Modulation of the pharmacokinetics of endogenous plasma uridine by 5-(phenylthio)acyclouridine, a uridine phosphorylase inhibitor: implications for chemotherapy.

PURPOSE: The purpose of this investigation was to evaluate the ability of oral PTAU, 5-(phenylthio)acyclouridine, to increase the concentration of endogenous plasma uridine. PTAU is a new potent and specific inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. METHODS: PTAU was administered to mice orally and parenterally. The plasma levels of PTAU as well as those of uridine and its catabolite uracil were measured by HPLC, and pharmacokinetic analysis was performed. RESULTS: PTAU was fully adsorbed after oral administration (over 100% oral bioavailability) and no PTAU metabolites were detected. PTAU administered orally had no apparent toxicity at doses up to 120 mg/kg per day for 5 days. Parenteral administration of PTAU at 30, 45 and 60 mg/kg increased the concentration of endogenous plasma uridine (1.8 +/- 0.2 microM) by approximately six-, seven-, and nine-fold, respectively. Plasma uridine concentration remained higher than control values until 8 h after PTAU administration. Similar results were obtained following oral administration of PTAU. The baseline concentrations of endogenous plasma uridine were increased by approximately six-, seven- and ten-fold by oral administration of PTAU at 30, 45 and 60 mg/kg, respectively, and remained higher than the controls until 8 h after PTAU administration. PTAU did not alter the concentration of endogenous plasma uracil. CONCLUSION: The effectiveness of the PTAU in elevating and sustaining high plasma uridine concentrations may be useful in rescuing or protecting the host from toxicities of various chemotherapeutic pyrimidine analogues as well as in the management of medical disorders that respond to the administration of uridine.

Effect of 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase, on plasma concentration of uridine released from 2',3',5'-tri-O-acetyluridine, a prodrug of uridine: relevance to uridine rescue in chemotherapy.

PURPOSE: The purpose of this investigation was to study the effects of combining oral 5-(phenylselenenyl)acyclouridine (PSAU) with 2',3',5'-tri-O-acetyluridine (TAU) on the levels of plasma uridine in mice. PSAU is a new lipophilic and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. PSAU has 100% oral bioavailability and is a powerful enhancer of the bioavailability of oral uridine. TAU is a prodrug of uridine and a far superior source of uridine than uridine itself. METHODS: Oral TAU was administered to mice alone or with PSAU. The plasma levels of uridine and its catabolites as well as PSAU were measured using HPLC and pharmacokinetic analysis was performed. RESULTS: Oral administration of 2000 mg/kg TAU increased plasma uridine by over 250-fold with an area under the curve (AUC) of 754 micromol x h/l. Coadministration of PSAU at 30 and 120 mg/kg with TAU further improved the bioavailability of plasma uridine resulting from the administration of TAU alone by 1.7- and 3.9-fold, respectively, and reduced the Cmax and AUC of plasma uracil. CONCLUSION: The exceptional effectiveness of PSAU plus TAU in elevating and sustaining a high plasma uridine concentration could be useful in the management of medical disorders that are remedied by administration of uridine, as well as the rescue or protection from host toxicities of various chemotherapeutic pyrimidine analogues.

Effect of administration of 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase, on the anti-tumor efficacy of 5-fluoro-2'-deoxyuridine against murine colon tumor C26-10.

