Reaction Phenotyping


Knowing how a drug candidate is eliminated by the human body is important in understanding the potential for drug-drug interactions. Compounds with a single route of elimination have a high victim potential, which is why the FDA requires reaction phenotyping studies.

CYP reaction phenotyping generally involves three types of analysis:
  • Metabolism by recombinant human CYP enzymes
  • Inhibition approach (antibody and/or chemical inhibition)
  • Correlation analysis

Each has its advantages and disadvantages, and a combination of approaches is highly recommended.

Sekisui XenoTech's experimental approach conforms to the scientific principles described in the FDA DDI draft guidance for industry (2012), the EMA guideline on the investigation of drug interactions (2012), established scientific practices, or current state of the art.

It is ideal to conduct a phenotyping study for the metabolite of interest (ex. quantify the metabolite formation). However, the study may be performed based on the substrate (drug candidate) disappearance when the study is performed at early stage of drug development and neither metabolic profiling has been elucidated nor authentic metabolite standards have been synthesized.

Analytical Method Development

XenoTech's Analytical Service Group will develop a robust analytical method for the study.

Determining  the Effect of Time, Protein and Substrate Concentrations ("Preliminary Evaluation")

Before proceeding with reaction phenotyping of the compound, we conduct an experiment to evaluate the effect of incubation time, protein concentration and substrate concentration on the conversion of the compound to its metabolite(s). This experiment will establish if metabolite formation is proportional to incubation time and protein concentration and help determine if the metabolites are primary metabolites or secondary metabolites. This expeiment is performed as a "Preliminary Evaluation" as part of a reaction phenotyping study to establish appropriate incubation conditions for the study.

Determining Michaelis-Menten Kinetic Constants

After the establishment of initial rate conditions (i.e., conditions under which metabolite formation is proportional to incubation time and protein concentration, and the metabolism of the substrate does not exceed 10%), an experiment is typically performed to determine the kinetic constants, namely Km and Vmax. These kinetic constants provide useful information to assess the importance of specific metabolic pathways of the compound. Km and Vmax are determined by incubating microsomes with a range of substrate concentrations, typically 0.1 to 10 times the crude Km value estimated in the experiment to determine initial rate conditions.

Recombinant Human CYP Enzymes

Recombinant human CYP enzymes are incubated individually with a test compound to assess the ability of a particular CYP enzyme to metabolize it. Additional experiments may be carried out to allow determination of Km and Vmax for each CYP enzyme that contributes to the reaction.

Antibody and Chemical Inhibition 

Antibody and chemical inhibition experiments involve an evaluation of the effect of selective CYP inhibition on the metabolism of a drug in pooled human liver microsomes. Chemical inhibitors can be non-selective when used at inappropriate concentrations; Sekisui XenoTech has extensive experience designing these experiments and avoiding such problems. The concern arising with chemical inhibitors is not a factor with those antibodies that have been shown to be selective CYP inhibitors.

Correlation Analysis

Correlation analysis involves determining the rate of drug metabolism by several samples of individual human liver microsomes, followed by correlating reaction rates with sample-to-sample variation of CYP enzyme activity in each individual sample. The FDA September 2006 Guidance for Industry recommends using at least 10 individual donors for this, with sufficient variance between activities to ensure adequate statistical power. This approach is successful because the variation in CYP enzyme activity from sample to sample varies greatly (up to 100-fold) and independently from one enzyme to the other. Differences in the rates of formation of the test article metabolites will be compared with the sample-to-sample variation in the following activities:
Enzyme Substrate
CYP1A2 Phenacetin O-dealkylation
CYP2A6 Coumarin 7-hydroxylation
CYP2B6 Bupropion hydroxylation
CYP2C8 Amodiaquine N-dealkylation
CYP2C9 Diclofenac 4´-hydroxylation
CYP2C19 S-Mephenytoin 4´-hydroxylation
CYP2D6 Dextromethorphan O-demethylation
CYP2E1 Chlorzoxazone 6-hydroxylation
CYP2J2 Ebastine hydroxylation
CYP3A4/5 Testosterone 6β-hydroxylation
CYP3A4/5 Midazolam 1´-hydroxylation
CYP4A11 Lauric acid 12-hydroxylation

UGT Reaction Phenotyping

Similar approaches are applicable to UGT Reaction Phenotyping. Metabolism can be carried out by recombinant human UGT enzymes and chemical inhibition (selective chemical inhibitors are not known for certain UGT enzymes). Correlation analysis is also available upon request.

For both CYP and UGT Reaction Phenotyping, the ideal approach is to quantify the formation of the metabolite of interest. However, when the metabolite standard is not available, a study may be performed based on the substrate disappearance. In addition, these studies may be performed with the use of radio-labeled compound. In such cases, metabolite quantification is feasible without an authentic metabolite standard because the calibration curve will be constructed with the radio-labeled parent compound to quantify both the substrate disappearance and the metabolite formation.
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