should be based on the pervious PK studies. The total study time should cover
at least three to five the plasma half-life (t1/2) of the compound. In the
traditional method, three rats/time point and 21–30 rats/study will be used
(such as at 0.5, 1, 4, 8, 24, 48, 72, 96, 124, and 168 h). In quantitative whole
body autoradiography (QWBA) method, 1 rat/time point and 5–6 rats/study
will be used (such as at 1, 12, 24, 48 72, and 168 h).
Radioactive dose level should produce no toxicity. The normal radioactivity
dose is 15–30mCi/rat for^14 C, and 150–200mCi/rat for^3 H. The route of
administration should be that proposed for clinical use. The dose and
formulation should be based on pervious toxicity studies.
Rats are sacrificed with an overdose of halothane anesthesia or under CO2,
and following tissues are collected and rinsed with water: adrenal glands,
bladder (urinary), blood, bone (femur), bone marrow (from femur), brain,
carcass, (residual), cecum, cecum contents/wash, eyes (both), heart, kidneys,
large intestine, large intestine contents/wash, liver, lungs, plasma, muscle
(pectoral), muscle (thigh), skin (pigmented), small intestine, small intestine
contents/wash, spleen, stomach, stomach contents/wash, testes, and thyroid/
parathyroid. Radioactivity in all tissue samples are determined by sample
oxidation in an oxidizer (Packard model 306). Samples are counted for up to
10 min in a scintillation counter (Packard model 2000CA).
For an QWBA method, the carcasses are immediately frozen in a hexane/
dry ice bath for approximately 5 min. The carcasses are drained, blotted dry,
and stored at approximately 70 C for at least 2 h. One frozen carcass/time
point is embedded in chilled 1.5–3% of carboxymethylcellulose and frozen into
a block. Carcasses are sectioned by using a cryomicrotome (such as Leica CM
3600) at a thickness (30–40mm, and temperature is maintained at approxi-
mately 20 C). Appropriate sections are collected on adhesive tape. All major
tissues, organs, and biological fluids are represented on five to six sections
collected in the sagittal plane. Tissue sections are placed on a holding frame
and stand and dried in the chamber of cryomicrotome maintained at
approximately 20 C. After freeze-drying, tissue sections are cut away from
the holding frame and mounted on a poster board.^14 Cor^3 H-labeled sections
are exposed to imaging plates for an appropriate time period (^14 C:12–20 h;^3 H:
1–2 weeks). WBA images from phosphor imaging plates are acquired (such as
by using the Fuji FLA 3000). Tissues, organs, and fluids are sampled, and
radioactivity concentrations in tissues are interpolated from the standard curve
as nanocuries per gram by using imaging analysis software (such as MCID,
Version 4.0, Imaging Research Inc). The tissues that can be analyzed by using
the QWBA method are extended from the list for traditional method, including
subfraction of a tissue.
For data presentation, tissue radioactivity levels are expressed as nanogram-
equivalents per gram tissue (Table 18.A1), and tissue/plasma ratios. The
maximum concentration (Cmax) and the time to reach maximum concentration
(Tmax) are obtained by visual inspection of the raw data. Pharmacokinetic
parameters, included half-life (t1/2), area under the concentration–time curve
598 ADME STUDIES IN ANIMALS AND HUMANS