Zhihong Lu

Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1

An antisense oligodeoxynucleotide phosphorothioate, namely gene expression modulator 91 (GEM 91), has been demonstrated to have significant anti-human immunodeficiency virus activity in various tissue culture models. The present study was undertaken to determine the pharmacokinetics and tissue distribution of GEM 91 in rats following i.v. bolus administration of 35S-radiolabeled GEM 91. Plasma disappearance curves for GEM 91-derived radioactivity could be described by the sum of two exponentials, with half-lives (mean +/- SEM) of 0.95 (+/- 0.07) and 47.57 (+/- 14.48) hr. Urinary excretion represented the major pathway of elimination of GEM 91, with 26.67 +/- 6.46% (mean +/- SD) of the administered dose excreted within 24 hr and 58.12 +/- 4.36% over 240 hr after GEM 91 administration. Fecal excretion was a minor pathway of elimination of GEM 91 with 1.4 +/- 0.62% (mean +/- SD) of the administered dose excreted over 24 hr and 8.54 +/- 0.64% over 240 hr. A wide tissue distribution of GEM 91 was observed. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidney, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the kidneys, liver, heart, and intestine. Analyses of the extracted radioactivities from plasma, kidney, and liver by gel electrophoresis showed the presence of both intact GEM 91 and degradative products with smaller molecular weights. Radioactivity in urine was found to be degradative metabolites of GEM 91. Based on the experimental data, pharmacokinetic parameters for GEM 91 in each tissue and biological fluids were calculated using computer-based two-compartmental i.v. bolus or absorption models. This study is important not only in providing the basis for future studies of GEM 91 in humans, but also in understanding the pharmacology and toxicology of antisense oligodeoxynucleotide phosphorothioates, in general.

Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects

Human pharmacokinetics of an antisense oligodeoxynucleotide phosphorothioate (GEM 91) developed as an anti—human immunodeficiency virus (HIV) agent was carried out in this study. 35S‐Labeled GEM 91 was administered to six HIV‐infected individuals by means of 2‐hour intravenous infusions at a dose of 0.1 mg/kg. Plasma disappearance curves for GEM 91—derived radioactivity could be described by the sum of two exponentials, with half‐life values of 0.18 ± 0.04 and 26.71 ± 1.67 hours. The radioactivity in plasma was further evaluated by polyacrylamide gel electrophoresis, showing the presence of both intact GEM 91 and lower molecular weight metabolites. Urinary excretion represented the major pathway of elimination, with 49.15% ± 6.80% of the administered dose excreted within 24 hours and 70.37% ± 6.72% over 96 hours after dosing. The radioactivity in urine was associated with lower molecular weight metabolites. No drug‐related toxicity was observed.

Population characteristics of hepatic dihydropyrimidine dehydrogenase activity, a key metabolic enzyme in 5-fluorouracil chemotherapy

Dihydropyrimidine dehydrogenase is the initial rate‐limiting enzyme in catabolism of 5‐fluorouracil, one of the most widely used cancer chemotherapeutic agents. Previous studies have shown the clinical importance of determination of dihydropyrimidine dehydrogenase in patients with cancer, particularly in those with deficiency of this enzyme who experience severe 5‐fluorouracil‐associated toxicity (including death) after 5‐fluorouracil treatment. In the present study, dihydropyrimidine dehydrogenase activity was determined in 138 donor liver samples. A normal distribution of human liver dihydropyrimidine dehydrogenase activity was shown. The mean enzyme activity was compared across different groups by age, gender, and race, with no significant differences observed except that the mean enzyme activity in women was slightly higher than that observed in men. With use of a polyclonal antibody generated against human liver dihydropyrimidine dehydrogenase, Western blot analysis showed that dihydropyrimidine dehydrogenase protein in the liver cytosol correlated with the activity of this enzyme.

Comparison of dihydropyrimidine dehydrogenase from human, rat, pig and cow liver : biochemical and immunological properties

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines and has an important role in cancer chemotherapy with fluoropyrimidine drugs. Recently, we purified and characterized this enzyme from human liver and raised a rabbit polyclonal antibody against the purified human enzyme (Lu et al., J Biol Chem 267: 17102-17109, 1992). In the present study, using this purification procedure, DPD was purified to homogeneity from three other mammalian species, i.e. pig, rat, and cow. Comparison of the biochemical properties of these purified enzymes was conducted. Molecular masses of DPD from human, pig, rat, and cow liver were: 210, 204, 210, and 216 kDa, respectively. DPD from all four species appeared to be composed of two subunits. The apparent pI values were 4.6, 4.8, 4.85, and 5.25, respectively. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out with the purified DPD preparation, suggesting species differences in kinetic parameters. Amino acid composition of these purified enzymes also demonstrated slight species differences. In the present study, a rabbit polyclonal antibody against rat liver DPD was raised. Using polyclonal antibodies against human and rat liver DPD, immunoblotting demonstrated cross-reactivity among the four species. In summary, purification and comparison of DPD from different mammalian species will provide a basis for further biochemical and molecular studies of this enzyme.

High-performance liquid chromatographic determination of 9-(3-pyridylmethyl)-9-deazaguanine (BCX-34) in biological fluids

9-(3-Pyridylmethyl)-9-deazaguanine (BCX-34), a new purine nucleoside phosphorylase inhibitor, has selective immunosuppressive activity with potential therapeutic value in T-cell-mediated disease. We now report a sensitive, specific and reproducible method for measurement of 9-(3-pyridylmethyl)-9-deazaguanine in biological fluids using high-performance liquid chromatography (HPLC). 9-(3-Pyridylmethyl)-9-deazaguanine was extracted from plasma using perchloric acid precipitation followed by passage through Sep-Pak C18 cartridges (average extraction efficiency, 64.6%). Standard curves were linear over the range of interest (28-1120 ng/ml in plasma and 200-4000 ng/ml in urine, r2 > 0.999). Within-day and between-day coefficients of variation were less than 8%. The limit of quantitation was 28 ng/ml in plasma and 200 ng/ml in urine. This HPLC method should be useful in future clinical studies with this drug.