In Vitro Cytochrome P450 Inhibition and Induction

Abstract

The assessment of in vitro inhibition and induction of the cytochrome P450 enzymes of the liver is a critical part of the drug discovery and development process in order to ensure that two or more drugs can be safely coadministered without alterations in exposure. Early assessment of potential candidates using high throughput approaches provides key direction in choosing the most promising chemical series to pursue. In later stage development, the use of in vitro data to assess the potential for clinical interactions is now a practice readily accepted by regulatory authorities. Inhibition of drug metabolizing enzymes can occur via two principal mechanisms, reversible inhibition and time dependent inhibition (mechanism-based inactivation). Clinically, either of these mechanisms can lead to reduced clearance of a coadministered drug and potentially toxic levels may be reached. Inducers of a drug metabolizing enzyme can increase the clearance of other drugs, or itself, resulting in a decreased therapeutic effect; they can also increase the bioactivation of drugs that can produce reactive intermediates, leading to hepatotoxicity. A number of in vitro models composed of human-derived microsomes, recombinantly expressed human drug metabolizing enzymes, human-derived cell lines, as well as fresh and cryopreserved human hepatocytes, are increasingly in use to evaluate inhibition and induction. In this review, the authors' understanding of currently utilized enzyme inhibition and induction methodologies are presented and the authors provide recommendations regarding which assay types offer the greatest advantage during the drug development process.

Identification of Key Licorice Constituents Which Interact with Cytochrome P450: Evaluation by LC/MS/MS Cocktail Assay and Metabolic Profiling

“…Analytes were recognized by the mass-to-charge ratio (m/z) of their pseudomolecular ion and a specific fragment ion. By matching these ion pairs, SRM mode ensures the specificity of target analytes and excludes the inference from other substrates/metabolites which could not be separated by HPLC (21,22).…”

“…The incubation mixture also contained an nicotinamide adenine dinucleotide phosphateoxidase (NADPH)-generating system (1 mM of NADP, 6 mM of G-6-P, and 2 unit/mL of G-6-P-DE), P450 probe substrates (20/10/40/6/2/40 μM of PHE/OME/TOL/DEX/MID/TES), and licorice compounds or extracts (0.1-200 μM, three to five concentrations for each sample). Compounds 8,14,22,24,25,27,30, and 34 were diluted in a mixture of MeOH and DMSO (1:1, v/v). The other compounds and the four extracts were dissolved in methanol.…”

“…Chemical structures of licorice compounds (isolated from Glycyrrhiza uralensis Fisch.). The compounds were 4-hydroxybenzoic acid (1), phloretic acid (2), liquiritin apioside (3), liquiritin (4), isoliquiritin apioside (5), isoliquiritin (6), ononin (7), 4′,7-dihydroxyflavone (8), liquiritigenin (9), licoricesaponin A3 (10), licopyranocoumarin (11), echinatin (12), naringenin (13), genistein (14), licorice-saponin G2 (15), licorice-saponin E2 (16), 3-methylkaempferol (17), davidigenin (18), glycyrrhizic acid (19), isoliquiritigenin (20), 7,4′-dihydroxy-3′-methoxyisoflavan (21), formononetin (22), glycycoumarin (23), semilicoisoflavone B (24), kumatakenin (25), licoisoflavone A (26), licoricone (27), isolicoflavonol (28), lupiwighteone (29), glycyrol (30), glyurallin A (31), licoflavonol (32), topazolin (33), glycyrin (34), gancaonin I (35), angustone A (36), isoangustone A (37), gancaonin G (38), 6,8-diprenylgenistein (39), and 18β-glycyrrhetinic acid (40). Api apinose, GluA glucuronic acid, Glc glucose, Prenyl isoprenoid.…”

“…If the test compound did not inhibit the CYP isozymes, the probe substrates would be metabolized to their corresponding metabolites, which can then be measured by LC/MS/MS (9,22). The probe compounds, viz.…”

Similarities and Differences in the Expression of Drug-Metabolizing Enzymes between Human Hepatic Cell Lines and Primary Human Hepatocytes

“…Genetic polymorphisms, including single nucleotide polymorphism, copy number variation, and insertion and deletion variation, contribute greatly to DMET expression profiles, drug metabolism, and clinical impacts (Zhou et al, 2008(Zhou et al, , 2009). In addition, environmental factors such as exogenous inducers and inhibitors may produce more heterogeneous DMET expression/activity and drug responses (Hewitt et al, 2007b;Walsky and Boldt, 2008). Donor variations in the responses to inducers and inhibitors (i.e., gene-environment interactions) further complicate the selection of primary hepatocytes for pharmacological and toxicological studies.…”