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ZHOU ET AL.
NAC. The substrate mixture was generated as described above. The concen-
tration of 2Ј,3Ј-didehydrodideoxyribose used in the microsomal incubation was
approximate 40 M. The concentrations of microsomal proteins, NADPH, and
phosphate buffer were 1 mg/ml, 1 mM, and 100 mM, respectively. For
incubation performed in the presence of either GSH or NAC, the concentra-
tions of both the reagents used were 5 mM. The reaction was initiated by
addition of NADPH to the incubation mixture. The incubations were carried
out in microcentrifuge tubes at 37°C for 30 min in a shaking water bath at a
final volume of 200 l. Incubations were stopped by cooling on ice after
addition of 200 l of acetonitrile. The incubation mixture was then centrifuged
acid, were detected in the plasma and urine of monkeys, and thymine
could have been incorporated into endogenous molecules through
pathways of purine and pyrimidine metabolism (Cretton et al., 1993).
Both the recovery of dose in expired air of rats and the poor recovery
in the monkeys suggested that labeling the thymine ring was not ideal
for further absorption, distribution, metabolism, and elimination
(ADME) studies.
The total fate of stavudine, including mass balance, routes of
excretion, metabolic pathways, and exposure to stavudine-related
material in circulation, has not been completely established in hu- at 14,000 rpm for 2 min in an Eppendorf model 5417C centrifuge (Eppendorf
AG, Hamburg, Germany). The supernatants were removed and evaporated to
near dryness under a stream of nitrogen. The residues were reconstituted in 50
l of water and kept at Ϫ20°C until analysis. Incubations with [1Ј-14C]stavu-
dine (40 M) or with thymine (40 M) were performed in parallel. Incubation
in the absence of the microsomal protein and incubation in absence of the
cofactor NADPH were used as negative controls.
TD Study in Rats and Sample Preparation. Experiments were performed
according to the Guide for the Care and Use of Laboratory Animals (Institute
of Laboratory Animal Resources, 1996). The study consisted of eight groups
of three male Long-Evans rats. [1Ј-14C]Stavudine in deionized water was
mans. The present study investigated the pharmacokinetics and dis-
position of [1Ј-14C]stavudine after a single 80-mg (100 Ci) oral dose
to six healthy human subjects. The decision to label the ribose ring
instead of the thymine ring was based on the animal data described
above where loss of label was observed. Before conducting the human
ADME study, a tissue distribution (TD) study in Long-Evans rats was
conducted with [1Ј-14C]stavudine to ensure the projected radiation
exposure in humans after a 100-Ci oral dose is below the safety
exposure limit (International Commission on Radiological Protection,
2008). As part of the rat study, expired air was collected to confirm administered at a target dose level of 5 mg/kg (100 Ci/kg) as a single oral
gavage dose to overnight-fasted animals. The animals in group 1 did not
receive test article and were euthanized to provide control samples. The
remaining animals were euthanized at 1 h (group 2), 4 h (group 3), 12 h (group
4), 24 h (group 5), 48 h (group 6), 96 h (group 7), and 168 h (group 8) after
dosing. Animals in group 6 were placed in glass metabolism cages, and their
expired 14CO2 was trapped in potassium hydroxide solution (6 M) and col-
lected at 0 to 6, 6 to 12, 12 to 24, and 24 to 48 h postdose. Blood samples were
the metabolic stability of the label. Here we present data from both the
TD study in rats and the human ADME study. Also presented are
metabolites that arise from metabolism of the ribose ring that could be
easily followed because of the label on the ribose ring.
Materials and Methods
Chemicals and Biologicals. Stavudine and its internal standard, d4-stavudine collected just before euthanasia. A portion of blood samples was centrifuged
(6-[D]-1-(5-(hydroxymethyl)-2,5-dihydrofuran-2-yl)-5-[CD3]methylpyrimidine-
for 10 min at 1000g and 5°C for plasma. After euthanasia of the animals, 21
2,4(1H,3H)-dione), were synthesized chemically at Bristol-Myers Squibb Re- major tissues and gastrointestinal contents were excised from each animal.
