thioinosine and nitrobenzylthioinosine-5--monophosphate

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Topics: Animals; Antimetabolites, Antineoplastic; Biological Transport; Cells, Cultured; Clofarabine; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Lamivudine; Male; Rats, Sprague-Dawley; Telomerase; Testis; Thioinosine; Thionucleotides

Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum.. We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real-time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol-induced EAAT2 expression.. An ENT1-specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up-regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol-induced EAAT2 up-regulation.. Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.

Topics: Animals; Astrocytes; Cell Line; Central Nervous System Depressants; Dose-Response Relationship, Drug; Equilibrative Nucleoside Transporter 1; Ethanol; Excitatory Amino Acid Transporter 2; Glutamic Acid; Mice; RNA, Small Interfering; Thioinosine; Thionucleotides; Time Factors

Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB-NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB-NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.

Topics: Animals; Antiparasitic Agents; Cells, Cultured; Drug Therapy, Combination; Enzyme Inhibitors; Leishmania; Leishmaniasis; Macrophages; Mice; Mice, Inbred BALB C; Schistosoma mansoni; Thioinosine; Thionucleotides; Trypanosoma brucei gambiense; Tubercidin

One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

Topics: Animals; Drug Combinations; Drug Resistance; Genetic Therapy; Genetic Vectors; Glucuronates; Green Fluorescent Proteins; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Luminescent Proteins; Macaca mulatta; Recombinant Proteins; Tetrahydrofolate Dehydrogenase; Thioinosine; Thionucleotides; Transduction, Genetic; Trimetrexate

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). Administration of the maximum tolerated dose of trimetrexate (TMTX) with the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose-5'-monophosphate (NBMPR-P) inhibited 32Dp210 tumor progression in mice engrafted with transgenic tyrosine-22 DHFR marrow and improved survival of tumor-bearing animals as long as drug administration was continued. NBMPR-P coadministration was necessary for maximal tumor inhibition, as administration of TMTX alone delayed but did not prevent tumor progression. The chemoprotection afforded by engraftment with transgenic tyrosine-22 DHFR marrow was necessary for effective chemotherapy, as normal mice lacking transgenic marrow could not tolerate the higher TMTX dose (60 mg/kg/day) administered to mice with transgenic marrow, and the decreased dose of TMTX with NBMPR-P tolerated by normal tumor-bearing animals did not inhibit tumor progression or improve animal survival. We conclude that TMTX with NBMPR-P inhibits tumor progression in the 32Dp210 model of CML in animals engrafted with drug-resistant tyrosine-22 DHFR transgenic marrow, and that based on this model the introduction of a drug-resistant DHFR gene into marrow combined with TMTX and NBMPR-P administration may provide an effective treatment for CML.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Bone Marrow Transplantation; Disease Models, Animal; Disease Progression; Drug Resistance, Neoplasm; Drug Synergism; Female; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Mice; Mice, Inbred C3H; Mice, Transgenic; Tetrahydrofolate Dehydrogenase; Thioinosine; Thionucleotides; Trimetrexate

Currently, low levels of stable gene transfer into hematopoietic tissues of large animals and humans continues to limit the clinical application of gene therapy. One strategy for overcoming this problem is to selectively expand, in vivo, the population of successfully gene-modified cells. Recent work has shown that nucleoside transport inhibition in combination with antifolates can be used to select in vivo for hematopoietic stem cells expressing drug-resistant dihydrofolate reductase (DHFR). In this study we investigated whether trimetrexate (TMTX) and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose phosphate (NBMPR-P) can be used to select for tyr22-variant DHFR expressing transgenic hematopoietic cells in a murine bone marrow transplant model. Our results indicate that 40 mg/kg TMTX and 20 mg/kg NBMPR-P can be used in combination to expand transgene-positive progenitor cells 3- to 4-fold immediately following drug administration. In addition, long-term progenitor populations were expanded 2- to 3-fold in primary recipients, to approximately 5 months following drug administration. Secondary transplants conducted with marrow from primary recipients 5 months following drug administration revealed a statistically significant selective expansion of transgene-positive cells in the spleens and peripheral blood of these animals. No such expansion was observed in groups of mice treated with TMTX alone or NBMPR-P alone. We conclude that TMTX + NBMPR-P can be used to selectively expand transgenic tyr22-variant DHFR expressing murine hematopoietic stem cells in vivo.

Topics: Animals; Blotting, Southern; Bone Marrow Transplantation; Female; Folic Acid Antagonists; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Leukocyte Count; Mice; Mice, Transgenic; Spleen; Tetrahydrofolate Dehydrogenase; Thioinosine; Thionucleotides; Trimetrexate

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.

