Discovery and Characterization of a Novel Series of Chloropyrimidines as Covalent Inhibitors of the Kinase MSK1.

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.

(-)-N-3-Benzylphenobarbital Is Superior to Omeprazole and (+)-N-3-Benzylnirvanol as a CYP2C19 Inhibitor in Suspended Human Hepatocytes.

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.

Application of Azamulin to Determine the Contribution of CYP3A4/5 to Drug Metabolic Clearance Using Human Hepatocytes.

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 (3 µM) was found to significantly inhibit CYP3A4/5 (>90%), whereas other P450 enzymes were not affected (less than 20% inhibition). Because human hepatocytes were used as a test system, the effect of azamulin on other key drug-metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, flavin monooxygenase, and sulfotransferase) 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 3 µM) inhibits both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocyte 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. SIGNIFICANCE STATEMENT: Accurate estimation of CYP3A4/5 contribution in drug clearance is essential to anticipate risk of drug-drug interactions and select the appropriate candidate for clinical development. The present study validated the use of azamulin as selective CYP3A4/5 inhibitor in suspended human hepatocytes and demonstrated that this novel approach provides a direct and accurate determination of the contribution of CYP3A4/5 (fraction metabolized by CYP3A4/5) in the metabolic clearance of new chemical entities.

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 µm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction.

Assessment of cytochrome P450 (1A2, 2B6, 2C9 and 3A4) induction in cryopreserved human hepatocytes cultured in 48-well plates using the cocktail strategy.

1. A fast, straightforward and cost-effective assay was validated for the assessment of CYP induction in cryopreserved human hepatocytes cultured in 48-well plates. The cocktail strategy (in situ incubation) was used to assess the induction of CYP1A2, CYP2B6, CYP2C9 and CYP3A4 by using the recommended probe substrate, i.e. phenacetin, bupropion, diclofenac and midazolam, respectively. 2. Cryopreserved human hepatocytes were treated for 72 h with prototypical reference inducers, ß-naphthoflavone (25 µM), phenobarbital (500 µM) and rifampicin (10 µM) as positive controls for CYP induction. The use of a cocktail strategy has been validated and compared to the classical approach (single incubation). The need of using phase II inhibitor (salicylamide) in CYP induction assay was also investigated. 3. By using three different batches of cryopreserved human hepatocytes and our conditions of incubations, we showed that there was no relevant drug-drug interaction using the cocktail strategy. The same conclusions were observed when a broad range of enzyme activity has to be assessed (wide range of reference inducers, i.e. EC50-Emax experiment). In addition, the interassay reproducibility assessment showed that the day-to-day variability was minimal. 4. In summary, the study showed that the conditions used (probe substrates, concentration of probe substrate and time of incubation) for the cocktail approach were appropriate for investigations of CYP induction potential of new chemical entities. In addition, it was also clear that the use of salicylamide in the incubation media was not mandatory and could generate drug-drug interactions. For this reason, we recommend to not use salicylamide in CYP induction assay.