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CYP Enzyme Inhibition
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).
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
Dedicated pool of 16 individuals, mixed gender, human liver microsomes
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.
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
Direct, time-dependent and metabolism-dependent IC50 values are determined from seven concentrations of the drug candidate.
Ki determinations: determine Ki 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. KI and Kinact determinations: determine KI and Kinact values for the inactivation of an enzyme by a drug candidate using multiple drug concentrations and pre-incubation time points.