Effects of 5-benzylacyclouridine, an inhibitor of uridine phosphorylase, on the pharmacokinetics of uridine in rhesus monkeys: implications for chemotherapy.

The effects of subcutaneous administration of 5-benzylacyclouridine (BAU), a uridine phosphorylase (UrdPase, EC 2.4.2.3) inhibitor, on uridine concentration in plasma and urine were evaluated in rhesus monkeys. Administration of BAU at 50, 100 and 250 mg/kg increased the plasma uridine baseline concentration 1.5-, 2.9-, and 3.2-fold, respectively. The basis for this moderate perturbation of plasma uridine by BAU was investigated using a tracer dose of 500 microCi 3H-uridine. Administration of 3H-uridine alone led to its rapid catabolism to uracil and dihydrouracil. Administration of 83.3 mg/kg BAU with 500 microCi 3H-uridine resulted in a 2.5-fold enhancement of 3H-uridine plasma levels and a substantial decrease in the plasma levels of uridine catabolites, suggesting inhibition of UrdPase activity by BAU in rhesus monkeys. Coadministration of 83.3 mg/kg BAU with 83.3 mg/kg uridine also reduced the plasma concentration of uracil and dihydrouracil, but it did not increase plasma uridine concentration above that of control animals receiving 83.3 mg/kg uridine alone. In animals receiving uridine alone at 83.3 or 25 mg/kg, approximately 10% of the administered dose was recovered in the urine within 6 h, with unchanged uridine being the major component. In contrast, administration of 83.3 mg/kg BAU increased the excretion of unchanged uridine to more than 32% of the total dose administered, even when the urinary excretion ratio of uracil to uridine was reduced ten-fold. Administration of multiple doses (three times per day) of BAU alone (83.3 mg/kg) or in the presence of uridine (83.3 mg/kg) did not enhance plasma uridine concentration further. In addition, uridine pharmacokinetics were associated with a time-dependent relationship as evidenced by an increased total plasma clearance, renal clearance and volume of distribution, resulting in a substantial decrease in uridine peak concentration with time. These results indicate that administration of BAU inhibits UrdPase activity in rhesus monkeys as manifested by decreased uracil and dihydrouracil plasma levels, as well as a lower urinary excretion ratio of uracil to uridine, as compared to control animals. However, plasma levels of unchanged uridine were not substantially enhanced by BAU in spite of the large increase in urinary excretion of unchanged uridine. This phenomenon was also observed when uridine was coadministered with BAU, suggesting that plasma uridine concentration in monkeys may be strongly regulated by the renal system as evidenced by the "spillover" of excess plasma uridine into urine. In addition, the pharmacokinetics of uridine were dose-independent, but time-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro and in vivo disposition and metabolism of 3'-deoxy-2',3'-didehydrothymidine.

The disposition and metabolic fate of 3'-deoxy-2',3'-didehydrothymidine (D4T) were evaluated both in isolated hepatocytes and in nonhuman primates. Rapid formation of thymine and beta-aminoisobutyric acid (BAIBA) occurred following incubation of hepatocytes with 10 microM [5(-3)H]D4T. Substantial levels of tritiated water were also detected. Exposure of cells to D4T in the presence of either 1 mM thymine or 10 microM benzyloxybenzyluracil, an inhibitor of dihydropyrimidine dehydrogenase, decreased intracellular BAIBA levels by approximately 89 and 63%, respectively. Concurrently, [3H]thymine levels increased two- to fivefold. These results are consistent with D4T being cleaved to thymine, which is then degraded to BAIBA. A similar metabolic disposition was observed in monkeys following administration of 25 mg of [5(-3)H]D4T per kg of body weight. BAIBA, thymine, and tritiated water were identified in plasma and urine. Approximately 50% of the administered dose was recovered in urine within 24 h, with the majority of the radioactivity representing unchanged drug. After administration intravenously or orally of 25 mg of [4(-14)C]D4T per kg of body weight to monkeys, a novel metabolite, designated X, in addition to unchanged D4T, thymine, and BAIBA, was also detected. The sum of the three metabolites and unchanged drug accounted for virtually all of the radioactivity in plasma and urine. Thymine and X exhibited kinetic profiles similar to that of D4T, with plasma elimination half-life of 2 to 3 h, whereas BAIBA levels remained constant for extended periods and declined slowly; this metabolite could be detected 24 h after intravenous drug administration. Mean oral bioavailability of D4T was high at approximately 70%. As observed in the [5(-3)H]D4T study performed in monkeys, approximately half of the administered [4(-14)C]D4T was recovered unchanged. The remainder was not recovered in urine or feces collected up to 30 days after drug administration. These data suggest that D4T metabolites are further metabolized by salvage pathways and/or converted to biological macromolecules.

