| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
CYP2E1 (marker substrate)
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
Chlorzoxazone is a well-established probe substrate for assessing CYP2E1 activity in vitro and in vivo, making its accurate quantification critical for drug-drug interaction studies, pharmacogenomics, and toxicology research. Chlorzoxazone is metabolized by CYP2E1 to 6-hydroxychlorzoxazone. The deuterated form (Chlorzoxazone-d3) is used for isotope tracing to accurately quantify the parent compound and its metabolite by LC-MS/MS, eliminating matrix effects and improving assay sensitivity. Chlorzoxazone-d3 itself has no intrinsic pharmacological or toxicological differences from the non-deuterated compound. |
| ln Vivo |
In vivo, chlorzoxazone (10 mg/kg) decreases alcohol, but not water, intake in a dose-dependent manner and reduces the propensity for rapid initial alcohol intake in rats with intermittent, but not continuous, access to alcohol. Formulations containing chlorzoxazone have been used in the treatment of pain and stiffness caused by muscle spasm. Chlorzoxazone-d3, as a stable isotope-labeled analog, is used in clinical and preclinical studies as an internal standard for pharmacokinetic studies to quantify CYP2E1 activity, rather than as a therapeutic agent itself.
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| Enzyme Assay |
For in vitro enzyme activity assays, human liver microsomes (HLMs) or recombinant CYP2E1 are incubated with chlorzoxazone (50-500 uM) as a probe substrate, along with an NADPH-regenerating system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP+, MgCl2) in potassium phosphate buffer (pH 7.4). The reaction is incubated at 37degC for 10-30 minutes. The reaction is stopped by adding acetonitrile containing an internal standard (e.g., Chlorzoxazone-d3). The metabolite, 6-hydroxychlorzoxazone, is quantified by LC-MS/MS. The assay can be used to determine enzyme kinetic parameters (Km, Vmax) and inhibition constants (IC50, Ki) for CYP2E1 inhibitors.
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| Cell Assay |
Primary human hepatocytes or CYP2E1-overexpressing cell lines (e.g., HEK293-CYP2E1) are cultured in appropriate media. Cells are treated with chlorzoxazone (50-500 uM) for 1-4 hours in culture medium. Chlorzoxazone-d3 can be added to the medium as an internal standard for quantification. The metabolite (6-hydroxychlorzoxazone) in the culture supernatant is quantified by LC-MS/MS. Cell viability is assessed by MTT or LDH assays. The assay can be used to screen for CYP2E1 inducers or inhibitors in a cellular context. Chlorzoxazone-d3 is used as an internal standard to correct for matrix effects and extraction recovery.
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| Animal Protocol |
In vivo, chlorzoxazone is used as a probe to assess CYP2E1 activity in humans and animal models. Typically, a single oral dose of chlorzoxazone (e.g., 250-500 mg in humans, or 10-20 mg/kg in rodents) is administered. Blood samples are collected at various time points (0-8 hours). Plasma concentrations of chlorzoxazone and its metabolite 6-hydroxychlorzoxazone are measured by LC-MS/MS. Chlorzoxazone-d3 is used as an internal standard in the bioanalytical method. The metabolic ratio (6-hydroxychlorzoxazone/chlorzoxazone) or clearance of chlorzoxazone is used as a measure of CYP2E1 activity in vivo. The assay is used to assess CYP2E1 phenotype, drug-drug interactions, and effects of disease states or genetic polymorphisms.
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| ADME/Pharmacokinetics |
Chlorzoxazone is rapidly absorbed after oral administration, with a plasma half-life of approximately 1 hour in humans. It is extensively metabolized by CYP2E1 to 6-hydroxychlorzoxazone, which is then conjugated and excreted in urine. Chlorzoxazone-d3 has identical pharmacokinetics to chlorzoxazone due to the same chemical structure except for three deuterium atoms, but it is used as an internal standard, not administered as a separate entity. Stable heavy isotopes (deuterium) have been incorporated into drug molecules as tracers for quantification during drug development.
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| Toxicity/Toxicokinetics |
Chlorzoxazone has a well-established safety profile as a muscle relaxant. It is generally well tolerated at therapeutic doses (250-500 mg three times daily in humans). Common side effects include drowsiness, dizziness, gastrointestinal disturbances, and liver enzyme elevations. Rare cases of hepatotoxicity have been reported. Chlorzoxazone-d3, as an isotope-labeled analog, is used in research at very low concentrations (as internal standard) and poses no significant toxicological risk beyond that of the parent compound. For research use only; not for human therapeutic use.
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| References |
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| Additional Infomation |
Chlorzoxazone-d3 (CAS: 1185173-60-7) is a stable isotope-labeled compound used as an internal standard in LC-MS/MS assays for CYP2E1 activity. The non-deuterated Chlorzoxazone is a centrally acting muscle relaxant, but the deuterated form is used exclusively as a research tool for drug metabolism and pharmacokinetic (DMPK) studies, pharmacogenomics, and drug-drug interaction research. Molecular weight: 172.58. Molecular formula: C7H4ClNO2 with 3 deuterium atoms. It acts on the spinal cord by depressing reflexes. It is a marker substrate for CYP2E1 in vitro and in vivo. Chlorzoxazone-d3 is for research use only and is not intended for diagnostic or therapeutic use. It is supplied as a high-purity powder.
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| Molecular Formula |
C7HD3CLNO2
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|---|---|
| Molecular Weight |
172.58
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| Exact Mass |
168.993
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| CAS # |
1185173-60-7
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| Related CAS # |
Chlorzoxazone;95-25-0
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| PubChem CID |
45038674
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| Appearance |
Light yellow to light brown solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.603
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| LogP |
2.19
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
11
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| Complexity |
185
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C1=C(C(=C(C2=C1OC(=O)N2)[2H])Cl)[2H]
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| InChi Key |
TZFWDZFKRBELIQ-CBYSEHNBSA-N
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| InChi Code |
InChI=1S/C7H4ClNO2/c8-4-1-2-6-5(3-4)9-7(10)11-6/h1-3H,(H,9,10)/i1D,2D,3D
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| Chemical Name |
5-chloro-4,6,7-trideuterio-3H-1,3-benzoxazol-2-one
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 5.7944 mL | 28.9721 mL | 57.9441 mL | |
| 5 mM | 1.1589 mL | 5.7944 mL | 11.5888 mL | |
| 10 mM | 0.5794 mL | 2.8972 mL | 5.7944 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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