PPARα (EC50 = 50 μM); PPARγ (IC50 = 500 μM)

Clofibrate is a PPAR agonist; its E50s for murine PPARα and PPARγ are 50 μM, approximately 500 μM, and for human PPARα and PPARγ are 55 μM, approximately 500 μM, respectively[1]. In two rat hepatoma cells treated with fatty acids (FA), clofibrate (0.5, 1, 2 mM) increases the expression of FABP1. When administered early, clofibrate significantly reduces ROS levels in FA-treated cells compared to when administered later[2].

Clofibrate (0.5%) elevates fetal serum concentrations and hepatic expression of FGF21, returning to baseline levels upon cessation of clofibrate treatment. Offspring administered with Clofibrate exhibit markedly increased expression of thermogenic genes (Ucp1, Cidea, Ppara Ppargc1a, Cpt1b) and UCP1 protein levels in response to a high-fat diet in the inguinal fat, but not in the epididymal or retroperitoneal fat (when combined with perirenal fat)[3].

In MEM/EBSS medium, cells are seeded at a density of 2.5 × 10⁴ cells/well (for WST-1, intracellular lipid droplet quantification and dichlorofluorescein (DCF) assay, 96-well plates) or 1 × 10⁵ cells/well (for Nile Red Staining, 12-well plates) and incubated overnight for adherence. The following day, fresh medium containing the fatty acid mixture oleate:palmitate (2:1) in the presence of 3% fatty acid-free bovine serum albumin is added to the cell culture medium. Fatty acid (FA) mixtures ranging from 0.5 to 3 mM are applied to cells for 24 to 48 hours at 37°C in a humidified incubator with 95% air and 5% CO₂. FABP1 levels in treated cell cultures are raised by clofibrate. After dissolving 500 μM of clofibrate in DMSO, it is added to the medium (final volume of DMSO < 0.1% v/v). Only DMSO is used to culture the control cells. One-day FA treatment, two-day FA treatment, early clofibrate intervention, and late clofibrate intervention are the four distinct cell treatments that are available[1].

C57BL/6JNarl mice, both male and female, are used in breeding. We use females whose parity ranges from 1 to 5. From breeding through parturition, pregnant females are fed either an experimental (CF) or control (C) diet. The CF diet is the C diet plus 0.5% clofibrate, while the C diet is based on an AIN-93M diet with a minor modification to include 21 kcal% fat from soybean oil. The presence of a vaginal plug (defined as pregnancy day 1) indicates the date of pregnancy. Following natural childbirth on day 19.5 ± 0.5 of pregnancy, all littermates are equally cared for by their mothers, who feed them the C diet for three weeks, adjusting the litter sizes to 8–10. Then, the babies are weaned onto a standard diet that isn't purified for four weeks, and finally, they are moved to a high-fat, butter-based daily fast for five weeks. In this investigation, only male progeny are employed, and two cohorts of progeny are identified based on the dietary habits of their mothers (CF or C). A controlled 12-hour light/dark cycle, unlimited access to food and water, and a temperature maintained at 23 ± 2°C are provided for each mouse. Weekly records are kept on body weight and feed consumption[3].

Absorption, Distribution and Excretion
Clofibrate is completely but slowly absorbed through the intestine. After oral administration, 95% to 99% of the dose is excreted in the urine as free and conjugated clofibrate; therefore, clofibrate absorption is almost complete. In the human body, clofibrate is completely absorbed through the intestine and appears in the plasma as deesterified p-chlorophenoxyisobutyric acid (CPIB); peak plasma CPIB concentrations are reached within 4 hours after oral administration…most CPIB…is bound to plasma albumin. The elimination of clofibrate (CPIB) occurs in two kinetic phases, with the slower exponential phase having a mean half-life of approximately 15 hours. Almost all the acid is excreted in the urine, with approximately 60% excreted as glucuronide. Clofibrate can cross the placenta into breast milk, and there are reports of elevated hepatic α-glycerophosphate dehydrogenase levels in newborn rats born to mothers administered clofibrate. Clofibrate is rapidly and completely absorbed after oral administration. In the human body, clofibrate's main metabolite, clofibrate acid, is excreted in the urine as a glucuronide conjugate; the plasma elimination half-life of clofibrate is 12-25 hours. Clofibrate is reported to be rapidly and almost completely absorbed in the gastrointestinal tract; approximately 95-99% of the oral dose is excreted in the urine as free and conjugated clofibrate. The drug is rapidly hydrolyzed to free clofibrate by serum enzymes. In healthy individuals, the mean peak plasma clofibrate concentrations 4-6 hours after a single oral dose of 500 mg, 1 g, or 2 g of clofibrate were 49-53, 89, or 151 μg/ml, respectively. For more complete data on the absorption, distribution, and excretion of clofibrate (7 items in total), please visit the HSDB record page. Metabolites/Metabolites: Hepatic and Gastrointestinal Tract: Rapidly deesterified during gastrointestinal and/or first-pass metabolism to the active form clofibrate (chlorophenoxyisobutyric acid [CPIB]). In the human body, clofibrate...appears in the plasma as deesterified p-chlorophenoxyisobutyric acid...almost all of its acidic form is excreted in the urine, of which approximately 60% is excreted as glucuronide. An acyl-linked acidic metabolite of clofibrate has been identified in human urine. This is the first acyl-linked thiol urate found in the human body. The authors propose that clofibrate acyl glucuronide is an electrophilic metabolite that reacts with thiol groups, and therefore may be the cause of clofibrate hepatotoxicity in humans. Liver and Gastrointestinal Tract: During gastrointestinal and/or first-pass metabolism, clofibrate is rapidly deesterified to the active form, clofibrate (chlorophenoxyisobutyric acid [CPIB]). Half-life: The average half-life in normal volunteers is 18 to 22 hours (range 14 to 35 hours), but the half-life in the same subject can vary by up to 7 hours at different times.

