0,0.25,0.5,1,2,4,6,8,12,24,48,72,96,120,144,and168hpostdosefor
PK analysis.
18.2.4.3 Human Blood (10 mL) for at least four time points is obtained by
venipuncture from healthy male and female subjects following a single dose of
50–100mCi^14 C-labeled drug. Urine (0–12, 12–24, and 24 h interval thereafter
throughout the study) and feces (24 h intervals 0 h to the end of the study) are
obtained. In addition, another series of blood samples (5 mL) are collected at
various time points such as 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144,
and 168 h postdose for PK analysis. Bile is collected over 3–8 h postdose from,
for example, 4–8 subjects if needed (Wang et al., 2006). An intravenous dose of
cholecystokinin is used to stimulate gallbladder contraction at 7 h postdose in
humans during bile collection.
The pH of the biological samples may need to be adjusted by adding acetic
acid (2–5%, v/v), for example, to stabilize metabolites such as acylglucuronide
metabolites after sample collection prior to sample freezing at 20 C. The
blood is collected in tubes containing K 3 EDTA and centrifuged within 30 min
of collection to harvest plasma (10 min, 1300 gat 4C). Fecal homogenates
are prepared by mixing feces with appropriate amounts of water or
water:ethanol (50 : 50, v/v) following by homogenization, and aliquots of fecal
homogenates are analyzed for total radioactivity determination.
Pooled urine (0 h to end of study) and pooled fecal homogenate (0 h to end
of study) samples are prepared by mixing, respectively, urine (2% by weight)
and fecal homogenates (1% by weight) obtained from all subjects for each
collection interval. A pooled plasma sample is prepared by combining equal
volumes of plasma (1–2 mL) from all subjects for each collection time point.
Pooled plasma, urine, and feces are analyzed for radioactivity distribution
(metabolite profiling) and metabolite identification by high performance liquid
chromatography (HPLC) and liquid chromatography/mass spectrometry
(LC/MS).
18.3 Sample Analysis
18.3.1 Sample Preparation: Plasma, Urine, Bile, and Feces
In general, depending on the amount of radioactivity in sample, different
approaches may be used in processing the sample. If sufficient radioactivity is
present, the sample may be diluted before analysis or analyzed (HPLC and/or
LC/MS) directly following centrifugation (for urine and bile only). For samples
that contain limited amount of radioactivity, concentration of the sample after
extraction or protein precipitation (in plasma) is needed. It is important to
check recovery of radioactivity after concentration. The procedures used in the
muraglitazar study (Wang et al., 2006; Zhang et al., 2006, 2007) are generalized
and described below as an example.
580 ADME STUDIES IN ANIMALS AND HUMANS