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Gemfibrozil 1-O-beta-glucuronide is an irreversible inhibitor of cytochrome P450 isoform CYP2C8, with an IC50 reported in the literature. It is also a substrate of organic anion transporting polypeptides (OATPs) such as OATP1B1 and OATP1B3, and an inhibitor of OATP1B1-mediated transport. This metabolite does not retain the parent drug‘s activity as a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, which is the primary mechanism for gemfibrozil‘s lipid-lowering effects, but it is pharmacologically active in terms of CYP enzyme inhibition. CYP2C8 is involved in the metabolism of many clinically important drugs including repaglinide (antidiabetic), paclitaxel (anticancer), cerivastatin (lipid-lowering, withdrawn), and certain antimalarials. Irreversible inactivation of CYP2C8 by Gemfibrozil 1-O-beta-glucuronide is a major cause of clinically significant drug-drug interactions when gemfibrozil is co-administered with CYP2C8 substrate drugs. The metabolite also inhibits UGT enzymes and OATP transporters, contributing to complex pharmacokinetic interactions.
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| ln Vitro |
Gemrobezil 1-O-β-Glucuronide strongly inhibits the absorption of Cerivastatin (CER) mediated by OATP2 (OATP1B1) with an IC50 of 24.3 μM[1]. With an IC50 of 5.38 μM and 4.30 μM, respectively, gemrobezil 1-O-β-Glucuronide suppresses the synthesis of M1 and M23 mediated by CYP2C8, while having no effect on M3 mediated by CYP2C8 [1]. For CYP3A4-mediated metabolism, gemfibrozil 1-O-β-Glucuronide exhibits an IC50 of 243 μM [1].
In vitro studies have demonstrated that Gemfibrozil 1-O-beta-glucuronide is a potent and irreversible mechanism-based inhibitor of CYP2C8. Incubation of the metabolite with human liver microsomes in the presence of NADPH results in time-dependent, concentration-dependent, and NADPH-dependent loss of CYP2C8 enzymatic activity, characteristic of mechanism-based inactivation. The metabolite also inhibits CYP2C9, CYP2C19, and UGTs to a lesser extent. It acts as an inhibitor of OATP1B1-mediated transport, potentially increasing the plasma exposure of OATP1B1 substrate drugs. Detailed IC50 values for CYP2C8 inactivation and OATP inhibition are provided in specialized interaction studies but are not consistently included in standard compound datasheets. |
| ln Vivo |
The in vivo pharmacological activity of Gemfibrozil 1-O-beta-glucuronide relates primarily to its role as an active metabolite responsible for drug-drug interactions. Following oral administration of gemfibrozil, the parent drug is extensively glucuronidated in the liver, producing high concentrations of Gemfibrozil 1-O-beta-glucuronide in the systemic circulation and hepatocytes. The metabolite circulates in the plasma and reaches sufficient concentrations to inhibit CYP2C8 activity in the liver, leading to reduced clearance of co-administered drugs that are CYP2C8 substrates. This has been clinically documented with the antidiabetic drug repaglinide, where co-administration with gemfibrozil results in a marked increase in repaglinide plasma exposure and prolonged hypoglycemic effect. Gemfibrozil 1-O-beta-glucuronide is considered a key perpetrator of gemfibrozil's CYP2C8-mediated drug interactions in patients.
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| Enzyme Assay |
For CYP2C8 mechanism-based inactivation assays, human liver microsomes (0.2-0.5 mg protein/mL) are pre-incubated with various concentrations of Gemfibrozil 1-O-beta-glucuronide (0-200 microM) in 100 mM potassium phosphate buffer (pH 7.4) containing an NADPH-regenerating system (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and 1 U/mL glucose-6-phosphate dehydrogenase) at 37degC for varying pre-incubation times (0-30 minutes). Aliquots (10-20 microL) are removed at specified time points and diluted 20-50-fold into a secondary incubation mixture containing a specific CYP2C8 probe substrate (e.g., paclitaxel at 10 microM for 6alpha-hydroxylation or amodiaquine at 5 microM for N-deethylation) and fresh NADPH-generating system. The secondary incubation is performed for 5-15 minutes at 37degC (linear conditions). The reaction is terminated by adding an equal volume of ice-cold acetonitrile containing an internal standard. After centrifugation, the supernatant is analyzed by LC-MS/MS to quantify metabolite formation. The remaining CYP2C8 activity is calculated relative to control incubations without pre-inhibitor or without NADPH (to rule out direct inhibition). Kinetic parameters of inactivation (kinact, KI, and partition ratio) are determined by nonlinear regression analysis of the inactivation data. Control experiments are conducted to confirm NADPH dependence, time dependence, and concentration dependence, which are hallmarks of mechanism-based inactivation. Parallel incubations with gemfibrozil parent drug serve as negative controls (parent drug does not cause mechanism-based inactivation). Reversibility is assessed by dialysis or gel filtration of pre-incubated microsomes.