Topics: Alanine; Animals; Biological Transport; Cell Line; Conserved Sequence; Dogs; Equilibrative Nucleoside Transporter 1; Genetic Vectors; Glycine; Guanosine; Humans; Inosine; Membrane Transport Modulators; Membrane Transport Proteins; Mutagenesis, Site-Directed; Point Mutation; Saccharomyces cerevisiae; Thioinosine; Thionucleotides; Transfection; Uridine

Adenosine levels increase in brain during cerebral ischemia, and adenosine has receptor-mediated neuroprotective effects. This study was performed to test the hypothesis that nitrobenzylthioinosine (NBMPR), a selective and potent inhibitor of one adenosine transporter subtype termed ENT1, or es, can protect against ischemic neuronal injury by enhancing adenosine levels and potentiating adenosine receptor-mediated effects, including attenuation of the cellular production and release of tumor necrosis factor-alpha (TNF-alpha). In rats, the phosphorylated prodrug form of NBMPR, NBMPR-phosphate, or saline was administered by intracerebroventricular injection 30 min before forebrain ischemia. Seven days following the ischemic episode, rats were killed, and neuronal damage in the CA1 region of the hippocampus was assessed. The number of pyramidal neurons was significantly (p < 0.001) greater in the NBMPR-P treatment group. A trend toward protection was still evident at 28 days postreperfusion. Adenosine increased significantly during ischemia to levels eight- to 85-fold above basal. NBMPR-P treatment did not cause statistically significant increases in ischemic adenosine levels; however, this treatment tended to increase adenosine levels in all brain regions at 7 min postreperfusion. Ischemia-induced expression of TNF-alpha was not altered by NBMPR-P treatment, and the nonselective adenosine receptor antagonist 8-(p-sulfophenyl) theophylline did not abolish the neuroprotective effects of NBMPR-P treatment. These data indicate that NBMPR can protect CA1 pyramidal neurons from ischemic death without statistically significant effects on adenosine levels or adenosine receptor-mediated inhibition of the proinflammatory cytokine TNF-alpha.

Topics: Adenosine; Affinity Labels; Animals; Cerebral Ventricles; Gene Expression Regulation; Injections, Intraventricular; Ischemic Attack, Transient; Male; Neurons; Prodrugs; Prosencephalon; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion; Thioinosine; Thionucleotides; Transcription, Genetic; Tumor Necrosis Factor-alpha

One of the main impediments to effective gene therapy of blood disorders is the resistance of human hematopoietic stem cells to stable genetic modification. We show here that a small minority of retrovirally transduced stem cells can be selectively enriched in vivo, which might be a way to circumvent this obstacle. We constructed two retroviral vectors containing an antifolate-resistant dihydrofolate reductase cDNA transcriptionally linked to a reporter gene. Mice were transplanted with transduced bone marrow cells and then treated with an antifolate-based regimen that kills unmodified stem cells. Drug treatment significantly increased the percentage of vector-expressing peripheral blood erythrocytes, platelets, granulocytes, and T and B lymphocytes. Secondary transplant experiments demonstrated that selection occurred at the level of hematopoietic stem cells. This system for in vivo stem-cell selection provides a means to increase the number of genetically modified cells after transplant, and may circumvent an substantial obstacle to successful gene therapy for human blood diseases.

Topics: Animals; Blood Cells; Drug Resistance; Female; Folic Acid Antagonists; Genes, Reporter; Genetic Therapy; Genetic Vectors; Hematopoietic Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Retroviridae; Selection, Genetic; Tetrahydrofolate Dehydrogenase; Thioinosine; Thionucleotides; Transformation, Genetic; Trimetrexate

Transfer of drug resistance genes to hematopoietic cells is being studied as a means to protect against the myelosuppression associated with cancer chemotherapy and as a strategy for the in vivo selection and amplification of genetically modified cells. The goal of this study was to test if retroviral-mediated gene transfer of a dihydrofolate reductase (DHFR) variant (L22Y) could be used for in vivo selection of transduced myeloid cells and to determine what proportion of transduced cells was required for protection from myelosuppression. Based on previous work suggesting that selection with antifolates may also require inhibition of nucleoside transport mechanisms, mice transplanted with DHFR-transduced bone marrow cells were treated with trimetrexate and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine riboside phosphate. In vivo selection of transduced myeloid progenitors was seen in the bone marrow and in circulating mature peripheral blood cells following drug treatment. These results show that the novel combination of the L22Y-DHFR cDNA, trimetrexate and nitrobenzylmercaptopurine riboside phosphate can be used to select for transduced myeloid cells, and that this approach warrants further study in large animal models. A bicistronic vector containing a human CD24 reporter gene was used to determine the number of modified cells needed for chemoprotection. Partial protection from neutropenia was seen when greater than 10% of myeloid cells expressed the vector, and high levels of protection were obtained when the proportion exceeded 30%. These results suggest that gene transfer may be useful for myeloprotection in certain pediatric cancers, but that more efficient gene transfer will be required to apply this approach to adult cancer patients.