search and Development (Princeton, NJ). [1-14C]D-Ribose (51 mCi/mmol, pack-
aged in water/MeOH, 5:1, at concentration of 16.7 mCi/ml and radiochemical
purity Ͼ97%) was purchased from ViTrax (Placentia, CA). Formic acid and
ammonium acetate were purchased from Mallinckrodt Baker, Inc. (Phillipsburg,
Disposition Study in Humans and Sample Preparation. This was an
open-label single-dose study. An 8-ml oral solution of [1Ј-14C]stavudine (80
mg, 100 Ci) was administered to six healthy male subjects. An 80-mg dose
represents the total daily dose used in clinical practice for the treatment of HIV
NJ). NADPH, GSH, and N-acetyl-L-cysteine (NAC) were purchased from Sigma- infection. Blood samples were collected for analysis of stavudine and total
Aldrich (St. Louis, MO). Carbo-Sorb E 14CO2 trapping solution, Hionic Fluor, and
Permafluor Eϩ liquid scintillation mixture were obtained from PerkinElmer Life
and Analytical Sciences (Waltham, MA). Ecolite liquid scintillation mixture was
radioactivity (TRA) determinations at 0, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, and
48 h and every 24 h for the subsequent study period (0–168 h). Additional
blood samples at 1, 2, 6, 12, 24, and 48 h were collected for biotransformation
purchased from MP Biomedicals (Solon, OH). Deionized water was prepared with studies. Plasma was prepared from the blood samples by centrifuging for 10
a Milli-Q plus ultrapure water system (Millipore Corporation). Pooled human liver min at 1000g and 5°C. Urine was collected at 6-h intervals for the first day and
microsomes were purchased from BD Gentest (Woburn, MA). All the other over 24-h intervals until end of the study period (0–168 h) for analysis of
chemicals used were reagent grade or better.
stavudine, TRA determination, and biotransformation studies. Feces were
Synthesis of [1
-14C]Stavudine. [1Ј-14C]Stavudine was synthesized from collected at 24-h intervals over the 168-h study period for TRA determination
its 14C-labeled precursor [1-14C]D-ribose based on a synthetic procedure re- and biotransformation studies.
ported by Discordia (1996). The synthesis of [1Ј-14C]stavudine was accom-
plished in 10 chemical steps in a total yield of 20.5% with specific activity of
15.53 Ci/mg, chemical purity of 99.3%, and radiochemical purity of 99.6%.
Pooled plasma, urine, and fecal samples were prepared for biotransforma-
tion studies. Plasma samples at 1, 2, 4, and 6 h were pooled by combining
equal volumes of individual plasma samples at a given time point. Pooled urine
Acidic Hydrolysis of [1
-14C]Stavudine. Hydrochloric acid solution (9% and fecal samples were prepared by combining 1% by weight of urine and
w/w) was prepared by mixing 1 volume of hydrochloric acid (approximately fecal homogenates excreted during each collection interval from all six sub-
36% w/w) with 3 volumes of water. Portions (1 ml each) of [1Ј-14C]stavudine jects over 168 h postdose.
methanol stock (15.6 Ci/ml) were transferred to a 5-ml glass tube and dried
under a stream of N2 gas. Hydrolysis reactions were started by adding 1 ml of
9% hydrochloric acid into glass tubes. The glass tubes were kept in a shaking
water bath at 90°C for 2 h. The reaction mixtures were evaporated to dryness
Liquid Scintillation Counting for TRA Determination. Radioactivity in
samples obtained from the rat TD study and the human disposition study was
determined with a Model LS 6500 liquid scintillation counter (Beckman
Coulter, Inc., Fullerton, CA) or a Tri-Carb 3100TR liquid scintillation analyzer
on a Savant SpeedVac SPD2010 Concentrator (Thermo Fisher Scientific, (PerkinElmer Life and Analytical Sciences). Before radioanalysis, tissue sam-
Waltham, MA) at room temperature. The residue was suspended in 100 l of ples, gastrointestinal content samples, and fecal samples were homogenized
100 mM phosphate buffer, pH 7.4, and kept at Ϫ20°C until incubated with with water to form a 20% (w/w) homogenate using a PT 45-80 probe homog-
human liver microsomes. The reaction was monitored by liquid chromatogra- enizer (Kinematica Polytron; Kinematica, Littau-Lucerne, Switzerland). Ali-
phy/mass spectrometry (LC/MS) and LC/radioactivity detection. Approxi- quots of blood (approximately 0.05 g), homogenized tissue (0.02–0.05 g),
mately 70% of [1Ј-14C]stavudine was hydrolyzed to [1Ј-14C]2Ј,3Ј-didehydro- gastrointestinal content (0.2–0.3 g), and homogenized fecal (0.2–0.3 g) sam-
dideoxyribose after 2-h incubation. No further purification steps were ples were placed in cones and pads, dried, and combusted. The combustions
performed on the final reaction mixtures. [1Ј-14C]2Ј,3Ј-Didehydrodideoxyri- were performed using
a model 307 or model 387 Sample Oxidizer
bose coexisted with its byproduct thymine and its precursor [1Ј-14C]stavudine. (PerkinElmer Life and Analytical Sciences). The resulting 14CO2 was trapped
Incubation with Human Liver Microsomes. Human liver microsomes in Carbo-Sorb E (PerkinElmer Life and Analytical Sciences). After the 14CO2
were incubated with a substrate mixture of [1Ј-14C]stavudine, [1Ј-14C]2Ј,3Ј- trapping, Permafluor Eϩ scintillation mixture (PerkinElmer Life and Analyt-
didehydrodideoxyribose, and thymine, in presence or absence of either GSH or ical Sciences) was added, and the mixture was analyzed. Each small organ