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Metabolism-Based Drug-Drug Interaction Studies

Due to the possibility of associated drug interactions and hepatotoxicity, the USFDA has placed new emphasis on metabolic drug-drug interaction studies for determining whether a new chemical entity (NCE) is likely to affect the metabolic elimination of drugs already on the market or vice versa.  To address this requirement, CellzDirect offers CYP450 induction, CYP450 inhibition, and in vitro phase IV drug interaction studies.

Cytochrome P450 (CYP450) Induction 

CYP450 enzymes can be induced as a result of drug exposure, which may cause increased formation of toxic metabolites and/or decreased systemic levels of a co-administered drug, and accordingly make that drug less effective or more toxic.  Primary cultures of human hepatocytes are widely accepted as the gold standard for determining enzyme induction potential of drugs in vitro.  CellzDirect's scientific staff have been instrumental in advancing the science and technologies relating to the use of human hepatocytes for assessing enzyme induction.

In a typical study, fresh human hepatocytes from three separate donors are exposed daily for 2-3 days with test article at clinically-relevant concentrations.  Drug-induced  changes in CYP 450 inmmunoreactive protein or mRNA are measured by western immunoblot or quantitative PCR (Taqman®), respectively.  Changes in CYP450 enzyme activities are determined either by placing probe substrates directly onto intact monolayers or using isolated microsomes.  The relative potency of a NCE is compared to that of known positive controls in accordance with the USFDA guidance on drug-drug interactions.

Ex vivo analysis of enzyme induction can also be performed by measuring cytochrome P450 activities in microsomes prepared from liver tissue harvested from animals treated with an NCE and appropriate controls.

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CYP450 Inhibition (Direct and Time-Dependent)

Inhibition of a specific drug-metabolizing enzyme can decrease the metabolic clearance of a co-administered drug leading to elevated blood concentrations, which may cause toxicity or enhanced effects.  In an IND submission, the USFDA requires the determination of the potential for a drug candidate to inhibit CYP450 enzymes.

Inhibition of CYP450 activity can be evaluated by incubating a NCE at multiple concentrations with pooled human microsomes or hepatocytes and known CYP450-specific substrates.  The effect of the NCE on turnover of CYP450-specific substrates is determined by LC-MS/MS analysis of metabolite formation, and an IC50 value is determined.  For assessment of time-dependent inhibition a pre-incubation of the test compound with pooled microsomes in the presence and absence of NADPH is performed prior to determining the effect on turnover of probe substrates. If significant inhibition (i.e. IC50 ≤ 50 µM) is observed after initial evaluation, then the Ki (or kinact and KI for time-dependent inhibition) for the relevant CYP450 isoforms is typically determined.

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In Vitro Phase IV Drug Interaction Studies

Drug interactions involving a NCE may lead to changes in the metabolism of co-administered compounds . Likewise, inhibition by a co-administered compound may lead to changes in the metabolism of the NCE.  If these interactions are not identified early in drug development, the result can be exposure to toxic drug levels in some patients, resulting in a “black box” warning on the product label or withdrawal from the market.  

In a typical in vitro Phase IV study, the rate of disappearance of certain marketed compounds is measured in the presence and absence of a NCE in human liver microsomes or hepatocytes.  The effect of administering the marketed compounds with the test compound also is examined by monitoring the disappearance of the test compound in the same incubates.  The goal is to predict whether patients taking the marketed compounds might be susceptible to a metabolic interaction with the test compound.  To this end, methods can be developed and validated for each marketed compound in human hepatocytes.  Concentrations of the marketed compounds and test compound are chosen based on relevant clinical or PK data.

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Metabolism-Based Drug-Drug Interaction Studies As Described by the Draft FDA Guidance (2006)

"Many metabolic routes of elimination, including most of those occurring through the P450 family of enzymes, can be inhibited or induced by concomitant drug treatment. Observed changes arising from metabolic drug-drug interactions can be substantial — an order of magnitude or more decrease or increase in the blood and tissue concentrations of a drug or metabolite — and can include formation of toxic and/or active metabolites or increased exposure to a toxic parent compound. These large changes in exposure can alter the safety and efficacy profile of a drug and/or its active metabolites in important ways. This is most obvious and expected for a drug with a narrow therapeutic range (NTR), but is also possible for non-NTR drugs as well (e.g., HMG CoA reductase inhibitors). It is important that metabolic drug-drug interaction studies explore whether an investigational agent is likely to significantly affect the metabolic elimination of drugs already in the marketplace and likely in medical practice to be taken concomitantly and, conversely, whether drugs in the marketplace are likely to affect the metabolic elimination of the investigational drug. Even drugs that are not substantially metabolized can have important effects on the metabolism of concomitant drugs. For this reason, metabolic drug-drug interactions should be explored, even for an investigational compound that is not eliminated significantly by metabolism."