Topics: Adult; Amino Acid Substitution; Animals; Antigens, CD; Bone Marrow Cells; CD24 Antigen; Encephalomyocarditis virus; Female; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Genetic Variation; Genetic Vectors; Harvey murine sarcoma virus; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Membrane Glycoproteins; Mice; Recombinant Proteins; Tetrahydrofolate Dehydrogenase; Thioinosine; Thionucleotides; Transfection; Trimetrexate

Antifolates such as methotrexate (MTX) and trimetrexate (TMTX) are widely used in the treatment of cancer and nonmalignant disorders. Transient, yet sometimes severe myelosuppression is an important limitation to the use of these drugs. It has previously been shown that clonogenic myeloid progenitors and colony-forming units-spleen are resistant to antifolates, suggesting that myelotoxicity occurs late in hematopoietic development. The goal of this study was to define the mechanisms by which primitive hematopoietic cells resist the toxic effects of antifolate drugs. To test the hypothesis that myeloid progenitors may salvage extracellular nucleotide precursors to resist TMTX toxicity, a defined liquid culture system was developed to measure TMTX toxicity in expanding progenitor populations. These in vitro experiments showed that both human and murine progenitors can resist TMTX toxicity by importing thymidine and hypoxanthine from the serum. As predicted from these findings, several drugs that block thymidine transport sensitized progenitors to TMTX in vitro, although to differing degrees. These nucleoside transport inhibitors were used to test whether progenitors and hematopoietic stem cells (HSCs) could be sensitized to TMTX in vivo. Treatment of mice with TMTX and nitrobenzylmercaptopurineriboside phosphate (NBMPR-P), a potent transport inhibitor, caused significant depletions of clonogenic progenitors within the bone marrow (20-fold) and spleen (6-fold). Furthermore, NBMPR-P administration dramatically sensitized HSCs to TMTX, with dual-treated mice showing a greater than 90% reduction in bone marrow repopulating activity. These studies demonstrate that both myeloid progenitor cells and HSCs resist TMTX by using nucleotide salvage mechanisms and that these pathways can be pharmacologically blocked in vivo using nucleoside transport inhibitors. These results have important implications regarding the use of transport inhibitors for cancer therapy and for using variants of dihydrofolate reductase for in vivo selection of genetically modified HSCs.

Topics: Animals; Antimetabolites, Antineoplastic; Biological Transport; Cell Death; Drug Resistance, Neoplasm; Female; Hematopoietic Stem Cells; Humans; Mice; Mice, Inbred C57BL; Thioinosine; Thionucleotides; Thymidine; Trimetrexate

Nucleoside transport inhibitors that cross the blood-brain barrier may be able to potentiate the neuroprotective effects of adenosine. We tested whether nitrobenzylthioinosine (NBMPR) crosses the blood-brain barrier in three types of experiments. First, intravenous injection of [3H]NBMPR and [14C]sucrose was performed. Brain volume of distribution and brain delivery were greater for [3H]NBMPR than for [14C]sucrose. Second, rats were injected intraperitoneally with NBMPR 5'-monophosphate (NBMPR-P), a prodrug form of NBMPR, or vehicle. Perchloric acid extracts of brains from rats treated with NBMPR-P inhibited [3H]NBMPR binding in competition binding assays nearly 3-fold more than extracts from brains of vehicle-treated animals. Third, cerebrospinal fluid (CSF) extracted from rats treated with NBMPR-P (10 mg/kg i.p.) contained 24.1 +/- 4.4 nM NBMPR while levels were undetectable in CSF from vehicle-treated rats. From these data, we conclude that NBMPR crosses the blood-brain barrier.

Topics: Animals; Binding, Competitive; Blood-Brain Barrier; Brain; Injections, Intraperitoneal; Injections, Intravenous; Male; Rats; Rats, Sprague-Dawley; Sucrose; Thioinosine; Thionucleotides

Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5'-phosphate of nitrobenzylthioinosine, a potent inhibitor of the es equilibrative nucleoside transport (NT) system. NBTGR-P, the 5'-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of "cured" mice than were the same treatment regimens without NBMPR-P.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Drug Synergism; Female; Hindlimb; Leukemia L1210; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Transplantation; Nervous System Diseases; Paralysis; Prodrugs; Thioinosine; Thionucleotides; Vidarabine

