Susceptibility to metabolism is a common issue with the tert-butyl group on compounds of medicinal interest. We demonstrate an approach of removing all the fully sp 3 C−Hs from a tert-butyl group: replacing some C−Hs with C−Fs and increasing the s-character of the remaining C−Hs. This approach gave a trifluoromethylcyclopropyl group, which increased metabolic stability. Trifluoromethylcyclopropyl-containing analogues had consistently higher metabolic stability in vitro and in vivo compared to their tert-butyl-containing counterparts.

Time-dependent inhibition (TDI) of cytochrome P450 enzymes is an important cause of drug-drug interactions. The standard approach to characterize the kinetics of TDI is to determine the rate of enzyme loss, k obs , at various inhibitor concentrations, [I], and replot the k obs versus [I] to obtain the key kinetic parameters, K I and k inact . In our companion manuscript (Part 1; Nagar et al., 2014) in this issue of Drug Metabolism and Disposition, we used simulated datasets to develop and test a new numerical method to analyze in vitro TDI data. Here, we have applied this numerical method to five TDI datasets. Experimental datasets include the inactivation of CYP2B6, CYP2C8, and CYP3A4. None of the datasets exhibited Michaelis-Menten-only kinetics, and the numerical method allowed use of more complex models to fit each dataset. Quasi-irreversible as well as partial inhibition kinetics were observed and parameterized. Three datasets required the use of a multiple-inhibitor binding model. The mechanistic and clinical implications provided by these analyses are discussed. Together with the results in Part 1, we have developed and applied a new numerical method for analysis of in vitro TDI data. This method appears to be generally applicable to model in vitro TDI data with atypical and complex kinetic schemes.

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.

This article reviews the use of a selection of in vitro assays implemented at Novartis and intends to address exposure and safety in early drug discovery. The authors' own experience, based on a large number of 'real' drug discovery compounds, is described to reflect on what has worked, where improvement is needed and how to best use the data for decision making. Possible strategies are discussed, and guidelines are provided on how to organise assays, extract value and contribute knowledge from the data.

Dear Editor,High-throughput analysis of in vitro cytochrome p450 inhibition samples using mass spectrometry coupled with an integrated liquid chromatography/autosampler system A significant amount of effort in drug discovery is devoted to the study and understanding of drug-drug interactions (DDI), because the effectiveness and/or toxicity of pharmaceutical drugs can be profoundly influenced by the coadministration of other agents. DDI is preferentially assessed by means of an in vitro assay whereby the inhibitory potential of a test drug is measured with human liver microsomes as the CYP450 enzyme source, along with a known probe enzyme substrate, e.g., midazolam with CYP3A4/5. 1 The production of the specific probe substrate metabolite is monitored and compared to the production of metabolite in the absence of the test drug. These assays can be readily performed in multi-well format, and automated using the latest generation of sample preparation robotics. Consequently, the timeliness of results delivery concerning DDI is predominantly dictated by the analysis of samples and data processing, and for this reason breakthroughs in analysis technology have been of recent interest. Some notable examples in the area of mass spectrometry include ultra-performance liquid chromatography, 2 laser diode thermal desorption (LDTD), 3 matrix-assisted laser desorption/ionization (MALDI), 4 direct analysis in real time (DART), 5 and column switching liquid chromatography/ mass spectrometry (LC/MS). 6 All of these approaches take advantage of the relatively simple nature of the sample matrix to completely eliminate or drastically reduce the need for chromatography, thereby dramatically improving sample turnaround time compared to traditional LC/MS, while preserving data quality. In this work, an integrated chromatography/autosampler system 7,8 is evaluated that achieves a 7 s cycle time, and is evaluated for DDI sample analysis with respect to precision, carryover, matrix effects and correlation of results to existing LC/MS methodology.Experimental details consisting of the reagents used, sample preparation procedures and experiments performed can be found in the Supporting Information. An Applied Biosystems/MDS Sciex API 4000 triple-quadrupole mass spectrometer (Concord, ON, Canada) operated in positive ion, multi-reaction monitoring (MRM) mode was used for all experiments. The declustering potentials and collision energies were individually optimized for all three sets of analytes and internal standards, which corresponded to the CYP P450 isoforms used in the DDI assay. 1 0 -Hydroxybufuralol (m/z 278.3!186.2) was monitored to evaluate the