The effect of co-administration of 5-(phenylselenenyl)acyclouridine (PSAU), a new uridine phosphorylase (UrdPase, EC 2.4.2.3) inhibitor, on the efficacy of 5-fluoro-2'-deoxyuridine (FdUrd) was tested against murine colon C26-10 tumor xenografts. In contrast to our previous results with human tumors, co-administration of PSAU with FdUrd decreased instead of increasing the efficacy of FdUrd against tumor growth. However, co-administration of PSAU with FdUrd (300 mg/kg/day) protected the mice completely from the 83% mortality induced by the same dose of FdUrd alone. Enzyme studies indicated that UrdPase in colon C26-10 tumors is responsible for the catabolism of FdUrd to 5-fluorouracil (FUra), as colon C26-10 tumors do not have thymidine phosphorylase (dThdPase, EC 2.4.2.4). In contrast, colon C26-10 tumors had extraordinarily high UrdPase activity (300 micromol/min/mg protein), which was at least 200-fold higher than the highest UrdPase activity in any of the human xenografts we tested previously. Furthermore, the activities of UrdPase and orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) were 192- and 2-fold higher, respectively, while that of dihydrouracil dehydrogenase (EC 1.3.1.2) was 1000-fold lower in the tumor than in the host liver. It is suggested that FdUrd exerts its anticancer effects against colon C26-10 tumors mainly through the catabolism of FdUrd to FUra by UrdPase, which then could be anabolized to 5-fluorouridine 5'-monophosphate (FUMP) by OPRTase and ultimately to other toxic 5-fluorouridine nucleotides, hence inducing the observed FdUrd toxic effects. Co-administration of PSAU with FdUrd inhibited UrdPase and the catabolism of FdUrd to FUra. This would result in the observed reduction of the antitumor efficacy of FdUrd. In addition, the increase in plasma uridine concentration induced by PSAU as well as the catabolism of FUra by the high dihydrouracil dehydrogenase activity in the liver also may have circumvented any residual FUra toxic effects against the host. These results clearly demonstrate that the anticancer efficacy of the combination of UrdPase inhibitors and FdUrd is not general and is dependent largely on the type of tumor under treatment and the mode of FdUrd metabolism in these tumors.

5-phenylthioacyclouridine: a potent and specific inhibitor of uridine phosphorylase.

5-Phenylthioacyclouridine (PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phenylthiouracil) was synthesized as a highly specific and potent inhibitor of uridine phosphorylase (UrdPase, EC 2.4.2.3). PTAU has inhibition constant (K(is)) values of 248 and 353 nM towards UrdPase from mouse and human livers, respectively. PTAU was neither an inhibitor nor a substrate for thymidine phosphorylase (EC 2.4.2.4), uridine-cytidine kinase (EC 2. 7.1.48), thymidine kinase (EC 2.7.1.21), dihydrouracil dehydrogenase (EC 1.3.1.2), orotate phosphoribosyltransferase (EC 2.4.2.10), or orotidine 5'-monophosphate decarboxylase (EC 4.1.2.23), the enzymes that could utilize the substrate (uridine or thymidine) or products (uracil or thymine) of UrdPase. Different isomers of 5-tolylthiouracil also were synthesized and tested as inhibitors of UrdPase. The meta-substituted isomer was 3- to 4-fold more potent as an inhibitor of UrdPase than the para- or ortho-substituted isomers. These data indicate that the hydrophobic pocket in the active site of UrdPase adjacent to the 5-position of the pyrimidine ring can accommodate the meta-substituted 5-phenyluracils better than the other isomers, leading to improved inhibition. Therefore, it is anticipated that the potency of PTAU can be increased further by the addition of certain hydrophobic groups at the meta position of the phenyl ring. PTAU has potential usefulness in the therapy of cancer and AIDS as well as other pathological and physiological disorders that can be remedied by the administration of uridine.

Modulation of plasma uridine concentration by 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase: relevance to chemotherapy.

