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Nonspecific binding of (a) midazolam and (b) ketoconazole. Fraction unbound, fu, is the ratio of the free concentration, Sf, to the total concentration, St. Circles, triangles, and plus marks represent concentrations of 1, 2, and 7.5 µmol/l of midazolam. Circles and triangles represent observed data at 1 and 100 µmol/l of ketoconazole. Lines indicate predicted binding at the different concentrations of midazolam (solid line: 1 µmol/l; dashed line: 2 µmol/l; dotted line: 7.5 µmol/l) or ketoconazole (solid line: 1 µmol/l; dashed line: 100 µmol/l).
Source publication
In vitro screening for drug–drug interactions is an integral component of drug development, with larger emphasis now placed on the use of in vitro parameters to predict clinical inhibition. However, large variability exists in K i reported for ketoconazole with midazolam, a model inhibitor–substrate pair for CYP3A. We reviewed the literature and ex...
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Citations
... Therefore, for unbiased kinetic determinations the unbound substrate concentration should be used for determinations of Michaelis-Menten kinetics [70]. However, if no data on the fraction unbound is available, as it is the case for VRC, low HLM concentrations should be chosen, having a smaller influence on the unbound fraction-as has been shown for midazolam [73]-and thus a smaller influence on the determined kinetics. Fifth and last, the observation of metabolite formation is generally favorable compared to substrate depletion [32]. ...
The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization.
... However, for this approach to be successful, the inhibitor should be potent, selective, metabolically stable to be able to attain >90% inhibition of the targeted enzyme over the incubation period. Based on the limited literature data available so far (Stresser et al., 2004;Parmentier et al., 2017), the potent CYP3A inhibitor azamulin, has potentially better properties to meet these criteria compared to the gold standard CYP3A inhibitor, ketoconazole, which mainly suffers from a lack of selectivity (Liu et al., 2007), high non-specific binding (Tran et al., 2002;Quinney et al., 2013) and metabolic instability (Stresser et al., 2004). ...
Early determination of CYP3A4/5 contribution to the clearance of new chemical entities is critical to inform on the risk of drug-drug interactions with CYP3A inhibitors and inducers. Several in vitro approaches (recombinant P450 enzymes, correlation analysis, chemical and antibody inhibition in human liver microsomes) are available but they are usually labor intensive and/or suffer from specific limitations. In the present study, we have validated the use of azamulin as a specific CYP3A inhibitor in human hepatocytes. Azamulin (3µM) was found to significantly inhibit CYP3A4/5 (>90%) while other CYP450 enzymes were not affected (less than 20% inhibition). Since human hepatocytes were used as test system, the effect of azamulin on other key drug metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, FMO, SULT) was also investigated. Apart from some UGTs showing minor inhibition (~20-30%), , none of these non-P450 enzymes were inhibited by azamulin. Use of CYP3A5-genotyped human hepatocyte batches in combination with CYP3cide demonstrated that azamulin (at 3µM) is inhibiting both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocytes assay. Results showed that the effect of azamulin on the in vitro intrinsic clearance of these known CYP3A4/5 substrates was predictive of the in vivo CYP3A4/5 contribution. Overall, the study showed that human hepatocytes treated with azamulin provide a fast and accurate estimation of CYP3A4/5 contribution in metabolic clearance of new chemical entities.
AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat.
2. Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition.
3. AFQ056 lung clearance (CLlung) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction.
4. While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.
Keywords: CYP1A1, AFQ056, lung slices, lung metabolism, extrahepatic metabolism, rat lung clearance
