Reaction Phenotyping

Reaction phenotyping studies assess the impact of enzyme isoform-specific inhibitors on the disappearance of a parent test article incubated in pooled human liver microsomes. Incubation with recombinant individual drug metabolizing enzyme isoforms provides confirmation of enzyme activities. Assessing the enzymes responsible for metabolizing a test article can highlight potential liabilities such as metabolism routes catalyzed by single enzymes or by polymorphically-expressed enzymes.

Regulatory considerations for reaction phenotyping studies

The investigation of CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6 and CYP3A) is recommended as an initial investigation to better understand the clearance pathways of the drug according to the drug-drug interaction (DDI) guidelines. If these major CYP enzymes are not involved in the metabolism of the drug, then other Phase I and Phase II enzymes should be evaluated including:

  • CYP enzymes (CYP2A6, CYP2J2, CYP4F2, and CYP2E1)
  • Non-CYP enzymes including aldehyde oxidase (AO), carboxylesterase (CES), monoamine oxidase (MAO), flavin monooxygenase (FMO), xanthine oxidase (XO), and alcohol/aldehyde dehydrogenase (ADH/ALDH)
  • Conjugative enzymes including UDP glucuronosyl transferases (UGT) and sulfotransferases (SULTs)

Reaction phenotyping provides information regarding the fraction metabolized of test articles and can be used to assess victim DDI potential, inform clinical study design, predict individual variability in pharmacokinetics and evaluate the impact of genetic polymorphism.


Methods

  • Test system: Pooled human hepatic microsomes and recombinant individually expressed human enzymes with required cofactors
  • CYP isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. (Other CYPs, UGTs and drug metabolizing enzymes are also available).
  • Isoform-specific chemical inhibitors and recombinant enzymes are used for confirmation.

The test article is pre-incubated for least 5 minutes with microsomes in phosphate buffer. Metabolism of the test article is initiated by the addition of NADPH or other appropriate cofactor. Incubations are terminated by the addition of an organic solvent and samples are then analyzed for the test article and/or its metabolites using liquid chromatography-mass spectrometry (LC-MS).

Phase I - Optimization of In Vitro Incubation Conditions

The test article is incubated in triplicates with a range of microsome concentrations (typically 0.1 – 1 mg protein/mL) for four time points up to 60 minutes in the presence of NADPH or other appropriate cofactor.

Control incubations are performed in the absence of a cofactor. Incubation conditions of microsomal concentration and time point that produce a linear rate of test article disappearance are used to define follow up experiments.

Phase II - Kinetic Analysis of Test Article Metabolism

The rates of how quickly the test article disappears are monitored in the incubation samples with at least eight concentrations of the test article in triplicates under optimized conditions. Data are analyzed using Michaelis-Menten curve fitting, and kinetic parameters Km and Vmax are determined for the test article.

Phase III - Inhibition Analysis Using CYP-specific Chemical Inhibitors

The test article is incubated in triplicate at a concentration <Km with pooled human hepatic microsomes in the absence or presence of the enzyme-selective chemical inhibitors. Control incubations are performed in the absence of the inhibitor using vehicle solvent only.

Phase IV - Metabolism Using Recombinant Human CYPs

The test article is incubated in triplicate at a concentration <Km with a range of recombinant human CYPs and UGTs, and other DME (see above). The relative concentration of the test article at 0 and 60 minutes is measured. 

Deliverables

These assays will identify enzymes responsible for and the extent of metabolism of the test article in vitro. Kinetics of the test article disappearance and/or metabolite formation are determined.  In vitro metabolism assessment identifies the major enzymes involved and alerts you to potential in vivo clearance mechanism liabilities prior to first-in-human testing.