degradations can take place in both blood and liver, since the latter also
expresses several classes of hydrolyases, such as carboxylesterases (Satoh and
Hosokawa, 1998; Satoh et al., 2002). The metabolic instability due to
hydrolysis could be potentially detected in the liver subcellular fractions,
particularly S9 fractions. Therefore, the potential instability in the blood is
usually minor for typical drug-like compounds to the overall metabolism.
13.2.2 Experimental Procedures
13.2.2.1 Assay Conditions
Buffers Tris–HCl and potassium phosphate buffer (or phosphate-buffered
saline, PBS) are used for the microsomal stability assays, while PBS is often the
choice for the metabolism in hepatocyte suspensions.
i. Tris–HCl buffer (10 , or 500 mM) and potassium phosphate buffer (10 ,
or 1 M): Both buffers can be purchased commercially or prepared (Ackley
et al., 2004), and may require a slight pH adjustment prior to use. The
stock solutions are usually stored at 4C after being filtered, if necessary.
ii. NADPH-regenerating system: The reagents are usually obtained
commercially. The system in the final reaction mixtures contains
NADP+(1.3 mM), glucose-6-phosphate (Glc-6-PO 4 ) (3.3 mM), MgCl 2
(3.3 mM), and glucose-6-phosphate dehydrogenase (G6PDH; 0.4 U/
mL).Enzymes
i. Liver subcellular fractions: The microsomal preparations, S9 fractions,
and cytosolic fractions are prepared using differential ultracentrifuga-
tion procedures (Raucy and Lasker, 1991). Liver microsomal prepara-
tions contain the majority of the drug-metabolizing enzymes,
particularly cytochromes P450 (CYPs), and are the most common
enzyme sources forin vitrometabolic studies. CYPs play a preeminent
role in drug metabolism. For example, the metabolic clearance of nearly
half of the drugs on the market is contributed by CYP3A4, the most
abundant hepatic CYP member in humans (Shimada et al., 1994). This
suggests that use of microsomal preparations as anin vitrosurrogate for
liver metabolism is a reasonable approximation. Liver microsomal
preparations also provide several practical benefits: a simple system that
provides clear-cut results; ease of use with high throughput adaptability;
a concentrated enzyme system, particularly for monooxidases (CYPs
and flavin-containing monooxygenases or FMOs), thereby permitting
marked turnover for unambiguous detection (if response enzymes are
microsomal); and a commercially steady high quality supply. Human
liver microsomes (HLM) are the most relevant and are often viewed asDETERMINATION OF METABOLIC STABILITY 415