Topics: Adenosine; Animals; Babesia bovis; Babesiosis; Biological Transport; Cattle; Cattle Diseases; Dilazep; Drug Therapy, Combination; Erythrocyte Membrane; Erythrocytes; Malaria; Malaria, Falciparum; Mice; Plasmodium falciparum; Pyrimidine Nucleosides; Thioinosine; Thionucleotides; Tubercidin

The efficacy of the highly selective antischistosomal combination chemotherapy with tubercidin (7-deazaadenosine) plus nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), [el Kouni et al., Proc Natl Acad Sci USA 80: 6667-6670, 1983; el Kouni et al., Biochem Pharmacol 36: 3815-3821, 1987] was examined against chronic and advanced stages of schistosomiasis in mice. Administration of four successive daily doses of tubercidin (5 mg/kg/day) plus NBMPR-P (25 mg/kg/day) to Schistosoma mansoni-infected mice beginning 5, 6, 7 and 8 weeks post-infection and monitored for 22 weeks was very effective against the parasite. It resulted in a marked increase in survivorship of treated mice. Repetition of the dose-regimen after a 10-day rest period was even more effective. However, survivorship of infected animals decreased with the delay of therapy. Early treatment (5 weeks post-infection) resulted in 100% survival compared to 13% only for untreated animals. If therapy was instituted at 8 weeks post-infection, only 70% of the treated mice survived. Treated animals appeared healthy and were found to have less splenomegaly and hepatomegaly. Combination therapy also caused a significant reduction in the number of worms as well as the number of eggs in the liver and small intestine. However, these differences diminished as the treatment was delayed. The number of eggs in the liver was reduced from an average of 120,000 eggs per liver in untreated animals to approximately 16,000 eggs per liver when treated at 5 weeks post-infection. When treatment was delayed to 8 weeks post-infection, the reduction in liver egg count was not as dramatic (88,000 eggs per liver). Similarly, the number of eggs was reduced in the intestine from 1,759 to an average of 58 and 860 eggs per cm2 of the intestine when the mice were treated at 5 and 8 weeks post-infection respectively. However, some worms survived and resumed egg production after an extended period of recuperation. Histological examination indicated that combination therapy was effective in preventing the formation of new egg granulomas but not on pre-existing granulomas.

Topics: Animals; Drug Therapy, Combination; Follow-Up Studies; Intestines; Life Expectancy; Liver; Mice; Schistosomiasis mansoni; Thioinosine; Thionucleotides; Tubercidin

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10 degrees C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.

Topics: Animals; Biological Transport; Erythrocytes; Hagfishes; Nucleosides; Thioinosine; Thionucleotides

Nitrobenzylthioinosine (NBMPR), a potent inhibitor of facilitated nucleoside transport in vitro and in vivo, and acyclothymidine (ACT), a potent inhibitor of pyrimidine nucleoside phosphorylase in vitro, have been used in an attempt to modulate the biodistribution of 125I-labelled iododeoxyuridine ([125I]IUdR). ACT or NBMPR-P (a water-soluble prodrug of NBMPR) were injected into BDF1 mice bearing implanted Lewis lung tumors, according to protocols which would provide high and low plasma levels of the inhibitor. Compared with controls, both inhibitors induced transient, marginal increases in hepatic, renal and blood levels of [125I]IUdR, and decreased levels in tumors at short time intervals after injection. It is concluded that there is a mild tumor-sparing effect when either NBMPR or ACT are administered together with single i.v. diagnostic doses of [125I]IUdR.

Topics: Animals; Idoxuridine; Inosine; Iodine Radioisotopes; Lung Neoplasms; Male; Mice; Neoplasm Transplantation; Radionuclide Imaging; Stimulation, Chemical; Thioinosine; Thionucleotides; Tissue Distribution; Uracil

Host toxicity of the dose regimen of tubercidin (7-deazaadenosine) plus nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) used in combination therapy of schistosomiasis (M. H. el Kouni, D. Diop, and S. Cha, Proc. Natl. Acad. Sci. USA 80:6667-6670, 1983; M. H. el Kouni, N. J. Messier, and S. Cha, Biochem. Pharmacol. 36:3815-3821, 1987) was examined in vivo in mice and in vitro with human bone marrow progenitor cells. Four successive daily intraperitoneal injections of tubercidin at 5 mg/kg per day produced 100% mortality in mice within 3 to 5 days following the first injection, with massive peritonitis and intestinal obstruction secondary to abdominal adhesions. Coadministration of NBMPR-P (25 mg/kg per day) protected the mice from the lethality of tubercidin and allowed the repetition of the regimen for a second time with 100% survival until the mice were sacrificed 22 days following the first injection. Blood chemistry, hematological studies, and histological examinations showed no evidence for injury to the liver, kidney, spleen, pancreas, mesentery, or peritoneal mesothelium. In vitro, tubercidin alone had a direct dose-dependent inhibitory effect on myeloid and erythroid human bone marrow progenitor cells, and consistent inhibition (50%) of granulocyte-macrophage CFU (CFU-GM) and erythroid burst-forming units (BFU-E) occurred at 2 to 3 nM tubercidin. At higher doses, BFU-E were more sensitive to tubercidin toxicity than CFU-GM. Complete inhibition (99%) of BFU-E colonies occurred at 10 nM tubercidin, while complete inhibition of CFU-GM occurred at 100 nM. NBMPR-P at 10 to 100 nM protected CFU-GM and BFU-E from tubercidin toxicity in a dose-dependent matter.

