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.
Reaction phenotyping generally involves three types of analysis: correlation analysis, antibody and chemical inhibition, and metabolism by recombinant human CYP enzymes. Each has its advantages and disadvantages, and a combination of approaches is highly recommended.
- Selecting the best test system
Using the structure of a compound and our knowledge of the enzymes likely to be involved in its metabolism, we select the most appropriate test system to obtain meaningful data about each drug candidate.
Read moreFor example, a compound with a simple alkane moiety will most likely undergo hydroxylation and/or dehydrogenation. Those reactions are commonly catalyzed by CYP enzymes, so NADPH-fortified liver microsomes would be an appropriate test system. On the other hand, a compound containing an aliphatic amine moiety in its structure could undergo a variety of metabolic pathways (e.g., N-dealkylation, N-oxidation, glucuronidation, sulfation, methylation, acetylation, etc.). In such case, we would recommend testing in hepatocytes, which contain essentially all liver drug metabolizing enzymes and endogenous cofactors. - Developing and evaluating analytical procedure
We then develop an analytical procedure to measure the rate of formation of the metabolites. We optimize conditions to maximize the detection of all possible metabolites in our study design, compare the metabolite profiles with blanks and use the resulting information to develop the best separation and detection techniques.
- Determining the effect of time, protein and substrate concentrations
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.
- Determining Michaelis-Menten kinetic values
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 20%), 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.
- 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 the 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. (See
Reaction Phenotyping Kit) 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.
- 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; 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. (
Click here to view Inhibitory Antibody products)
- Recombinant 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. (
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In addition to our patented Reaction Phenotyping Kit and publications on the conduct of reaction phenotyping studies, XenoTech's innovation in the area of reaction phenotyping is illustrated by our role in the discovery of the contribution of CYP4F enzymes in the metabolism of an antimalarial drug candidate. Read more
Michael Zhuo Wang et al.: CYP4F Enzymes Are the Major Enzymes in Human Liver Microsomes That Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289 [2,5-Bis(4-amidinophenyl)furan-bis-O-methlamidoxime]. Drug Metab Dispos, 34: 1985-1994, (2006). The enzymes typically evaluated for reaction phenotyping purposes were found not to be responsible for the hepatic metabolism of the drug candidate, which led to the discovery that CYP4F enzymes, previously thought to metabolize fatty acids and other endobiotics, can play a key role in the metabolism of certain drugs.