On the prediction of hepatic clearance using the diluted plasma in metabolic stability assay

It was suggested that in vivo hepatic clearance, CL(h), may be predicted rather accurately with the in vitro values of intrinsic clearance, CL(int), obtained using the microsomal incubation mix containing diluted plasma, and consequently calculated by the well-stirred model equation. Conceivably the improvement could be due to the direct account of plasma protein binding in the measured values of CL(int). It is shown in this article that the prediction of CL(h) done in this manner may not yield accurate results, both substantial underestimation or overestimation of the true value is possible. The procedure may be useful to reduce the overestimation of CL(h) for highly protein bound drugs, though the obtained value of CL(h) may be far off from the correctly calculated one. The accurate way of calculating CL(h), based on the value of CL(int) obtained in diluted plasma, is presented. It takes into account both the drug protein binding in diluted plasma and microsomal binding, as well as blood-plasma concentration ratio. The prediction of CL(h) by the suggested calculation using the experimental data on CL(int), measured at different plasma dilutions for several drugs, yields consistent (dilution independent) values of hepatic clearance. It does not seem possible to avoid the measurement of plasma protein binding, microsomal binding and blood-plasma concentration ratio for an accurate and consistent prediction of CL(h), even if the value of CL(int) were obtained in the pure (undiluted) plasma. In an early stage screening using plasma in the microsomal incubation mix may be beneficial for fast metabolizing drugs with relatively high protein binding. This would reduce a possible overestimation CL(h), and also lead to the increase of the half-life in the microsomal incubation, so that it could be measured more accurately.     

High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis

Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using well-known HLM-based MS probes. We provide consistently robust IC(50) values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC(50) values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)-warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC(50) results for five major CYP isoforms using our method compared to values reported in the literature.