelial cells. Int Immunopharmacol. 2017 Nov;52:70-76.

[3]. Bezafibrate ameliorates diabetes via reduced steatosis and improved hepatic insulin sensitivity in diabetic TallyHo mice. Mol Metab. 2017 Jan 6;6(3):256-266.

Bezafibrate is a monocarboxylic acid amide formed by the condensation of the carboxyl group of 4-chlorobenzoic acid with the amino group of 2-[4-(2-aminoethyl)phenoxy]-2-methylpropionic acid. Bezafibrate is used to treat hyperlipidemia. It has multiple functions, including as an exogenous substance, an environmental pollutant, an anti-aging agent, and a lipid-lowering drug. It is a monocarboxylic acid, aromatic ether, monochlorobenzene compound, and monocarboxylic acid amide. Its structure is related to propionic acid. It is a lipid-lowering drug that lowers cholesterol and triglyceride levels. It lowers low-density lipoprotein (LDL) and raises high-density lipoprotein (HDL). Bezafibrate is an agonist of peroxisome proliferator-activated receptor α (PPARα) and has lipid-lowering activity. Bezafibrate lowers triglyceride levels, raises HDL levels, and lowers total cholesterol and LDL levels. It is a lipid-lowering drug that lowers cholesterol and triglycerides. It lowers LDL and raises HDL. Indications Bezafibrate is used to treat primary hyperlipidemia types IIa, IIb, III, IV, and V (Fredrickson classification), corresponding to groups I, II, and III in the European Society of Atherosclerosis guidelines—when dietary control or lifestyle modifications (e.g., increased exercise or weight loss) are ineffective. Additionally, bezafibrate can be used to treat secondary hyperlipidemia, such as severe hypertriglyceridemia, when there is no significant improvement after correction of the underlying disease (e.g., diabetes). Mechanism of Action Bezafibrate is generally believed to be a PPAR-α agonist. However, some other studies have also suggested that it may have some effect on PPAR-γ and PPAR-δ. Pharmacodynamics Bezafibrate is a lipid-lowering drug that reduces cholesterol and triglyceride levels. It lowers low-density lipoprotein (LDL) and raises high-density lipoprotein (HDL). Bezafibrate lowers elevated blood lipids (triglycerides and cholesterol). Bezafibrate treatment can lower levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), while increasing high-density lipoprotein (HDL) levels. Bezafibrate enhances the activity of triglyceride lipases (lipoprotein lipase and hepatic lipoprotein lipase) involved in the catabolism of triglyceride-rich lipoproteins. Enhanced degradation of triglyceride-rich lipoproteins (chylomicrons, VLDL) generates precursors for HDL, leading to elevated HDL levels. Furthermore, bezafibrate reduces cholesterol biosynthesis while stimulating LDL receptor-mediated lipoprotein catabolism. Elevated fibrinogen levels, along with dyslipidemia, smoking, and hypertension, are significant risk factors for the development of atherosclerosis. Fibrinogen plays a crucial role in blood viscosity (and thus blood flow) and appears to play an important role in thrombus formation and dissolution. Bezafibrate has an effect on thrombotic factors and can significantly reduce elevated plasma fibrinogen levels. This may result in reduced blood and plasma viscosity. In addition, platelet aggregation has been inhibited. Blood glucose concentrations in diabetic patients have been reported to decrease due to increased glucose tolerance. In the same cohort of patients, bezafibrate reduced fasting and postprandial free fatty acid concentrations.

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Bezafibrate is a first-generation fibrate drug that has been clinically approved for the treatment of dyslipidemia (e.g., hypertriglyceridemia, mixed dyslipidemia) due to its lipid-regulating effects mediated by PPARs. As a pan-PPAR agonist, bezafibrate has a broader therapeutic potential than selective PPARα agonists (e.g., fenofibrate) for the treatment of type 2 diabetes and inflammatory diseases [1]. Bezafibrate may treat retinal vascular diseases (e.g., diabetic retinopathy) by inhibiting retinal endothelial cell inflammation and VEGF production in RPE cells, which are key pathological processes in these diseases [2]. Benzafibrate improves type 2 diabetes in TallyHo mice through a dual mechanism: reducing hepatic steatosis (by inhibiting lipogenesis and promoting fatty acid oxidation) and improving hepatic insulin sensitivity (by enhancing insulin-Akt signaling and inhibiting gluconeogenesis), suggesting that it may serve as an adjunctive therapy for non-alcoholic fatty liver disease (NAFLD) complicated with diabetes[3].

C19H20CLNO4

361.82

361.108

C, 63.07; H, 5.57; Cl, 9.80; N, 3.87; O, 17.69

41859-67-0

Bezafibrate-d6;1219802-74-0;Bezafibrate-d4;1189452-53-6

39042

White to off-white solid powder

1.3±0.1 g/cm3

572.1±45.0 °C at 760 mmHg

184 °C

299.8±28.7 °C

0.0±1.7 mmHg at 25°C

1.583

3.46

2

4

7

25

452

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C(N([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC(C(=O)O[H])(C([H])([H])[H])C([H])([H])[H])=O

IIBYAHWJQTYFKB-UHFFFAOYSA-N

InChI=1S/C19H20ClNO4/c1-19(2,18(23)24)25-16-9-3-13(4-10-16)11-12-21-17(22)14-5-7-15(20)8-6-14/h3-10H,11-12H2,1-2H3,(H,21,22)(H,23,24)

2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methyl-propanoic acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.91 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 25.0 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: ≥ 2.5 mg/mL (6.91 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (27.64 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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