Simultaneous phenotyping of CYP2E1 and CYP3A using oral chlorzoxazone and midazolam microdoses

Br J Clin Pharmacol. 2019 Oct;85(10):2310-2320. doi: 10.1111/bcp.14040. Epub 2019 Aug 9.


Aims: Chlorzoxazone is the paradigm marker substrate for CYP2E1 phenotyping in vivo. Because at the commonly used milligram doses (250-750 mg) chlorzoxazone acts as an inhibitor of the CYP3A4/5 marker substrate midazolam, previous attempts failed to combine both drugs in a common phenotyping cocktail. Microdosing chlorzoxazone could circumvent this problem.

Method: We enrolled 12 healthy volunteers in a trial investigating the dose-exposure relationship of single ascending chlorzoxazone oral doses over a 10,000-fold range (0.05-500 mg) and assessed the effect of 0.1 and 500 mg of chlorzoxazone on oral midazolam pharmacokinetics (0.003 mg).

Results: Chlorzoxazone area under the concentration-time curve was dose-linear in the dose range between 0.05 and 5 mg. A nonlinear increase occurred with doses ≥50 mg, probably due to saturated presystemic metabolic elimination. While midazolam area under the concentration-time curve increased 2-fold when coadministered with 500 mg of chlorzoxazone, there was no pharmacokinetic interaction between chlorzoxazone and midazolam microdoses.

Conclusion: The chlorzoxazone microdose did not interact with the CYP3A marker substrate midazolam, enabling the simultaneous administration in a phenotyping cocktail. This microdose assay is now ready to be further validated and tested as a phenotyping procedure assessing the impact of induction and inhibition of CYP2E1 on chlorzoxazone microdose pharmacokinetics.

Keywords: CYP2E1; CYP3A; drug interactions; microdosing; phenotyping.

Publication types

  • Randomized Controlled Trial

MeSH terms

  • Administration, Oral
  • Adult
  • Area Under Curve
  • Chlorzoxazone / administration & dosage*
  • Chlorzoxazone / pharmacokinetics
  • Cytochrome P-450 CYP2E1 / metabolism*
  • Cytochrome P-450 CYP3A / metabolism*
  • Dose-Response Relationship, Drug
  • Drug Interactions
  • Female
  • Humans
  • Male
  • Midazolam / administration & dosage*
  • Midazolam / pharmacokinetics
  • Middle Aged
  • Phenotype
  • Young Adult


  • Cytochrome P-450 CYP2E1
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • Chlorzoxazone
  • Midazolam