microcentrifuge at maximum speed. The supernatant is filtered through
a spin filter (0.22mm), and transferred into an HPLC injection vial. The
filtrate is analyzed using either a high performance liquid chromato-
graphy/ultraviolet/fluorescence (HPLC/UV/FL) or preferably a high
performance liquid chromatography/triple quadrupole mass spectro-
metry (LC/MS/MS).
Zero-time samples, containing the intact compounds (100%), are
prepared by mixing all reaction components on ice, followed by the
immediate addition of 250mL methanol containing the appropriate
internal standard. The samples are prepared for the analyses in the same
manner as described above.
iv. Metabolism in hepatocyte suspensions: The hepatocytes (either freshly
isolated or cryopreserved) are thawed and resuspended in PBS (1 )
following one of the protocols provided by the manufacturers; 1mLof
the compound stock (e.g., 250 DMSO stock) is mixed into 249mL PBS
containing 0.25–1 106 cells (or 1–4 106 cells/mL) in a test tube (Soars
et al., 2002). The suspension is incubated at 37C in a gently shaking
waterbath for up to 4 h, followed by reaction quenching and sample
preparation as described for HLM assays.13.2.2.2 Determination of Metabolic Stability Substrate depletion andin vitro
half-life (t1/2) are common descriptors for metabolic stabilities (Obach and Reed-
Hagen et al., 2002). These descriptive terms are useful, particularly for the ranking
of stabilities among a series of synthetic analogs (MacKenzie et al., 2002).
Substrate Depletion and In vitro Half-Life Studies to detect substrate
disappearance are readily undertaken. The results obtained are directly used
for estimatingt1/2(Dordal et al., 2005).
As mentioned, such experiments are frequently and increasingly carried out
in hepatocyte suspensions and in reaction mixtures containing liver subcellular
fractions, especially microsomal preparations. In a standard metabolic study,
the extent of linear increases of metabolic rates with enzyme concentrations
(e.g., 0.2–4 mg/mL), and with reaction periods (e.g., 0–90 min), should be pre-
determined. Such preliminary data, while essential for kinetic characteriza-
tions, may not be feasible in discovery, or may not be crucial when substrate
disappearance or half-life is determined. Therefore, it may be possible to
perform stability assays under a condition of default setting, that is, a 30-min
incubation at the protein concentration of 1 mg/mL (or any concentration
between 0.4 and 2.0 mg/mL) (Mandagere et al., 2002). To minimize the effects
of nonspecific protein or lipid binding, a potential variable (Kalvass et al.,
2001), lower protein concentrations are generally preferable. However, such
nonspecific binding is likelyin vitro, and even more soin vivo. Thus, the effects
of protein or lipid binding of substrates have not been completely resolved
(Andersson et al., 2004; Obach, 1999). The selection of the quantity of the
DETERMINATION OF METABOLIC STABILITY 419