oral route of administration is preferred for preclinical studies, either by gavage
or as an admixture in the animal diet, to provide a more relevant route of
exposure for compounds that will eventually be administered p.o. in humans,
especially those compounds that undergo significant first pass metabolism in
the gut epithelium or liver, or compounds that may require metabolic
activation to produce a metabolite that is an active inducer. Of course the
proper control groups must be included. These should include vehicle controls
for injection, or purified diet controls for dietary administration. Positive
control groups dosed with a known enzyme-specific inducer are also
recommended. For example, positive controls for CYP3A induction in
humanized mice should be administered known effective doses of a human
CYP3A-specific inducer such as rifampicin. In the case of humanized mice,
negative control animals that are hPXR/, or wild type mouse PXR+/+
control animals will help identify induction that is specifically human PXR-
mediated (Xie et al., 2000; Xie and Evans, 2002).
The following protocol might be used to investigate potential CYP3A4
induction by a test compound in humanized transgenic mice. For generation of
transgenic mice, readers are referred to Xie’s work (Xie et al., 2000). Details of
protocols for specific candidate molecules will depend on, among other factors,
chemical stability and solubility, toxicity and side effects of the putative
inducer, and the possibility that metabolites, rather than the parent candidate
molecule, might mediate induction. Animals are maintained on a 12 h light/
dark cycle under controlled temperature (22oC) and humidity (45%) with
ad libitumaccess to food and water. Each experimental group consists of four
mice; one group is designated as a vehicle control, one group as a positive
control (rifampin 5 mg/kg/day), and three groups are exposed to test
compound at low, moderate, and high doses. All treatments are administered
by gavage, in a dosing volume of 2–5 mL/kg, for 3–7 consecutive days. The
frequency of treatment will depend on the rates of clearance of the test
compound in its mode of administration. Normally, animals are treated once
daily, but this frequency may be adjusted when the pharmacokinetics of the
molecule are known. The goal is to maintain blood concentrations of the
compound at as constant levels as possible, over the entire treatment period.
The mice can also be dosed by intraperitoneal injection, but this route of
administration is not recommended for testing drug candidates that are
designed to be administered orally in therapeutic situations. At the end of the
treatment period, mice are euthanized by IP injection of pentobarbital, and
their livers are removed immediately, weighed and placed in a container
immersed in an ice bath. The liver tissues are divided to provide samples for
mRNA extraction and microsome preparation. Total RNA is extracted using
TRI REAGENTTMand 1-bromo-3-chloropropane, and is subsequently used
for quantitative real-time RT–PCR (Chomczynski and Sacchi, 1987). Mouse
liver microsomes are prepared using standard techniques (Matsuura et al.,
1991), and the microsomal pellets are suspended in storage buffer (pH 7.5,
K 2 HPO 4 5 mM, DTT 0.05 mM, EDTA 0.5 mM, glycerol 10% (v/v), and
ASSESSMENTS 551