thioinosine and mioflazine

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [3H]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a 'mixed' type inhibitor of [3H]nitrobenzylthioinosine binding (increased KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax of 44 pmol/mg protein, which is comparable to the number of [3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [3H]R75231 and actually caused an initial increase in the amount of site-bound [3H]R75231. Adenosine, on the other hand, enhanced the rate of [3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.

Topics: Adenosine; Animals; Binding, Competitive; Carrier Proteins; Cerebral Cortex; Erythrocyte Membrane; Half-Life; Humans; In Vitro Techniques; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Piperazines; Rabbits; Synaptosomes; Thioinosine; Uridine

The effects of coformycin, alpha,beta-methylene ADP, dipyridamole in the absence and presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), nitrobenzylthioinosine (NBTI), mioflazine and ouabain on the metabolic pathways of exogenously applied ATP and its metabolites in the frog innervated sartorius muscle were investigated. ATP catabolism yielded ADP, AMP, IMP, adenosine and inosine; the ecto-ATPase in situ was shown to be Ca(2+)- or Mg(2+)-activated with a Kmapp for ATP of 767 +/- 48 microM. AMP catabolism yielded IMP, adenosine and inosine; inosine was formed from either exogenous IMP or exogenous adenosine. Catabolism of AMP into IMP was blocked by coformycin, which enhanced adenosine and inosine formation from AMP. alpha,beta-Methylene ADP blocked adenosine formation from AMP and inosine formation from IMP; formation of IMP from AMP was enhanced by alpha,beta-methylene ADP. Complete blockade of AMP degradation was achieved with the simultaneous use of coformycin and alpha,beta-methylene ADP. Dipyridamole attenuated but did not completely block extracellular adenosine removal and inosine appearance in the bath. EHNA, applied in the presence of dipyridamole, did not cause any further attenuation of extracellular adenosine removal. Mioflazine, NBTI and ouabain did not affect adenosine disappearance from the bath. The results suggest that, in the frog innervated sartorius muscle, ATP can be sequentially catabolized into AMP which is then catabolized either into IMP or into adenosine. This extracellular degradation of AMP into IMP might then constitute a shunt-like mechanism to control the levels of adenosine formed from adenine nucleotides.

Topics: Adenine; Adenine Nucleotides; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cations; Coformycin; Dipyridamole; In Vitro Techniques; Inosine Monophosphate; Kinetics; Muscles; Piperazines; Rana ridibunda; Sodium-Potassium-Exchanging ATPase; Thioinosine

Topics: Animals; Biological Transport; Dilazep; Dipyridamole; Epinephrine; Erythrocytes; Humans; In Vitro Techniques; Nucleosides; Phosphates; Piperazines; Rabbits; Thioinosine

The in vivo nucleoside transport inhibitory effects of 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), used as its 5'-monophosphate derivative (NBMPR-P), dilazep, mioflazine and its derivatives soluflazine, R57974 and R75231, were investigated in BALB/c mice. The extent and duration of action were followed by assaying adenosine transport in blood cells sampled at time intervals following i.p. administration (ca. 20 mg/kg). Dilazep and R57974 were found to be short-acting inhibitors, while NBMPR-P and R75231 were similar in their action and caused essentially full inhibition of adenosine transport over a 4- to 5-h period. Mioflazine and soluflazine were rather ineffective, causing only partial inhibition. R75231 was also active after oral administration which, when repeated three times in 4-h intervals, resulted in essentially full transport inhibition up to 20 h following the initial dose. Effects of NBMPR-P, R57974 and dilazep on adenosine transport in blood cells were also measured in blood cells of hamsters after i.p. administration of the same doses. All three drugs caused full transport inhibition, but the action of dilazep and R75231 showed reversal within about 30 min and 2 h, respectively, while NBMPR-P caused full inhibition for at least 3-4 h. These results demonstrate the potential of the mioflazine derivative R75231 to be useful in vivo, possibly even after p.o. administration, for host protection against the actions of cytotoxic nucleosides used in experimental antiparasitic therapy or other studies requiring suppression of nucleoside transport.

