Drug Metabolizing Enzyme (DME) Phenotyping, Inhibition, and Induction

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Cytochrome P450 Phenotyping

Establishing the major routes of clearance is essential for the development and safe clinical use of new drugs as it enables an understanding of potential drug-drug interactions (DDI) and risks associated with the metabolism by polymorphic enzymes or by different ethnic populations.

Our cytochrome P450 (CYP) reaction phenotyping assay is designed to identify the main CYP enzymes responsible for the primary metabolism of a drug candidate. The turnover (intrinsic clearance) of the test compound is determined after incubation with Bactosomes™ containing the recombinantly expressed human CYP isoforms 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and/or 3A5.

Confirmatory experiments should be performed in hepatocyte suspensions in the absence and presence of CYP-specific inhibitors to determine the quantitative contribution of CYP isoenzymes responsible for primary hepatic metabolism of the drug candidate (fractional metabolic clearance).

Both assays can be extended or customized to your needs, e.g., including elucidating the structure of metabolites formed after incubation with individual CYP isoforms or hepatocytes, ideally using a radiolabeled test compound.

Phenotyping assays for further drug metabolizing enzymes e.g., aldehyde oxidase (AO), monoamine oxidase (MAO), flavin-containing monooxygenase (FMO), alcohol and aldehyde dehydrogenase (ADH/ALDH), and carboxylesterase (CES) are available upon request.

Phenotyping assays for further drug metabolizing enzymes e.g., aldehyde oxidase (AO), monoamine oxidase (MAO), flavin-containing monooxygenase (FMO), alcohol and aldehyde dehydrogenase (ADH/ALDH), and carboxylesterase (CES) are available upon request.

Cytochrome P450 Inhibition

Inhibition of relevant drug metabolizing  CYPs are one of the most feared pharmacokinetic drug-drug interaction (DDI) mechanisms as it can alter both efficacy and safety. Hence, it needs to be investigated prior to the first dosage of a new chemical entity to patients, who may receive concomitant medications cleared by a potentially affected CYP pathway.

Our CYP inhibition assay is designed to evaluate the potential of the test compound exerting direct, time-dependent, and metabolism-dependent enzyme inhibition. Initial IC50-shift experiments are performed for the main drug-relevant CYP enzymes 1A, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A. Isoforms affected by direct, time-dependent and/or metabolism-dependent inhibition can be further investigated in terms of kinetic and mechanistic characterization of the inhibition (Ki, KI, and kinact).

In vitro inhibition assays for additional CYP isoforms are available upon request.

Cytochrome P450 Induction

The induction of drug metabolizing enzymes can lead to decreased systemic exposure of the inducing drug itself (auto-induction),  concomitant medications (potentially resulting in reduced or even lack of efficacy) or may increase the exposure towards active or toxic metabolites (causing adverse effects). Cytochrome P450 (CYP) 1A2, 2B6, and 3A4 are the key enzymes to detect induction effects on the three drug-drug interaction (DDI) relevant nuclear receptors, i.e., aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR).

Our in vitro assay is designed to assess the potential of a drug candidate to induce the expression of metabolizing CYP enzymes 1A2, 2B6, and 3A4 (optionally CYP2C8, 2C9, and 2C19). Primary cultures of human hepatocytes are treated with the test compound at different concentrations for at least two consecutive days. Its induction potential towards the CYP isoforms of interest can be evaluated by either quantification of mRNA expression levels (using bDNA technology) or enzyme activity, measuring the metabolic turnover of prototypical CYP marker substrates in relation to a vehicle control. If applicable, the concentration required for 50% (EC50) of the maximum induction (Emax) will be calculated. Comparison of the data generated to our internal relative induction score (RIS) to predict  itsclinical relevance can be provided upon request.

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