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Colony-Forming Units Assay; Female; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Inosine; Mice; Schistosomiasis; Thioinosine; Thionucleotides; Tubercidin

Schistosoma mansoni incorporated tubercidin, nebularine, 9-deazaadenosine, 5'-deoxy-5'-iodo-2-fluoroadenosine, 7,9-dideaza-7-thiaadenosine and toyocamycin but not sangivamycin, 3'-deoxy-sangivamycin, or 1-methylformycin into their nucleotide pool after a 4-hr incubation in vitro. In contrast to mammalian systems, addition of nucleoside transport inhibitors nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), dilazep, or benzylacyclouridine had no significant effect on the pattern of incorporation of these adenosine analogues. Dipyridamole, on the other hand, reduced, but did not prevent, the incorporation of these analogues into the nucleoside 5'-triphosphate pool. These results suggest that the transport of purine nucleosides in schistosomes is different from that of their mammalian hosts. Therefore, coadministration of a specific nucleoside transport inhibitor with tubercidin, nebularine, 9-deazaadenosine, 5'-deoxy-5'-iodo-2-fluoroadenosine, toyocamycin, or 7,9-dideaza-7-thiaadenosine may result in high selective toxicity against schistosomes, as was the case with the combination of tubercidin plus NBMPR-P [el Kouni et al., Proc. natn. Acad. Sci. U.S.A. 80, 6667 (1983); el Kouni et al., Biochem. Pharmac. 34, 3921 (1985)], by protecting the host but not the parasite from the toxicity of these analogues.

Topics: Adenosine; Animals; Biological Transport; Dilazep; Dipyridamole; Kinetics; Nucleosides; Schistosoma mansoni; Structure-Activity Relationship; Thioinosine; Thionucleotides; Uracil

In contrast to their effects on mammalian cells, the nucleoside transport inhibitors nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) dilazep, benzylacyclouridine (BAU), and to a lesser extent, dipyridamole have no significant effect on the in vitro uptake of adenosine analogues by Schistosoma mansoni [el Kouni and Cha, Biochem. Pharmac. 36, 1099 (1987)]. Coadministration of either NMBPR-P or dilazep with potentially lethal doses of tubercidin (7-deazaadenosine), nebularine or 9-deazaadenosine protected mice from the toxicity of these adenosine analogues. Dipyridamole caused partial protection, whereas BAU did not protect the animals from this toxicity. Toyocamycin caused delayed mortality (after 16 weeks) which could not be prevented by coadministration of NBMPR-P. In S. mansoni infected mice, treated with the combination of NBMPR-P and 9-deazaadenosine was not effective against the parasite. On the other hand, the combinations of NBMPR-P or dilazep with either tubercidin or nebularine were highly toxic to the parasite but not the host. Combination therapy caused a marked reduction in the number of pairing of worms. Effectiveness of combination therapy could also be noted by a drastic decrease in the number of eggs in the liver and small intestine. All eggs found were dead, indicating a direct effect on ovigenesis. Although dipyridamole was less effective than NBMPR-P or dilazep in protecting the host from the toxicity of tubercidin or nebularine, the combinations with dipyridamole produced similar significant therapeutic effects in animals that survived. Mice receiving the combination of tubercidin (or nebularine) plus NBMPR-P or dilazep, as well as those that survived the combination with dipyridamole, appeared healthy and were found to have normal size livers and spleens. These results suggest that highly selective toxicity against schistosomes can be achieved by coadministration of various nucleoside transport inhibitors with adenosine analogues.