Topics: Adenosine; Administration, Oral; Animals; Biological Transport; Cardiovascular Agents; Cricetinae; Dilazep; Injections, Intraperitoneal; Mesocricetus; Mice; Mice, Inbred BALB C; Piperazines; Species Specificity; Thioinosine

The potency of mioflazine and related drugs (Janssen Pharmaceutica, Belgium) as inhibitors of adenosine transport in isolated erythrocytes from several species were measured and compared with those of dilazep and 6-(4-nitrobenzylmercapto)purine ribonucleoside (NBMPR). [8-3H]Adenosine was used as the permeant at 1 microM and incubation times were 10 s, and assays were conducted in the presence and absence of varying doses of potential transport inhibitors. The species investigated included mouse, hamster, rabbit, baboon and man. Dilazep was the most potent compound throughout with an IC50 of about 2 nM. In the mouse and hamster mioflazine and its derivatives were considerably less potent (IC50 values greater than 200 nM) with the exception of R57974 with IC50 values of about 150 and 60 nM in mouse and hamster, respectively. In the man and baboon the derivatives had IC50 values in the same order of magnitude as NBMPR (less than 100 nM), and in the rabbit they had potencies close to that of NBMPR, ranging between 10-60 nM. Nucleoside transport inhibitors are of potential importance as host protectors during treatment of parasitic infections with cytotoxic nucleosides. Present data indicate that mioflazine and its derivatives are not very potent in some of the preferred animal models for parasitic infections (mouse, hamster) but are more effective in primates such as man and baboon.

Topics: Adenosine; Animals; Cricetinae; Dilazep; Dimethyl Sulfoxide; Erythrocytes; Humans; In Vitro Techniques; Male; Mesocricetus; Mice; Mice, Inbred BALB C; Papio; Piperazines; Rabbits; Species Specificity; Thioinosine

The sensitivity of facilitated-diffusion and Na(+)-dependent nucleoside transporters to inhibition by a series of novel compounds related to lidoflazine and mioflazine was investigated. Uridine transport by rabbit erythrocytes, which proceeds solely by the nitrobenzylthioinosine (NBMPR)-sensitive facilitated-diffusion system, was inhibited with apparent Ki values of less than 10 nM by lidoflazine, mioflazine, soluflazine and R73-335. These compounds also blocked site-specific [3H]NBMPR binding to rabbit erthrocyte membranes in a competitive fashion. The NBMPR-sensitive system in rat erythrocytes was also inhibited by lidoflazine, mioflazine, soluflazine and R73-335 but was two to three orders of magnitude less sensitive to inhibition than the system in rabbit erythrocytes (apparent Ki 7.3, 2.4, 5.7 and 0.1 microM, respectively). Lidoflazine, mioflazine and R73-335 exhibited a similar potency for the NBMPR-sensitive and -insensitive nucleoside transporters in rat erythrocytes. In contrast, soluflazine was 20- to 100-fold more potent as an inhibitor of the NBMPR-insensitive nucleoside transport component in rat erythrocytes (IC50 of 0.08-0.2 microM) compared to the NBMPR-sensitive nucleoside carrier in these cells (IC50 approximately 10 microM). None of the test compounds were potent inhibits of Na(+)-dependent uridine transport in bovine renal brush-border membrane vesicles. These results indicate that lidoflazine, mioflazine, soluflazine and R73-335 are selective inhibitors of nucleoside transport in animal cells and that the potency of these compounds as nucleoside transport inhibitors is species dependent.

Topics: Affinity Labels; Animals; Biological Transport; Cardiovascular Agents; Cattle; Cells, Cultured; Erythrocytes; Humans; Kidney; Lidoflazine; Nucleosides; Piperazines; Placenta; Rabbits; Rats; Sodium; Thioinosine; Uridine

The potency of nucleoside transport inhibitors, including 6-[(4-nitrobenzyl)-mercapto]purine ribonucleoside (NBMPR), dilazep, mioflazine and its derivatives soluflazine and R57974 as inhibitors of the binding of [3H(G)]NBMPR to intact erythrocytes and respective ghost membranes from human, mouse and hamster was determined. There was no close agreement between the IC50 profiles for the different inhibitors when comparing values obtained for intact cells and membranes from each species, and there was no consistent profile of differences when considering individual drugs and comparing their actions in the three species. Present data also were compared with potency values obtained previously with the same drugs directly in nucleoside transport inhibition studies with erythrocytes from the same species as well as with [3H(G)]NBMPR binding studies in isolated liver and lung membranes from hamster. The overall conclusion from this and previous studies is that the evaluation of relative potencies in screening of potential nucleoside transport inhibitors is best carried out at the level actual nucleoside transport studies in intact cells, since [3H(G)]NBMPR binding studies yield discrepant data.