Reduction of 3'-azido-2',3'-dideoxynucleosides to their 3'-amino metabolite is mediated by cytochrome P-450 and NADPH-cytochrome P-450 reductase in rat liver microsomes.

Using in vitro liver systems, we previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) is reduced to a highly toxic metabolite, 3'-amino-3'-deoxythymidine (AMT) through a NADPH-dependent system. This pathway also occurs for other 3'-azido-2',3'-dideoxynucleosides (3'-azido ddNs), indicating that reduction to a 3'-amino metabolite is a general catabolic route of this class of compounds. This study was undertaken to understand the enzymatic reaction responsible for this catabolic pathway. Rat liver microsomes were exposed to 1 mM [3H]AZT or 1 mM [3H]AzddU, and incubated under various conditions. Reduction to the 3'-amino derivative was enhanced 5-fold by the addition of NADPH. When FAD or FMN was combined with NADPH, AMT and AMddU formation was enhanced 2-fold. Addition of equimolar FAD and FMN enhanced azido reducing activity by 3-fold and 5-fold when compared with NADPH alone for AZT and AzddU, respectively. Exposure to carbon monoxide inhibited 3'-amino formation approximately 60%, consistent with involvement of cytochrome P-450 (P-450). This inhibitory effect was not detected in the presence of combined flavin and NADPH; in control incubations that contained these cofactors but no microsomes, AMT or AMddU formation was not observed. This suggests that a flavoprotein, possibly NADPH-cytochrome P-450 reductase (P-450 reductase), is also involved in azido reduction. Preincubation with various P-450 ligands resulted in variable inhibition; reduction of AZT and AzddU was decreased approximately 20-80%.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of 3'-azido-3'-deoxythymidine to 3'-amino-3'-deoxythymidine in human liver microsomes and its relationship to cytochrome P450.

The formation of 3'-amino-3'-deoxythymidine (AMT) in patients receiving 3'-azido-3'-deoxythymidine (zidovudine) and the potential role of this metabolite in zidovudine-induced toxicity was recently demonstrated by our laboratory. This study evaluated the formation of AMT versus cytochrome P450 (P450) content, cytochrome B5 (B5) content and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase activity in human liver microsomes obtained from 24 different donors. Significant interindividual differences in total P450 content and P450 reductase activity were observed, whereas no variation was observed in B5 content. Of particular importance, metabolism of zidovudine to AMT varied widely and correlated with P450 content but not with B5 content or P450 reductase activity. The apparent values for the Michaelis-Menten constant and the maximum rate of metabolism of the reaction were 46.1 mmol/L and 3.5 nmol/min/mg microsomal protein. These large variations of AMT levels as a function of P450 suggest that major interindividual differences may be observed in the pharmacokinetics and formation of this metabolite that may affect the pharmacodynamic properties of zidovudine. Potential drug-drug interactions may occur with therapeutic agents that interact with or induce P450 (zidovudine).

Catabolic disposition of 3'-azido-2',3'-dideoxyuridine in hepatocytes with evidence of azido reduction being a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides.

3'-Azido-2',3'-dideoxyuridine (AzddU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in vitro with low bone marrow toxicity. Although AzddU is currently being evaluated in clinical trials, its catabolic disposition is unknown. Pharmacokinetic studies in rhesus monkeys have demonstrated that a 5'-O-glucuronide is excreted in urine. The present study examined the catabolic disposition of AzddU is isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AzddU, low intracellular levels of two catabolites, identified as 3'-azido-2',3'-dideoxy-5'-beta-D-glucopyranosyluridine (GAzddU) and 3'-amino-2',3'-dideoxyuridine (AMddU), were detected. Studies using rat microsomes demonstrated that GAzddU formation was only detected in the presence of uridine 5'-diphosphoglucuronic acid, and that the rate of AMddU formation increased significantly in the presence of NADPH. Under similar conditions, reduction of the 3'-azido function was also demonstrated herein with 3'-azido-2',3'-dideoxycytidine (AzddC), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeC) and 3'-azido-2',3'-dideoxyguanine (AzddG), suggesting that enzymatic reduction to a 3'-amino derivative is a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides at the hepatic site.

Clinical pharmacokinetics of 3'-azido-3'-deoxythymidine (zidovudine) and catabolites with formation of a toxic catabolite, 3'-amino-3'-deoxythymidine.