Topics: Adenosine; Animals; Biological Transport; Dilazep; Dipyridamole; Drug Therapy, Combination; Female; Mice; Purine Nucleosides; Ribonucleosides; Schistosoma mansoni; Schistosomiasis; Thioinosine; Thionucleotides; Toyocamycin; Tubercidin; Uracil

Nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), a water-soluble form of the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) was administered by i.v. injection to normal mice and BDF1 mice with implanted Lewis Lung carcinomas. Tritiated 5-Fluoro-2'-deoxyuridine (3H-FUdR) was injected either alone (control), 10 min before (I + 10), 10 min (I - 10) after, 60 min (I - 60) after, or simultaneously (I = 0) with the transport inhibitor. Tissue distributions of tritium were determined after intervals of 1, 2 and 4 h. The per cent of injected radioactivity (% dose) in liver was increased by all NBMPR-P protocols. Kidney radioactivity was similarly affected, with maximum increases (from 9.3 +/- 3.4 to 24.1 +/- 5.2% of the injected dose/g) after 1 h in the I - 60 animals. No statistically significant changes in the distribution of radioactivity in tumor, spleen, marrow or blood were induced by doses of NBMPR-P. Elevated levels of tritium radioactivity in blood were accompanied by similar increases in renal and hepatic radioactivity. The apparent increase in the tumor uptake of 3H-FUdR (from 1.4 +/- 0.2 to 5.7 +/- 2.3% dose/g) was not statistically significant at the 95% confidence limit. In general, NBMPR-P induced a relative tumor-sparing effect and at the same time increased uptake of 3H-FUdR by the liver and kidney, or delayed its clearance from these organs. There was no evidence to suggest that any advantage would be gained by using NBMPR-P treatment in conjunction with radiolabelled FUdR for tumor diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Floxuridine; Inosine; Lung Neoplasms; Male; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Radionuclide Imaging; Thioinosine; Thionucleotides; Tissue Distribution; Tritium

5-[125I]Iodo-2'-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be matabolically inert in these cells. Linear initial velocities were obtained at 20 degrees C for 125IdUrd transport, yielding a Km of 73 +/- 18 microM (n = 6). Low-affinity inhibitors of 125IdUrd transport, such as adenosine (Ki = 32 +/- 2 microM, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with 125IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 microM 125IdUrd, all at 20 degrees C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of 125IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use 3H- or 14C-labeled nucleoside permeants.

Topics: Adenosine; Biological Transport, Active; Erythrocytes; Humans; Idoxuridine; In Vitro Techniques; Iodine Radioisotopes; Kinetics; Nucleosides; Thioinosine; Thionucleotides

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Animals; Blood Glucose; Energy Metabolism; Erythrocytes; Inosine; Swine; Thioinosine; Thionucleotides

Coadministration of nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) with high doses of tubercidin by i.p. injection into Schistosoma japonicum infected mice beginning 5 weeks post-infection was highly toxic to the parasite but not the hose. Combination therapy resulted in a striking reduction in the number of worms, and the few worms that could be found were stunted. Combination therapy also caused a drastic reduction in the number of eggs in the livers (from 86,500 to 2,800 eggs/liver) and intestines (from 2,200 to 74 eggs/cm2), and 95% of eggs that were found were dead, indicating the termination of oviposition. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal size livers and spleens. These results demonstrate that by combining NBMPR-P with tubercidin high selective toxicity against S. japonicum can be achieved, as was shown previously with S. mansoni.

Topics: Animals; Drug Therapy, Combination; Female; Inosine; Kidney; Mice; Mice, Inbred Strains; Ribonucleosides; Schistosomiasis japonica; Thioinosine; Thionucleotides; Tubercidin

HeLa cells as well as human and mouse erythrocytes possess membrane sites which bind the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), reversibly but tightly (KD, 10(-9)-10(-10) M). Site-specific binding of the ligand correlates with inhibition of nucleoside transport. The present study showed that the 5'-phosphate of NBMPR, NBMPR-P, was not transport inhibitory. Upon exposure to [35S]NBMPR-P or [G-3H]NBMPR-P, HeLa cells retained the isotopic labels virtually exclusively in the form of NBMPR. The dephosphorylation of [G-3H]NBMPR-P by HeLa cells, assayed by the production of extracellular [G-3H]NBMPR, was competitively inhibited by AMP, but was not affected by the presence of 5 microM NBMPR, a concentration sufficient to completely occupy the transport inhibitory sites. Thus, the sites at which dephosphorylation of NBMPR occurs in HeLa cells are separate from and function independently of the high affinity sites which bind NBMPR.

