Walter A. Korfmacher

The lead optimization paradigm includes a team of experts that has a multitude of parameters to consider when moving from an initial lead compound through the lead optimization phase to the development phase. While in the past the team may have had only a medicinal chemist and a pharmacologist, the current team would often include experts in the areas of drug metabolism and pharmacokinetics (DMPK) as well as chemical toxicity. This review provides an overview of the some of the recent advances in the areas of DMPK screening plus a discussion of some of the assays that can be used to begin to screen for toxicity issues. The focus of this review is the major potential problem areas: oral bioavailability, half-life, drug-drug interactions and metabolism and toxicity issues.

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.

Hydroxyproyl‐ β ‐cyclodextran (HPBCD), methyl cellulose (MC), Tween 80 and PEG400 are commonly used in dosing formulations in pharmacokinetic (PK) studies during the early drug discovery stage. A series of studies was designed to evaluate the potential matrix effects of these dosing vehicles when the samples are assayed by high‐performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS). These dosing vehicles were dosed into the rats via either an intravenous (IV) or an oral route (PO) and plasma samples were collected for a 24‐h post‐dose period. Five test compounds with CLog P values ranging from 0.9 to 5.4 were spiked into the collected rat plasma. After protein precipitation, these samples were analyzed using a generic fast‐gradient HPLC/MS/MS method. Three popular mass spectrometers (Thermo‐Finnigan Quantum with ESI and APCI, AB‐Sciex API 3000 with ESI and APCI, and Waters‐Micromass Quattro Ultima with ESI) were used to test these plasma samples. Results indicated that there was no observed matrix effect for all five compounds when 20% HPBCD or 0.4% MC was used as the vehicle in either the IV or the PO route, respectively. In addition, 0.1% Tween 80 dosed either IV or PO caused significant ion suppression (50–80%, compared to results obtained from plasma samples free from vehicles) for compounds that eluted at the beginning of the chromatogram. Also, PEG400 when used in an oral formulation caused significant ion suppression (30–50%) for early eluting compounds. These matrix effects were not only ionization mode (ESI or APCI) dependent, but also source design (Thermo‐Finnigan, AB‐Sciex or Waters‐Micromass) dependent. Overall, the APCI mode proved to be less vulnerable to matrix effects than the ESI mode. Some possible mechanisms of these matrix effects are proposed and simple strategies to avoid these matrix effects are discussed. Copyright © 2005 John Wiley & Sons, Ltd.

The rapid increase in the rate at which new chemical entities can be synthesized by medicinal chemists and tested in vitro for potency using high-throughput screening has provided opportunities for lead selection and optimization of thousands of compounds in major pharmaceutical companies. This opportunity coincides with the realization that drug metabolism input at the lead optimization stage is crucial for the development of compounds, with increased likelihood of success when tested in preclinical safety studies and in phase I trials. This has led to new discovery and optimization paradigms, in which drug metabolism is playing an important role.

This report addresses the continuing need for increased throughput in the evaluation of new chemical entities (NCEs) in terms of their pharmacokinetic (PK) parameters by describing an alternative procedure for increasing the throughput of the in vivo screening of NCEs in the oral rat PK model. The new approach is called ‘cassette‐accelerated rapid rat screen’ (CARRS). In this assay, NCEs are dosed individually (n = 2 rats/compound) in batches of six compounds per set. The assay makes use of a semi‐automated protein precipitation procedure for sample preparation in a 96‐well plate format. The liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API‐MS/MS) assay is also streamlined by analyzing the samples as ‘cassettes of six’. Using this new approach, a threefold increase in throughput was achieved over the previously reported ‘rapid rat screen’. Copyright © 2001 John Wiley & Sons, Ltd.