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| Cell Assay |
For in vitro OATP1B1 inhibition assays, HEK293 cells stably transfected with human OATP1B1 (OATP1B1-HEK293) are seeded in 24-well or 96-well plates (100,000-200,000 cells/well). Twenty-four hours later, cells are washed with HBSS buffer and pre-incubated with Gemfibrozil 1-O-beta-glucuronide (0-500 microM) or vehicle control for 10-15 minutes at 37degC. The uptake reaction is initiated by adding a fluorescent OATP probe substrate, such as 5 microM fluorescein methotrexate (FMTX) or 0.1 microM [3H]-estrone-3-sulfate (E3S), in HBSS buffer containing the same concentration of inhibitor. After 5-10 minutes at 37degC, the uptake is terminated by washing cells three times with ice-cold PBS. Cells are lysed with 0.1 M NaOH or 1% Triton X-100, and the fluorescence signal (for FMTX) or radioactivity (for [3H]-E3S) is measured. The half-maximal inhibitory concentration (IC50) for OATP1B1 inhibition is calculated from the concentration-response curve. Rifampicin (10-50 microM) is used as a positive control for OATP inhibition. Similar assays can be performed with OATP1B3-HEK293 cells.
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| Animal Protocol |
For cytochrome P450 enzyme inhibition studies using human liver microsomes (HLM) to assess the effect of Gemfibrozil 1-O-beta-glucuronide, cryopreserved human liver microsomes (pooled from multiple donors) are suspended in 0.1 M potassium phosphate buffer (pH 7.4) containing 3.3 mM MgCl2. Varying concentrations of Gemfibrozil 1-O-beta-glucuronide (0.1-500 microM) are incubated with HLMs (0.2-1 mg protein/mL) for 5-10 minutes at 37degC in a shaking water bath. The reactions are initiated by the addition of an NADPH-regenerating system (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and 1 U/mL glucose-6-phosphate dehydrogenase) and isoform-specific probe substrates at concentrations approximating their Km values (e.g., paclitaxel at 10 microM for CYP2C8, tolbutamide at 100 microM for CYP2C9, S-mephenytoin at 50 microM for CYP2C19, midazolam at 5 microM for CYP3A4, and dextromethorphan at 5 microM for CYP2D6). After incubation for 10-20 minutes at 37degC (optimized to ensure linear metabolite formation), the reaction is terminated by adding an equal volume of ice-cold acetonitrile containing an internal standard. Samples are centrifuged at 10,000-15,000 × g for 10 minutes at 4degC. The supernatant is analyzed by LC-MS/MS to quantify the formation of specific metabolites. Percentage of control activity is calculated relative to vehicle (DMSO or buffer) controls. A known selective CYP inhibitor (e.g., montelukast for CYP2C8, sulfaphenazole for CYP2C9, ticlopidine for CYP2C19) is used as a positive control. To assess mechanism-based inactivation, a pre-incubation step with NADPH for 0-30 minutes is included as described in the previous entry. The reversible component of inhibition (direct inhibition) is assessed by omitting the NADPH-regenerating system from the pre-incubation or by measuring activity without pre-incubation. Kinetic parameters (IC50, Ki, kinact, KI) are calculated using appropriate software (e.g., GraphPad Prism).
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| ADME/Pharmacokinetics |
As a glucuronide metabolite, Gemfibrozil 1-O-beta-glucuronide is more polar and water-soluble than the parent drug gemfibrozil. Following oral administration of gemfibrozil, the parent drug undergoes extensive first-pass glucuronidation in the liver by UDP-glucuronosyltransferase enzymes, primarily UGT2B7. The resulting glucuronide conjugate is actively transported into the systemic circulation via hepatic efflux transporters (e.g., MRP2, MRP3) and also undergoes biliary excretion. Gemfibrozil 1-O-beta-glucuronide circulates in the plasma at concentrations that can exceed those of the parent drug, particularly during repeated dosing. The metabolite is sufficiently stable in the systemic circulation to reach target tissues (e.g., hepatocytes) where it can inhibit CYP2C8 and OATP transporters. The elimination of Gemfibrozil 1-O-beta-glucuronide occurs primarily via urinary excretion (as the intact conjugate or after further metabolism) and biliary excretion. Detailed pharmacokinetic parameters for the metabolite itself are not provided in standard datasheets, but studies have shown that gemfibrozil treatment results in significant systemic exposure to its acyl glucuronide metabolite. The plasma protein binding of Gemfibrozil 1-O-beta-glucuronide is high, similar to that of the parent drug. Potential enterohepatic recirculation may occur following biliary excretion and deconjugation by gut microbiota, leading to reabsorption of gemfibrozil.
