Drug Metabolism in Drug Design and Development Basic Concepts and Practice

transfer, dilution, and incubation. Gemini software version 4.0 was used for
programming the operation. A general instruction of the automated assay is
depicted in Scheme 16.2 (Yao et al., 2007). All analytical methods were
conducted by LC/MS/MS. The LC/MS/MS consists of a triple quadrupole
mass spectrometer (Sciex, API-365, Biosystems MD, Toronto, Canada)
equipped with a turbo ion spray ionization source, two Shimadzu LC-
10ADvp Solvent Delivery Modules (pumps), an SCL-10ADvp controller
(Shimadzu, Columbia, MD), a DGU-14 solvent degasser (Shimadzu,
Columbia, MD), and an autosampler (Perkin–Elmer serious 200, Norwalk,
CT). Data were collected and processed using Sciex Analyst 1.1 data collection
and integration software on an IBM compatible computer. A centrifuge
equipped with a 96-well plate rotor was used to conduct the filtration process
to separate precipitated proteins. Table 16.1 shows the LC/MS/MS analyses of
individual CYP marker substrates in pooled human liver microsomes.

16.2.3 Optimization of Kinetic Reaction

Phosphate buffer (100 mM, pH 7.4) containing 1 mM EDTA was prepared by
a dilution of 400 mM mono and dibasic potassium phosphate stock solutions
(stored at 4C) and stored at ambient temperature. Frozen stocks of HLM
were used and the remaining was discarded after the first thawing. NADPH
stock, at 10 mM in phosphate buffer was made fresh daily. Stock solutions of
analytes (i.e., metabolites) were prepared in solvent and stored at 20 Cor
4 C. Internal standards were dissolved in either DMSO or 50% acetonitrile in
water.
To measure accurate kinetic parameters, all reaction rates should be linear
to protein concentrations (0.1–0.25 mg/mL) and incubation times (up to
20 min). Substrate consumption was limited to less than 20%. Because the
HLM contains multiple CYPs an isoform-mediated metabolite(s) is (are)
monitored to measure the enzyme activity. If the metabolite detection is highly
sensitive, a low protein concentration is recommended for the highly protein
bound compounds to minimize protein binding. To obtain aKmvalue eight
substrate concentrations are routinely chosen, which are set around theKm
(0.1–3Km).Kmis determined by a nonlinear regression on a velocity versus

Incubation Reaction termination

Quantitation Protein removal

Reagent addition and

sample dilution in 96-

well plate (200μL)

Incubation

reaction

Acetonitrile

precipitation (+ IS)

Preparation

LC/MS/MS Filtration (filter plate)

SCHEME 16.2 High throughput screening of CYP inhibition by automated TECAN.

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