FDA- and EMA-recommended studies
Cytotoxicity and stability assays
CYP mRNA and enzyme induction
Inhibition of cytochrome P450 (CYP) and UGT enzymes is a major cause of clinically relevant drug-drug interactions.
The inhibitory potential of a test article is assessed by determining its effect on the metabolism of selective probe substrates for human CYP enzymes in pooled human hepatic microsome-based incubations. Inhibitory enzyme kinetics can be further characterized and the resultant data used to predict whether a clinically significant DDI may occur following administration of the drug.
These studies are recommended by both FDA and EMA drug-drug interactions (DDI) guidelines to generate data on both reversible (direct) and irreversible (time-dependent) cytochrome P450 inhibition before going to first-in-human trials. Inhibition data is used in determining the requirement and scope of clinical DDI studies.
Assays using 8 concentrations of test article are incubated in the absence and presence of NADPH for 30 minutes prior to dilution into a probe substrate assay mixture. If notable time-dependent inhibition is observed, additional kinetic parameters can be determined, including the inactivation constant (kinact) and the inhibition constant (KI). These parameters, determined experimentally by varying the pre-incubation time and inhibitor concentration, can help define the potential for drug-drug interactions.
Assays are performed in the absence and presence of 8 concentrations of test article to determine inhibition potential and to define an IC50 where possible.
Direct inhibition can be further characterized by determining the inhibition constant (Ki) and the type of inhibition observed, using 5 concentrations of probe substrate.
Cytochrome P450 |
Substrate |
Analyte |
Direct inhibition |
Time-dependent Inhibition |
|---|---|---|---|---|
CYP1A2 |
Phenacetin |
Acetaminophen |
Fluvoxamine |
Furafylline |
CYP2B6 |
Bupropion |
Hydroxybupropion |
Orphenadrine |
ThioTEPA |
CYP2C8 |
Amodiaquine |
Desethylamodiaquine |
Montelukast |
Gemfibrozil 1-O-β-glucuronide |
CYP2C9 |
Diclofenac |
4'-Hydroxydiclofenac |
Sulfaphenazole |
Tienilic Acid |
CYP2C19 |
Mephenytoin |
4'-Hydroxymephenytoin |
Nootkatone |
Esomeprazole |
CYP2D6 |
Dextrometorphan |
Dextrorphan |
Quinidine |
Paroxetine |
CYP3A4/5 |
Testosterone |
6β-Hydroxytestosterone |
Ketoconazole |
Erythromycin |
CYP3A4/5 |
Midazolam |
1'-Hydroxymidazolam |
Ketoconazole |
Troleandomycin |
These assays will provide IC50 values for direct or irreversible inhibition of CYP enzymes. If significant direct inhibition is observed, the inhibition constant (Ki) may be determined. If significant time-dependent inhibition is observed, the mechanism-based inactivation parameters (KI and kinact) may be determined. With these parameters, pharmacometrics may allow additional guidance when assessing the need for a clinical trial.
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