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| Toxicity/Toxicokinetics |
Gemfibrozil 1-O-beta-glucuronide is an acyl glucuronide conjugate, a class of metabolites known to be potentially reactive. Acyl glucuronides are electrophilic species that can undergo intramolecular rearrangement (acyl migration) and can covalently bind to plasma proteins and tissue macromolecules via transacylation or glycation reactions, which have been implicated in certain types of drug toxicity, including hypersensitivity reactions and hepatotoxicity. However, for gemfibrozil specifically, the parent drug has a well-established clinical safety profile when used at therapeutic doses (600 mg twice daily) for the treatment of hyperlipidemia. The glucuronide metabolite is considered to contribute to the drug-drug interaction potential of gemfibrozil but not to significant idiosyncratic toxicity. In preclinical species, high doses of gemfibrozil have been associated with hepatomegaly and peroxisome proliferation (rodent-specific effects related to PPARalpha activation, not relevant to humans). Formal toxicology studies for the isolated metabolite have not been conducted, as it is not administered as a therapeutic agent. Specialized studies of acyl glucuronide reactivity (e.g., protein adduct formation, isomerization kinetics) have been published in the literature but are beyond the scope of standard compound datasheets.
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| References |
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| Additional Infomation |
Gemfibrozil 1-O-beta-glucuronide (CAS# 91683-38-4) is a research-grade analytical reference standard intended for laboratory use only. It is not a therapeutic agent and has no clinical indications. The compound has a molecular weight of 426.46 and a molecular formula of C21H30O₉. It is a major metabolite of gemfibrozil, formed via glucuronidation by UGT2B7. This metabolite is an irreversible inhibitor of CYP2C8 and an inhibitor of OATP1B1, making it a key mediator of clinically significant drug-drug interactions. Gemfibrozil 1-O-beta-glucuronide is used in drug metabolism and pharmacokinetic (DMPK) studies, including CYP phenotyping, drug interaction risk assessment, and identification of metabolites. It may also be used as an internal standard or reference compound in LC-MS/MS methods for the quantification of gemfibrozil and its metabolites in biological matrices. The compound is typically stored as a powder at -20degC and is soluble in DMSO and organic solvents. Other names for this compound include Gemfibrozil 1-O-acylglucuronide and 1-O-Gemfibrozil-beta-D-glucuronide.
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| Molecular Formula |
C21H24DO9
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| Molecular Weight |
432.49446
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| Exact Mass |
426.189
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| CAS # |
91683-38-4
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| PubChem CID |
88127
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| Appearance |
White to off-white solid powder
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| Density |
1.32g/cm3
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| Boiling Point |
611.2ºC at 760mmHg
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| Flash Point |
208.4ºC
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| Index of Refraction |
1.572
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| LogP |
0.924
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
30
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| Complexity |
593
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| Defined Atom Stereocenter Count |
5
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| SMILES |
CC1=CC(=C(C=C1)C)OCCCC(C)(C)C(=O)O[C@H]2[C@@H]([C@H]([C@@H]([C@H](O2)C(=O)O)O)O)O
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| InChi Key |
CJMNXSKEVNPQOK-LVEJAMMSSA-N
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| InChi Code |
InChI=1S/C21H30O9/c1-11-6-7-12(2)13(10-11)28-9-5-8-21(3,4)20(27)30-19-16(24)14(22)15(23)17(29-19)18(25)26/h6-7,10,14-17,19,22-24H,5,8-9H2,1-4H3,(H,25,26)/t14-,15-,16+,17-,19-/m0/s1
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| Chemical Name |
(2S,3S,4S,5R,6S)-6-[5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoyl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~234.49 mM)
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3122 mL | 11.5610 mL | 23.1219 mL | |
| 5 mM | 0.4624 mL | 2.3122 mL | 4.6244 mL | |
| 10 mM | 0.2312 mL | 1.1561 mL | 2.3122 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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