PURPOSE: The purpose of this investigation was to evaluate the efficacy of oral 5-(phenylselenenyl)-acyclouridine (PSAU) in increasing endogenous plasma uridine concentration as well as its ability to improve the bioavailability of oral uridine. PSAU is a new potent and specific inhibitor of uridine phosphorylase (Urd-Pase, EC 2.4.2.3), the enzyme responsible for uridine catabolism. This compound was designed as a lipophilic inhibitor in order to facilitate its access to the liver and intestine, the main organs involved in uridine catabolism. METHODS: Oral PSAU was administered orally to mice alone or with uridine. The plasma levels of PSAU as well as uridine and its catabolites were measured using high-performance liquid chromatography and pharmacokinetic analysis was performed. RESULTS: PSAU has an oral bioavailability of 100% and no PSAU metabolites were detected. PSAU has no apparent toxicity at high doses. Oral administration of PSAU at 30 and 120 mg/kg increased baseline concentration of endogenous plasma uridine (2.6 +/- 0.7 microM) by 3.2- and 8.7-fold, respectively, and remained three- and six-fold higher, respectively, than the controls for over 8 h. PSAU, however, did not alter the concentration of endogenous plasma uracil. Co-administration of PSAU with uridine elevated the concentration of plasma uridine over that resulting from the administration of either alone, and reduced the peak plasma concentration (C(max)) and area under the curve (AUC) of plasma uracil. Co-administration of PSAU at 30 mg/kg and 120 mg/kg improved the low bioavailability of oral uridine (7.7%) administered at 1,320 mg/kg by 4.8- and 4.2-fold, respectively, and reduced the AUC of plasma uracil from 1,421 to 787 micromol/h x l and 273 micromol/h x l, respectively. Similar results were observed when PSAU was co-administered with lower doses of uridine. Oral PSAU at 30 mg/kg and 120 mg/kg improved the bioavailability of oral 330 mg/kg uridine by 5.2- and 8.9-fold, and that of oral 660 mg/kg uridine by 6.4- and 9.0-fold, respectively. However, the reduction in the AUC values of plasma uracil was less dramatic than that seen when the high dose of 1,320 mg/kg uridine was used. CONCLUSION: The effectiveness of the PSAU plus uridine combination in elevating and sustaining high plasma uridine concentration may be useful to rescue or protect from host toxicity of various chemotherapeutic pyrimidine analogs as well as in the management of medical disorders that are remedied by administration of uridine.

Synthesis and biological properties of 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil.

A novel 5-o-carboranyl-containing nucleoside, 5-o-carboranyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (6, CFAU), was synthesized as a potential intracellular neutron capture agent. This compound was prepared in five steps starting from 5-iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (1). The desired carboranyl derivative was obtained by addition of decaborane [as the bis(propionitrile) adduct] to the protected acetylenic nucleoside precursor followed by debenzoylation. The synthesis of CFAU was also performed by glycosylation of the suitably protected 5-o-carboranyluracil with the appropriate 2-fluoroarabinosyl derivative. This compound was evaluated for its cytotoxicity in human lymphocytes, monkey cells, and rat and human gliomas cells, as well as for antiviral activity against human immunodeficiency virus and herpes simplex virus type 1. Its biological activity was compared to 5-o-carboranyl-1-(2-deoxyribofuranosyl)uracil in these cell culture systems, human bone marrow cells, and mice. The results obtained to date suggest that CFAU has suitable characteristics as a sensitizer for boron neutron capture therapy.

Cellular pharmacology and biological activity of 5-carboranyl-2'-deoxyuridine.

PURPOSE: The intracellular uptake and metabolism of 5-carboranyl-2'-deoxyuridine was investigated in primary human lymphocytes and in a T lymphoblastoid cell line using unlabeled and tritium labeled compound. The cytotoxicity and antiviral activity of the compound and stability to enzyme degradation was determined. METHODS AND MATERIALS: A novel method for radiolabeling the 5-carboranyl moiety of pyrimidine nucleosides was developed. Cells were exposed to unlabeled and tritium labeled 5-carboranyl-2'-deoxyuridine and the intracellular uptake and egress of the compound determined by high pressure liquid chromatography. The viability and growth of normal and malignant cells, including human and rat gliomas, in the presence of the compound was determined. RESULTS: Substantial levels of 5-carboranyl-2'-deoxyuridine-5'-monophosphate are formed intracellularly and this major metabolite can be detected in cells 48 h after removal of the parent compound from the medium. No significant phosphorylation to the 5'-diphosphate or triphosphate of 5-carboranyl-2'-deoxyuridine was detected. Furthermore, radiolabeled 5-carboranyl-2'-deoxyuridine was not incorporated into deoxyribonucleic acid. 5-carboranyl-2'-deoxyuridine was essentially nontoxic to human lymphocytes as well as human or rat glioma cells, and had no marked effect in human lymphocytes acutely infected with human immunodeficiency virus type 1. CONCLUSION: The results demonstrate for the first time that 5-carboranyl-2'-deoxyuridine is phosphorylated intracellularly and suggest that it should be considered for further studies as a potential sensitizer for boron neutron capture therapy.

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.

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.