This study investigated pharmacokinetics and metabolism of 3'-azido-3'-deoxythymidine (zidovudine) in patients after a 1-hour intravenous infusion of 2.5 mg/kg zidovudine with a radiolabeled tracer amount of [5-3H]-zidovudine. In addition to unchanged drug and its 5'-O-glucuronide (zidovudine glucuronide), two novel catabolites of zidovudine were detected as 3'-amino-3'-deoxythymidine (AMT), and its 5'-O-glucuronide (GAMT). The AMT apparent plasma elimination half-life (2.70 +/- 0.7 hours) was longer than that of zidovudine (1.20 +/- 0.30 hours) and zidovudine glucuronide (1.60 +/- 0.5 hours). The zidovudine/AMT plasma peak concentration and area under the concentration-time curve ratios were approximately 8 and 5, respectively. Urinary recovery of radioactivity was essentially complete within 24 hours. AMT glucuronide was not detected in urine or plasma, and only low levels of this catabolite were detected in bile. In contrast, AMT was not detected in bile. The substantial levels of AMT in the plasma of patients after zidovudine administration suggests that this catabolite may affect the pharmacodynamic properties of zidovudine in relation to its activity against human immunodeficiency virus replication and cytotoxicity to host cells.

Comparative effects of 3'-azido-3'-deoxythymidine and its metabolite 3'-amino-3'-deoxythymidine on hemoglobin synthesis in K-562 human leukemia cells.

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits hemoglobin (Hb) synthesis and globin gene transcription in butyric acid-induced K-562 leukemia cells, suggesting that these effects may play a role in the AZT-induced anemia observed in patients [Mol. Pharmacol. 38:797-804 (1990)]. The recent discovery by our group of a novel metabolite of AZT. 3'-amino-3'-deoxythymidine (AMT), which exhibits a high degree of toxicity toward human hemopoietic cells [Mol. Pharmacol. 39:258-266 (1991); Antimicrob. Agents Chemother. 35:801-807 (1991)], has led us to explore potential effects of this AZT metabolite on Hb production, globin mRNA expression, and heme synthesis in butyric acid-induced K-562 human erythroleukemia cells. AMT inhibited Hb synthesis by approximately 21%, as measured by benzidine staining, at concentrations as low as 25 microM, with slightly increased inhibition at higher AMT concentrations. The inhibition of Hb production by AMT was substantially lower, compared with that of AZT. AMT inhibited globin mRNA steady state levels in a dose-dependent manner to a similar extent as did the parent drug, with approximately 50% inhibition by each compound at a concentration of 100 microM. Nuclear run-on transcription assays demonstrated that inhibition by AMT of globin mRNA synthesis was associated with a decreased rate of globin-specific gene transcription. Globin mRNA stability was not affected by either 100 microM AZT or AMT, as measured after blockage of transcription with actinomycin D. To gain insight into potential mechanism(s) responsible for the different quantitative effects of AZT and AMT on Hb synthesis, the effect of each compound on induction of heme synthesis in K-562 cells was determined. Although heme induction was not affected by AMT, a significant inhibition approximating 20% was observed in the presence of 100 microM AZT. In addition, AZT down-regulated mRNA steady state levels under conditions where heme synthesis was inhibited by succinylacetone. These data suggest that inhibition by AZT of globin gene expression is a direct effect and is not secondary to inhibition of heme synthesis. This study emphasizes the role of AMT in the pharmacodynamic properties of AZT, in relation to its toxicity, and suggest that both AMT and AZT may be involved in the inhibition of erythroid differentiation observed in vivo, through changes in gene expression.

Modulation of 3'-azido-3'-deoxythymidine catabolism by probenecid and acetaminophen in freshly isolated rat hepatocytes.

Metabolic studies of 3'-azido-3'-deoxythymidine (AZT) in humans have demonstrated that this compound is primarily eliminated as a 5'-O-glucuronide, 3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine (GAZT), accounting for approximately 80% of the administered dose. Recently, we characterized the complete catabolic pathway of AZT in freshly isolated rat hepatocytes in suspension, demonstrating extensive formation of three catabolites, including GAZT, 3'-amino-3'-deoxythymidine (AMT), and 3'-amino-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine (GAMT). The present study evaluated the effects of probenecid (PROB) and acetaminophen (ACET), two agents which are also metabolized by UDP-glucuronyltransferase, on the metabolism and transmembrane distribution of AZT in rat hepatocytes. Pre-exposure of cells to 350 microM PROB 30 min prior to the addition of 10 microM [3H]AZT decreased intracellular GAZT levels by approximately 10-fold. Interestingly, AMT formation was enhanced approximately 1.5-fold in the presence of PROB, probably resulting from increased AZT availability. In contract, pre-exposure to 50 microM ACET 30 min prior to addition of 10 microM [3H]AZT did not substantially alter AZT glucuronidation. Additionally, decreased AZT catabolism by PROB did not contribute to the formation of 5'-phosphorylated derivatives of AZT. Agents which undergo glucuronidation may thus not necessarily affect AZT conversion to GAZT, and their potential interactions should be investigated using in vitro systems prior to co-administration with AZT.

