CYP Enzyme Inhibition

Assay SelectionTest SystemIncubation ConditionsAutomation and AnalysisResultsFollowup Studies

Click Here to download XenoTech's poster (ISSX 2009) regarding the pitfalls of using a dilution step when examining metabolism-dependent inhibition or time-dependent inhibition!

XenoTech's study design evaluates drug candidates as direct, time-dependent and metabolism-dependent inhibitors of CYP enzymes in human liver microsomes. Our study design allows us to screen for inhibition by metabolites and features low protein concentrations and short incubation times to minimize artifacts caused by protein binding, metabolic instability of the test article and excessive metabolism of the marker substrate. We offer various options for follow-up studies to support the further characterization of those drug candidates that are identified as potent direct or metabolism-dependent inhibitors (for example: Ki determinations, XTRA: XenoTech's Reversibility Assay, KI/kinact determinations).

Download XenoTech's Drug Inhibition Informational Flyer Here!

Assay Selection

CYP1A2      Phenacetin O-dealkylation
CYP2A6      Coumarin 7-hydroxylation
CYP2B6      Efavirenz 8-hydroxylation
CYP2B6      Bupropion hydroxylation
CYP2C8      Amodiaquine N-dealkylation
CYP2C8      Paclitaxel 6-hydroxylation
CYP2C9      Diclofenac 4´-hydroxylation
CYP2C19    S-Mephenytoin 4´-hydroxylation
CYP2D6      Dextromethorphan O-demethylation
CYP2E1      Chlorzoxazone 6-hydroxylation
CYP3A4/5   Testosterone 6β-hydroxylation
CYP3A4/5   Nifedipine oxidation
CYP3A4/5   Midazolam 1´-hydroxylation
CYP3A4/5   Atorvastatin ortho-hydroxylation
CYP4A11    Lauric acid 12-hydroxylation

Test System

Dedicated pool of 16 individuals, mixed gender, human liver microsomes

More Information

XenoTech’s inhibition assays are typically performed with a dedicated pool of human liver microsomes characterized to provide Km and Vmax values for each marker substrate reaction.  This allows the same pool of human liver microsomes (and often the same experimental conditions, for example, protein concentration and incubation time) to be used to study all CYP enzymes of interest. 

Incubation Conditions

Typical IC₅₀ incubation conditions

Protein concentration: ≤0.1 mg/mL

Incubation time: 5 minutes

Pre-incubation time (in the presence and absence of NADPH): 30 minutes

The following plots represent the effect of protein concentration, incubation time and pre-incubation time on the IC₅₀ value.

Automation and Analysis

Tecan liquid-handling system or manual incubations available
Validated LC/MS/MS methods
High-throughput Shimadzu autosamplers
Deuterated internal standards
Automated data retrieval and processing
Comprehensive, publication-quality reports
Available as GLP or non-GLP

Results

Direct, time-dependent and metabolism-dependent IC₅₀ values are determined from seven concentrations of the drug candidate.

Followup Studies

Direct Inhibition

K_i determinations: determine K_i value for direct inhibition with multiple concentrations of the drug candidate and marker substrate

Metabolism-Dependent Inhibition (MDI)

1. XTRA (XenoTech Reversibility Assay): our unique approach to determine qualitative, mechanistic inhibition information about your compound’s metabolites. This assay determines if the metabolism-dependent inhibition is due to:

      -formation of a more potent reversible inhibitory metabolite after ultracentrifugation
      -formation of a quasi-irreversible metabolite inhibitor complex (MIC formation)
      -irreversible inactivation (indicates possible covalent binding)

Click Here to download XenoTech's XTRA Poster presented at ISSX in 2008.

2. MIC Formation: evaluation of a drug candidate for spectral changes associated with metabolite inhibitory complex formation.

3. K_I and K_inact determinations: determine K_I and K_inact values for the inactivation of an enzyme by a drug candidate using multiple drug concentrations and pre-incubation time points.

Click on the chart above to expand