Topics: Adenosine; Animals; Binding, Competitive; Carrier Proteins; Cricetinae; Dilazep; Erythrocyte Membrane; Erythrocytes; Hot Temperature; Humans; Hydrogen-Ion Concentration; Inosine; Membrane Proteins; Mice; Nucleoside Transport Proteins; Piperazines; Rats; Thioinosine

Adenosine transport by rat and guinea pig synaptosomes was studied to establish the basis for the marked differences in the potency of some transport inhibitors in these species. An analysis of transport kinetics in the presence and absence of nitrobenzylthioinosine (NBTI) using synaptosomes derived from several areas of rat and guinea pig brain indicated that at least three systems contributed to adenosine uptake, the Km values of which were approximately 0.4, 3, and 15 microM in both species. In both species, the system with the Km of 3 microM was potently (IC50 of approximately 0.3 nM) and selectively inhibited by NBTI. This NBTI-sensitive system accounted for a greater proportion of the total uptake in the guinea pig than in the rat and was inhibited by dipyridamole, mioflazine, and related compounds more potently in the guinea pig. Preliminary experiments with other species indicate that adenosine transport in the mouse is similar to that in the rat, whereas in the dog and rabbit, it is more like that in the guinea pig. In the rat, none of the systems appeared to require Na+, but the two systems possessing the higher affinities for adenosine were inhibited by veratridine- and K(+)-induced depolarization. The transport systems were active over a broad pH range, with maximal activity between pH 6.5 and 7.0. Our results are consistent with the possibility that adenosine transport systems may be differentiated into uptake and release systems.

Topics: Adenosine; Animals; Biological Transport; Brain; Dipyridamole; Dogs; Female; Guinea Pigs; Hydrogen-Ion Concentration; Kinetics; Male; Membrane Potentials; Mice; Piperazines; Potassium; Rabbits; Rats; Rats, Inbred Strains; Sodium Chloride; Species Specificity; Synaptosomes; Thioinosine; Tissue Distribution

A new series of compounds related to the nucleoside transport inhibitors, lidoflazine and mioflazine, is introduced. The influence of these derivatives on nucleoside-specific transport proteins was studied in two ways. First, a rapid, non-radioactive assay was developed for the screening of this type of material for actual transport inhibition in human erythrocytes. The method is based on the dose-dependent reversal of the inhibition of inorganic phosphate release induced by inosine when human erythrocytes are suspended in a phosphate-free medium. It enables the estimation of the potency and specificity of this new series of nucleoside transport inhibitors, most of which are highly active (EC50 values as low as 13 nM). Second, the displacement of a radiolabeled transport inhibitor, [3H]nitrobenzylthioinosine, was examined. All compounds were capable of displacing specific [3H]nitrobenzylthioinosine binding to crude and solubilized plasma membranes of calf lung tissue, displaying affinities in the nanomolar range. Pseudo-Hill coefficients derived from the shape of the displacement curves were significantly greater than unity for most derivatives, in contrast to values of approximately unity obtained for dipyridamole and analogs. These findings were incorporated in a mathematical model describing the interaction of mioflazine analogs with the transport protein, suggesting that one molecule of mioflazine is capable of displacing two or more molecules of [3H]nitrobenzylthioinosine at a time. The consequences of this model regarding the nature of the transport protein are discussed.

Topics: Animals; Binding, Competitive; Cattle; Dipyridamole; Erythrocytes; In Vitro Techniques; Lidoflazine; Lung; Membranes; Nucleosides; Phosphates; Piperazines; Thioinosine

The binding of [G-3H]-6-(4-nitrobenzylmercapto)purine ribonucleoside [( G-3H]NBMPR) was investigated using a centrifugation assay with membrane preparations from hamster tissues including liver, lung, kidney and heart. Only liver and lung membranes showed high specific binding, with dissociation constants (Kd) values of 2.4 +/- 0.4 and 0.44 +/- 0.05 nM, and maximal binding (Bmax) of 3.7 +/- 0.4 and 1.04 +/- 0.01 pmol/mg, respectively. The binding of [G-3H]NBMPR was inhibited in a concentration dependent manner by unlabelled NBMPR, dilazep and a new group of chemically related nucleoside transport inhibitors, mioflazine, soluflazine and R57974, the latter being the most potent derivative. R57974 displaced bound [G-3H]NBMPR as effectively as unlabelled NBMPR suggesting a common binding site. The assay procedure used appears useful for the rapid screening of the effectiveness of nucleoside transport inhibitors which will be of value for the selection of inhibitors suitable for combination with cytotoxic nucleosides in the treatment of selected cancers or parasitic diseases.

Topics: Animals; Binding Sites; Cardiovascular Agents; Cell Membrane; Cricetinae; Dilazep; In Vitro Techniques; Inosine; Liver; Lung; Male; Mesocricetus; Piperazines; Thioinosine