Topics: 5'-Nucleotidase; Animals; Binding Sites; Cell Membrane; Erythrocytes; HeLa Cells; Humans; Inosine; Kinetics; Male; Mice; Nucleotidases; Protein Binding; Thioinosine; Thionucleotides; Uridine

N-Phosphonacetyl-L-aspartic acid (PALA) resistance may be due to the ability of tumor cells to utilize preformed circulating pyrimidine nucleosides, thereby overcoming the block of de novo pyrimidine biosynthesis which PALA causes. To test this hypothesis we examined the effects of PALA and nitrobenzylthioinosine (NBMPR) alone and in combination on B16 melanoma cells in vitro using a clonogenic assay and in vivo using growth delay. In medium containing purine and pyrimidine nucleosides at a final concentration of 28 microM, exposure to PALA (100 microM) alone or to NBMPR (10 microM) alone for periods up to 72 hr did not result in any cytotoxicity. However, exposures to PALA (100 microM) plus NBMPR (10 microM) resulted in a decrease in clonogenic survival to 0.011 at 72 hr. In medium without nucleosides, PALA (100 microM) exposure for 72 hr caused a similar decrease in survival to 0.015, whereas NBMPR (10 microM) had no effect on survival. The addition of uridine resulted in a concentration-dependent reversal of the cytotoxic effects of PALA. C57 Bl female mice bearing B16 melanoma were treated intraperitoneally daily for 4 days with PALA, the phosphate of NBMPR (NBMPR-P), or PALA plus NBMPR-P. PALA, 300 mg/kg daily X 4, resulted in a 6-day tumor growth delay but NBMPR-P, 100 mg/kg daily X 4, had no effect. PALA, 150 mg/kg daily X 4, plus NBMPR, 50 or 100 mg/kg daily X 4, resulted in a 6-day tumor growth delay also. These studies demonstrate that: (1) circulating pyrimidine nucleosides are determinants of the cytotoxic effects of PALA; (2) in vitro PALA and NBMPR combine to cause significant cytotoxicity whereas either agent alone has no effect; (3) in vivo the combination of PALA and NBMPR-P results in the same antitumor affect as PALA alone at twice the dose; and (4) due to an increase in animal toxicity, no therapeutic advantage could be demonstrated for the combination over PALA alone in vivo. We conclude that the cytotoxic effect of PALA is modulated by the levels of the preformed circulating nucleosides and that combining PALA with an inhibitor of salvage pyrimidine uptake would not increase the therapeutic efficacy of PALA because of an increase in toxicity.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Cell Line; Dose-Response Relationship, Drug; Female; Inosine; Melanoma; Mice; Mice, Inbred C57BL; Nucleosides; Organophosphorus Compounds; Phosphonoacetic Acid; Thioinosine; Thionucleotides

In isolated, perfused mouse livers, initial rates of uptake of [2-14C]pseudoisocytidine (PIC), measured during the first 15 secs of perfusion were markedly reduced when the perfusion medium contained 5 X 10(-6) M nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport. A similar inhibition of PIC uptake occurred when mice were treated with NBMPR-P (the 5'-monophosphate of NBMPR) at doses greater than 0.2 mg/kg ip injected 30 mins prior to the liver perfusion assay. However, in vivo studies showed that a late effect of NBMPR-P was enhancement in PIC levels in liver and other tissues in mice and rats, relative to levels in animals that had not received NBMPR-P. Increases in incorporation of PIC into RNA reflected the NBMPR-P-induced increases in tissue levels of PIC. NBMPR-P and other inhibitors of nucleoside transport may have therapeutic applications in manipulation of the pharmacokinetic behavior and toxicity of nucleoside drugs.

Topics: Animals; Biological Transport; Cell Membrane; Cytidine; Inosine; Kinetics; Liver; Male; Mice; Mice, Inbred Strains; Nucleosides; Perfusion; Rats; RNA; Thioinosine; Thionucleotides

Nitrobenzylthioinosine 5'-monophosphate (NBMPR-P) inhibits the transport of nucleosides, including tubercidin, in mammalian systems but not in Schistosoma mansoni. Administration of NBMPR-P with high doses of tubercidin (lethal doses if injected alone) by intraperitoneal injection into S. mansoni-infected mice was highly toxic to the parasite but not to the host. Combination therapy resulted in a striking decrease in the number and copulation of worms. The few worms that could be found were so stunted that it was difficult to identify their sex. Mice receiving the combination of tubercidin plus NBMPR-P appeared healthy and had normal-sized livers and spleens. Combination therapy also caused a drastic decrease in the number of eggs in the liver (from 32,500 to 1,800 eggs per liver) and in the intestine (from 1,295 to 2 eggs per cm2). All eggs found were dead, indicating the termination of oviposition. Very few granulomas were detected in livers of treated animals. Sections of these livers showed lesions containing dead worms and what appeared to be a process of regeneration of normal tissue around old granulomas. Thus, combination therapy reduced the number and the progress of the primary pathological lesions associated with schistosomiasis. These results demonstrate that through combination therapy, highly selective toxicity against a parasite can be achieved. The effectiveness, simplicity, and practicality of host protection afforded by this method may yield a promising chemotherapeutic approach for the treatment of schistosomiasis and other parasitic diseases.