Pharmacokinetics of 3'-azido-3'-deoxythymidine and its catabolites and interactions with probenecid in rhesus monkeys.

The pharmacokinetics and metabolism of 3'-azido-3'-deoxythymidine (AZT) were investigated in rhesus monkeys after subcutaneous administration of 33.3 mg of AZT per kg of body weight alone or in the presence of 100 mg of probenecid per kg. In addition to unchanged drug, two catabolites, 5'-O-glucuronide (GAZT) and 3'-amino-3'-deoxythymidine (AMT), were detected in plasma within 30 min. GAZT exhibited a kinetic profile similar to that of AZT, with an elimination half-life of approximately 1 h, while AMT was more variable, with an apparent half-life of 1.6 +/- 1.5 h. Approximately 90% of the total administered dose was recovered in urine within 24 h as AZT, GAZT, AMT, and the 5'-O-glucuronide of AMT. AZT and AMT demonstrated similar cerebrospinal fluid (CSF) penetration 1 h after AZT treatment, while GAZT poorly crossed the blood-brain barrier. Concomitant administration of probenecid greatly altered the pharmacokinetics of AZT, GAZT, and AMT, resulting in prolongation of their apparent elimination half-lives, increased concentrations in plasma, and marked reduction in renal clearances. In addition, the CSF/plasma concentration ratios for AZT and its catabolites were greatly increased, suggesting that probenecid inhibits efflux of AZT and its catabolites from CSF to plasma. The substantial levels of AMT in plasma suggest that this catabolite affects the pharmacodynamic properties of AZT in relation to its activity against human immunodeficiency virus replication and cytotoxicity to host cells. Enhanced AMT levels in plasma in the presence of probenecid may decrease the therapeutic efficacy of the AZT-probenecid combination.

Catabolism of 3'-azido-3'-deoxythymidine in hepatocytes and liver microsomes, with evidence of formation of 3'-amino-3'-deoxythymidine, a highly toxic catabolite for human bone marrow cells.

Metabolic studies in humans have demonstrated that 3'-azido-3'-deoxythymidine (AZT) is primarily eliminated as its 5'-O-glucuronide (GAZT). However, no detailed cellular metabolic studies have been reported on the complete catabolic fate of AZT at the hepatic site. Because the liver is probably the major site of AZT catabolism, the metabolism and transmembrane distribution of AZT were evaluated in freshly isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic, and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AZT, the predominant intracellular catabolite was GAZT, which reached a concentration of approximately 22 microM by 60 min. Additionally, under nonreducing conditions substantial levels of two previously unidentified AZT catabolites that were formed at the hepatic site and were distinct from any known anabolites or catabolites were also detected. These catabolites were identified as 3'-amino-3'-deoxythymidine (AMT) by fast atom bombardment mass spectrometry and 3'-amino-3'-deoxythymidine glucuronide (GAMT) through specific enzymatic hydrolysis. However, AMT was not a substrate for uridine 5'-diphosphoglucuronyltransferase and GAMT was found to be a reductive product of GAZT. Studies using rat and human liver microsomes demonstrated that the rate of formation of AMT and GAMT increased in the presence of NADPH, suggesting the involvement of a NADPH-dependent enzyme system. Studies using human hematopoietic progenitor cells demonstrated that AMT was 5- to 7-fold more toxic to human colony-forming units granulocyte-macrophage and burst-forming units erythroid than was AZT. This study provides the first detailed catabolic profile of AZT at the hepatic site and emphasizes the critical role that the liver plays in drug clearance. Formation of AMT, a highly toxic catabolite of AZT, raises a question regarding the role of AMT in the cytotoxic effects of AZT observed in patients.

Glucuronidation of 3'-azido-3'-deoxythymidine by rat and human liver microsomes.

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by rat and human liver microsomes has been studied in vitro. The AZT-glucuronide was preliminarily identified through specific hydrolysis by beta-glucuronidase and rigorous product identification was performed by high-field proton nuclear magnetic resonance and fast-atom-bombardment mass spectrometry. A beta-linked 5'-O-glucuronide was the exclusive product formed in liver microsomes. Rat and human liver microsomal uridine 5'-diphosphoglucuronyltransferase activities toward AZT were investigated. These studies revealed that AZT had a lower Km and a 5-6-fold higher relative catalytic efficiency for uridine 5'-diphosphoglucuronyltransferase in human as compared to rat liver microsomes which may play a role in the quantitative differences observed in the degree of AZT glucuronidation between rat and human.