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Enzyme inhibition

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.

  • FDA- and EMA-recommended studies

  • Cytotoxicity and stability assays

  • CYP mRNA and enzyme induction

Regulatory considerations for enzyme inhibition studies

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.

Method

  • Test System: Pooled human liver microsomes (HLM)
  • CYP Enzymes Evaluated: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,CYP2E1 and CYP3A4/5
  • UGT enzymes evaluated in direct inhibition assays upon request: UGT1A1, UGT1A3, UGT1A4, UGT11A6, UGT1A9, UGT2B7, UGT2B15 
  • Test Article Concentrations: 8 concentrations in triplicate
  • All sample preparations and incubations are performed using a Hamilton Microlab Star automated liquid handling system

 

CYP inhibition is measured in both direct and dependent assays. A CYP-selective substrate is used, at a concentration of substrate that achieves half the maximum reaction velocity (Km) for each CYP enzyme (see below). Known inhibitors are used as positive controls for both direct and metabolism-dependent inhibition assays. All incubations are terminated by addition of chilled acetonitrile, containing a stable-labeled internal metabolite standard specific to the CYP substrate.

Time-Dependent (Irreversible) Inhibition

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. 

Direct Inhibition

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 P450SubstrateAnalyteDirect inhibitionTime-dependent Inhibition
CYP1A2PhenacetinAcetaminophenFluvoxamineFurafylline
CYP2B6BupropionHydroxybupropionOrphenadrineThioTEPA
CYP2C8AmodiaquineDesethylamodiaquineMontelukastGemfibrozil 1-O-β-glucuronide
CYP2C9Diclofenac4'-HydroxydiclofenacSulfaphenazoleTienilic Acid
CYP2C19Mephenytoin4'-HydroxymephenytoinNootkatoneEsomeprazole
CYP2D6DextrometorphanDextrorphanQuinidineParoxetine
CYP3A4/5Testosterone6β-HydroxytestosteroneKetoconazoleErythromycin
CYP3A4/5Midazolam1'-HydroxymidazolamKetoconazoleTroleandomycin

 

Deliverables

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.