Topics: Animals; Biological Transport; Disease Models, Animal; Drug Therapy, Combination; Female; Inosine; Mice; Ribonucleosides; Schistosomiasis; Thioinosine; Thionucleotides; Tubercidin

The coadministration of tubercidin and ethidium to mice infected with Trypanosoma gambiense gave a better parasite clearance than either of the single drugs. The combination was also more toxic to the mice but the inclusion of nitrobenzylthioinosinate in the therapy significantly alleviated the toxicity of the drug combination. Nitrobenzylthioinosinate per se had no trypanocidal activity and did not affect the trypanocidal action of the drugs. The biochemical basis for the nitrobenzylthioinosinate action appears to be due to the reduction of access of the drugs to tissues or organs sensitive to the toxic drugs. The potential for the use of this compound with nucleoside analogue compounds in the therapy of African trypanosomiasis is suggested.

Topics: Animals; Drug Synergism; Drug Therapy, Combination; Ethidium; Inosine; Male; Mice; Ribonucleosides; Thioinosine; Thionucleotides; Trypanosoma brucei gambiense; Trypanosomiasis, African; Tubercidin

The i.v. administration of tubercidin, an analog of adenosine, in a single dose of 45 mg/kg caused death in about 90% of B10D2F1 mice so treated. Serum and urine analysis, as well as histological examination of tissues, related the lethality of tubercidin to hepatic injury, which was markedly reduced when mice were treated with the inhibitor of nucleoside transport, nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), at i.p. doses higher than 10 mg/kg 30 min prior to tubercidin injection. With high NBMPR-P doses (100 mg/kg, i.p.) followed by tubercidin injection (45 mg/kg, i.v.), kidney damage and high mortality occurred. The tissue distribution of 3H following (( G-3H]tubercidin administration paralleled hepatic or renal injury: NBMPR-P treatment decreased the content of tubercidin-derived 3H in liver and increased that in kidney. Furthermore, the half-life of the decline in tubercidin levels in serum during the first minute after[3H]tubercidin administration was longer in NBMPR-P-treated mice (26 sec) than in untreated mice (10 sec), with the result that 3H levels in serum were more than ten times higher in the former than in the latter at an early stage during the distribution of tubercidin. Within 15 min after i.p. administration, the tissue distribution of (( 3H]tubercidin was complete. The i.p. administration of tubercidin caused ascites and the appearance of amylase in the peritoneal fluid evidently because of peritonitis and pancreatic injury. Administration of NBMPR-P by the i.p. route, but not by the i.v. route, prevented these injuries and shifted the LD50 of i.p. injected tubercidin (5 mg/kg) to markedly higher values (a 4-fold increase with NBMPR-P at 100 mg/kg). The protection of mice by NBMPR-P against lethal injuries caused by i.p. injected tubercidin was consistent with the inhibition by NBMPR-P of tubercidin accumulation in mesentery and pancreas. The tissue specificity of the NBMPR-P influence on the tissue distribution of tubercidin may reflect differences in NBMPR-P pharmacokinetics and/or in properties of the nucleoside permeation mechanism among various tissues.

Topics: Animals; Female; Inosine; Kidney; Kinetics; Liver; Mice; Ribonucleosides; Thioinosine; Thionucleotides; Tissue Distribution; Tubercidin

Earlier reports from this laboratory showed that: (a) in the presence of nitrobenzylthioinosine (NBMPR), a potent, tightly bound inhibitor of nucleoside transport, cells proliferating in culture were protected against a number of cytotoxic nucleosides; and (b) mice were protected against potentially lethal dosages of nebularine (and other toxic nucleosides) by coadministration of NBMPR. The present study, which used nitrobenzylthioinosine 5'-phosphate (NBMPR-P), a readily soluble "prodrug" form of NBMPR, extended the in vivo protection studies and showed that the half-life of the protection effect was about 4 hr. In chemotherapy experiments, mice bearing transplanted neoplasms were treated with high dosages of nebularine together with protecting doses of NBMPR-P. When mice bearing leukemia L1010 were treated with a potentially lethal regimen of nebularine administered together with NBMPR-P, a substantial kill of leukemic cells resulted (some mice were long-term survivors). The therapeutic effect was optimal at dosage levels of the protecting agent in excess of those required in nonleukemic mice for protection against the lethal nebularine dosages used, suggesting that the therapeutic effect was due to the joint presence in the leukemic cells of a metabolite of NBMPR-P and nebularine; NBMPR-P protection of the leukemic host against nebularine lethality was necessary for the therapeutic effect to be manifested.

Topics: Animals; Biological Transport, Active; Drug Therapy, Combination; Inosine; Leukemia L1210; Mice; Purine Nucleosides; Ribonucleosides; Thioinosine; Thionucleotides