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Biological Half-Life
The average half-life in normal volunteers is 18 to 22 hours (range 14 to 35 hours), but the half-life of the same subject at different times can vary by up to 7 hours. The plasma elimination half-life of clofibrate is 12-25 hours. The elimination half-life of clofibrate in healthy adults is 12-35 hours (mean 12-22 hours), while the elimination half-life in patients with renal failure is 29-88 hours.

Toxicity Summary
Clofibrate increases the activity of extrahepatic lipoprotein lipase (LL), thereby promoting the breakdown of lipoprotein triglycerides. Chylomicrons are degraded, very low-density lipoprotein (VLDL) is converted into low-density lipoprotein (LDL), and LDL is converted into high-density lipoprotein (HDL). At the same time, the secretion of lipids in bile is slightly increased and eventually enters the intestine. Clofibrate also inhibits the synthesis of apolipoprotein B (the carrier molecule of VLDL) and promotes its clearance. In addition, as a fibrate, clofibrate is an agonist of peroxisome proliferator-activated receptor (PPAR-α) receptors in muscle, liver and other tissues [4]. This agonistic effect ultimately leads to changes in gene expression, thereby increasing β-oxidation, reducing triglyceride secretion, increasing high-density lipoprotein cholesterol (HDL) levels and increasing lipoprotein lipase activity. Hepatotoxicity
A small number of patients taking clofibrate may experience mild, transient elevation of serum transaminases, but the incidence of more than 3 times the normal value does not exceed 2%. These abnormalities are usually asymptomatic and transient, and resolve spontaneously even with continued clofibrate use. A small number of cases of clinically significant liver injury have been reported in patients taking clofibrate. The injury typically appears 2 to 3 months after treatment, and the pattern of elevated serum enzymes can be cholestatic or hepatocellular. Symptoms of immune-mediated hepatitis and autoantibodies are rare. Long-term clofibrate use is also associated with an increased incidence of gallstones, particularly in patients with chronic cholestatic liver disease (primary biliary cirrhosis). Probability Score: D (likely a rare cause of clinically significant liver injury). Protein Binding: Highly protein bound (95% to 97%). Toxicity Data: Mice, oral LD50 = 1220 mg/kg; Rabbits, oral LD50 = 1370 mg/kg; Rats, oral LD50 = 940 mg/kg.

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Interactions
One case report describes a patient with type IV hyperlipoproteinemia treated with clofibrate experiencing a recurrence of elevated serum cholesterol and triglyceride levels after taking oral contraceptives.
It is speculated that furosemide and clofibrate may compete for plasma albumin binding sites.
Other reports indicate that clofibrate can increase the release of posterior pituitary antidiuretic hormone, block pancreatic insulin and glucagon secretion in response to arginine, and reduce fasting blood glucose and serum insulin concentrations in diabetic patients. The effect of clofibrate on serum uric acid varies from person to person.
Clofibrate stimulates peroxisome fatty acid oxidation, increasing peroxide levels and thus enhancing the oxidation of ethanol by catalase. However, catalase plays a significant role in ethanol metabolism only in rare cases.
For more complete data on interactions of clofibrate (15 in total), please visit the HSDB records page.

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

[2]. Clofibrate Attenuates ROS Production by Lipid Overload in Cultured Rat Hepatoma Cells. J Pharm Pharm Sci. 2017;20(0):239-251.

[3]. Prenatal PPARα activation by clofibrate increases subcutaneous fat browning in male C57BL/6J mice fed a high-fat diet during adulthood. PLoS One. 2017 Nov 2;12(11):e0187507.

