Cytochrome P450 reaction phenotyping assay

Cytochrome P450 reaction phenotyping assay protocol

Test Compound Concentration 5 μM (different concentrations available) CYP Isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 Bactosomes™ or Supersomes™ Time Points 0, 5, 15, 30, 45 minutes Number of Replicates n = 1 per time point Negative Controls Without NADPH (45 minutes only) Control Bactosomes™/Supersomes™ (no CYP450 enzyme present) Positive Control Known probe substrate Compound Requirements 250 μL of 10 mM solution Analysis method LC-MS/MS Data Delivery Parent compound remaining at each time point for each isoform Half life Standard error of half life

Understanding which cytochrome P450 isoform is involved in the metabolism of a drug is important in predicting the propensity towards inter individual variability due to polymorphisms in enzyme expression and the tendency for drug-drug interactions.

Data from the Cloe Screen Cytochrome P450 Reaction Phenotyping assay

7 known substrates for the respective cytochrome P450 isoforms were screened in the Cloe Screen Cytochrome P450 Reaction Phenotyping assay. For the validation, the substrates were incubated with Bactosomes™ (bacterial membranes containing human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase) in the presence of NAPDH.

Questions and answers on cytochrome P450 reaction phenotyping

Please provide an overview of the Cloe Screen Cytochrome P450 Reaction Phenotyping assay.

Cytochromes P450 are a family of enzymes which play a major role in the metabolism of drugs. This screen is designed to identify which cytochrome P450 isoforms play a role in the metabolism of the compound of interest.

Test compound is incubated with expressed individual cytochrome P450 enzymes (Bactosomes™ or Supersomes™) at 37°C in the presence of the co-factor, NADPH, which initiates the reaction. The reaction is terminated by the addition of methanol. Following centrifugation, the supernatant is analysed by LC-MS/MS. The disappearance of test compound is monitored over a 45 minute time period. The test compound is also incubated in the presence of control Bactosomes™ or Supersomes™ (where no enzyme is present). The profile for the compound disappearance in the presence of the expressed enzyme is corrected for any disappearance observed in the presence of the control Bactosomes™ or Supersomes™. This ensures that only isoform-specific metabolism is determined. An example of a typical profile is detailed in Figure 3.

The ln peak area ratio (compound peak area/internal standard peak area) is plotted against time and the gradient of the line is determined.

The percent test compound remaining at each concentration (after correction for the disappearance in the presence of the control Bactosomes™ or Supersomes™) is also calculated and reported.

Why is it important to know which cytochrome P450 isoforms are involved in the metabolism of a drug?

There are several reasons why this screen is important. Firstly, the information may be used to predict potential drug-drug interactions with likely co-administered compounds. Secondly, certain cytochrome P450 enzymes are polymorphic (e.g., CYP2D6) and metabolism can vary considerably between individuals. If a compound is metabolised by an isoform which exhibits these characteristics, it may be detrimental, as plasma concentrations can vary considerably between subjects and determining an effective therapeutic range may be difficult.

How is the cytochrome P450 reaction phenotyping assay data used in drug discovery?

The data are interpreted in several ways. Firstly, clients may investigate the effect of structural changes on the extent of metabolism of the compound by a particular isoform. Secondly, if larger numbers of compounds are being screened, models can be built in order to predict whether a compound is likely to be metabolised by a particular isoform.

Drug metabolising systems which use cDNA expressed enzymes are artificial because the enzyme is not present in its native environment and is often over-expressed. With these systems, there is an absence of competing enzymes and reactions. It is therefore difficult to relate these data to either in vitro or in vivo measures of human clearance. The screen only identifies compounds which are capable of being metabolised by a particular isoform.

Typically, the reaction phenotyping assay will be performed for compounds which show medium or high clearance in the microsomal or hepatocyte stability assays in order to understand the mechanism of metabolism , or if metabolism by a particular isoform has been considered to be a problem in the past.

When should I consider my test compound to be metabolised by a particular cytochrome P450?

Typically, we would consider a compound to be metabolised by a particular cytochrome P450 isoform if the disappearance of the test compound is greater than 15% (or less than 85% of the test compound is remaining) at 45 minutes.

What controls are included in the cytochrome P450 reaction phenotyping assay?

Positive control compounds are included for each isoform. These are known isoform specific substrates, namely, ethoxycoumarin for CYP1A2, efavirenz for CYP2B6, amodiaquine for CYP2C8, diclofenac for CYP2C9, diazepam for CYP2C19, dextromethorphan for CYP2D6, and testosterone for CYP3A4. In addition we also run a minus NADPH control for both the control and expressed Bactosomes™ or Supersomes™ (as shown in Figure 3 – control dashed line).

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