Therapeutic Uses
Clofibrate is only indicated for patients with elevated VLDL and IDL levels who do not respond well to gemfibrozil or niacin (e.g., patients with familial type III hyperlipoproteinemia). Due to its limited effect on LDL and the availability of more effective LDL-lowering drugs, clofibrate has limited efficacy in patients with familial or polygenic hypercholesterolemia. In a primary prevention trial in men with asymptomatic hypercholesterolemia, clofibrate reduced plasma cholesterol levels by only 6% to 11%. In unselected patients, the efficacy of clofibrate is very limited, contrasting sharply with its efficacy in patients with familial type III hyperlipoproteinemia, where cholesterol and triglyceride levels are reduced by approximately 50% and up to 80%, respectively. In these patients, clofibrate mobilizes cholesterol deposits in tissues, accompanied by regression and disappearance of xanthoma. Clofibrate is ineffective against hyperchylomicronemia and does not affect high-density lipoprotein (HDL) concentrations. Therefore, clofibrate appears to have specific efficacy only in patients with familial type III hyperlipoproteinemia. Clinical evidence for the prevention of coronary disease mortality with clofibrate is not encouraging. Multiple completed clinical trials have not shown a clear benefit. One double-blind study…compared the efficacy of clofibrate to placebo in 10,000 men with plasma cholesterol concentrations in the top third of the distribution. Patients treated with clofibrate had a lower incidence of non-fatal myocardial infarction. …Non-cardiac mortality was higher in patients treated with clofibrate than in the control group, primarily attributed to an increased incidence of malignancies and complications from cholecystectomy. For more complete data on the therapeutic uses of clofibrate (9 types), please visit the HSDB records page.

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Drug Warning
Of the more than 30 recorded side effects, erectile dysfunction observed after clofibrate treatment is the fourth most common side effect…although the exact mechanism is unclear…
Response to clofibrate varies from person to person. Serum cholesterol and triglyceride levels should be monitored regularly (e.g., every 3–6 months) before starting clofibrate treatment and during treatment. If possible, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels should be measured, and LDL levels should be rechecked during the first few months of clofibrate treatment…
Liver function tests and complete blood counts should be performed regularly during clofibrate treatment…Some clinicians recommend continuous monitoring of plasma creatine kinase (CPK) levels during clofibrate treatment…
…This drug has limited efficacy in patients with type II hyperlipoproteinemia. Furthermore, because clofibrate may increase the levels of certain very low-density lipoproteins (VLDL), the effects of this drug should be monitored by continuous measurement of plasma lipoproteins. Excessive conversion of very low-density lipoprotein (VLDL) to low-density lipoprotein (LDL) suggests the need to discontinue this medication. For more complete data on clofibrate (10 of these), please visit the HSDB records page.

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Pharmacodynamics
Clofibrate is a lipid-lowering drug similar to gemfibrozil. It lowers elevated blood lipids by reducing triglyceride-rich very low-density lipoprotein (SF20-400). Serum cholesterol may decrease, especially in patients with elevated cholesterol due to elevated intermediate-density lipoprotein (IDL) caused by type III hyperlipoproteinemia. Several studies have found that clofibrate may lead to a decrease in cholesterol linoleate levels, but an increase in palmitate and oleate levels, the latter of which is considered to have atherogenic effects in experimental animals. The significance of this finding is currently unclear. In some patients treated with clofibrate or certain chemically and clinically similar analogues, the decrease in triglyceride levels may be associated with an increase in low-density lipoprotein cholesterol (LDL-C) levels. Elevated LDL-C levels were also observed in some patients with initially normal cholesterol levels. Animal studies have shown that clofibrate interferes with cholesterol biosynthesis before mevalonate is formed.

C12H15CLO3

242.7

242.07

C, 59.39; H, 6.23; Cl, 14.61; O, 19.78

637-07-0

Clofibrate;637-07-0

2796

Colorless to light yellow liquid

1.1±0.1 g/cm3

274.8±0.0 °C at 760 mmHg

118-119
< 25 °C

115.1±19.9 °C

0.0±0.5 mmHg at 25°C

1.505

3.32

0

3

5

16

232

0

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

KNHUKKLJHYUCFP-UHFFFAOYSA-N

InChI=1S/C12H15ClO3/c1-4-15-11(14)12(2,3)16-10-7-5-9(13)6-8-10/h5-8H,4H2,1-3H3

ethyl 2-(4-chlorophenoxy)-2-methylpropanoate

Clofibrate; Clofibrato; Ethyl clofibrate; Clofibratum; Atromid-S

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)

DMSO: ~7 mg/mL (~28.8 mM)
Ethanol: ~5 mg/mL (~20.6 mM)

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.

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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).

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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)

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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).

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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

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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.

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