[Efficiency of etoxidol in treating cardiovascular disorders caused by experimental cerebral ischemia].

A complex pharmacological study of the new cytoprotector drug etoksidol in animals with the disturbances of cerebral blood circulation showed that the intravenous introduction of the drug restores autonomous nomotopic driver of rhythm, conductivity in the atria and atrioventricular connection, and refractoriness of the atrioventricular connection, which were violated a result of sharp cerebral ischemia. The new drug does not suppress the inotropic function of the heart in cats and limits the dimensions of the zone of necrosis in rats with the myocardial infarction on the background of deficiency of the cerebral blood flow.

Inosine reduces ischemic brain injury in rats.

BACKGROUND AND PURPOSE: Purinergic nucleoside inosine elicits protection and regeneration during various injuries. The purpose of this study was to examine the protective effects of inosine against cerebral ischemia. METHODS: Adult Sprague-Dawley rats were anesthetized. Inosine, hypoxathine, or vehicle was administered intracerebroventricularly before transient right middle cerebral artery occlusion (MCAo). Animals were placed in behavioral chambers 2 days to 2 weeks after MCAo and then euthanized for tri-phenyl-tetrazolium chloride staining. Glutamate release was measured by microdialysis/high-performance liquid chromatography, and single-unit action potentials were recorded from neurons in the parietal cortex. RESULTS: Stroke animals receiving inosine pretreatment demonstrated a higher level of locomotor activity and less cerebral infarction. Intracerebroventricular administration of the same dose of hypoxanthine did not confer protection. Coadministration of selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) significantly reduced inosine-mediated protection. Inosine did not alter basal glutamate release, nor did it reduce ischemia-evoked glutamate overflow from cerebral cortex. However, inosine antagonized glutamate-induced electrophysiological excitation in cerebral cortical neurons. CONCLUSIONS: Inosine inhibits glutamate postsynaptic responses and reduces cerebral infarction. Its protective effect against ischemia/reperfusion-related insults may involve activation of adenosine A3 receptors.

AIT-082 and methylprednisolone singly, but not in combination, enhance functional and histological improvement after acute spinal cord injury in rats.

Extracellular non-adenine based purines are neuroprotective. Preliminary studies indicate that administration of the synthetic purine 4-[[3-(1,6 dihydro-6-oxo-9-purine-9-yl)-1-oxypropyl] amino] benzoic acid (AIT-082, leteprinim potassium) to rats immediately after acute spinal cord injury (SCI), improves functional outcome. The effects of potential new agents are often compared to methylprednisolone (MPSS). We evaluated the effects of AIT-082 and MPSS, separately and in combination, on the functional and morphological outcome of acute SCI in adult rats. After standardized T11-12 spinal cord compression rats were given intraperitoneally one of the following: vehicle (saline); MPSS (30 mg/kg or 60 mg/kg body weight, first dose 15 min after crush); AIT-082 (60 mg/kg body weight daily, first dose 15 min after crush); or AIT-082 plus MPSS. After 1, 3, or 21 days, the rats were perfused for histological analysis. AIT-082 administrations significantly reduced locomotor impairment from 121 days post-operatively. At 1 and 3 days post injury, AIT-082-treatment reduced tissue swelling, tissue loss and astrogliosis at the injured cords but did not alter the extent of hemorrhage and the number of macrophages and/or microglia. MPSS reduced hemorrhage and the number of macrophages and/or microglia, but did not alter astrogliosis. At 21 days, either AIT-082 or MPSS administration improved function and morphology similarly (less tissue loss and astrogliosis). In contrast, administration of AIT-082 and MPSS together abolished the beneficial effects observed when either drug was given individually. These results suggest that MPSS and AIT-082 may exert their beneficial effects through different and potentially antagonistic pathways.

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors.

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.

DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine.

Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death.

Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors.

The culture conditions under which hypoxanthine maintains a two-cell block in preimplantation mouse embryos were assessed. Hypoxanthine prevented embryo development past the two-cell stage at concentrations as low as 30 nM, and this inhibitory activity required the presence of D-glucose. The action of hypoxanthine plus D-glucose was reversed by glutamine and higher lactate. D-mannose substituted for D-glucose in supporting the inhibitory action of hypoxanthine, but L-glucose, D-fructose, and 2-deoxyglucose were much less effective. Other purine derivatives such as inosine and adenosine, but not xanthosine or uric acid, also blocked development at the two-cell stage at a concentration of 30 microM, and guanosine was inhibitory at higher doses. Assays of hypoxanthine phosphoribosyltransferase (HPRT) activity in lysates of four-cell embryos determined that the drugs 6-mercapto-9-(tetrahydro-2-furyl)-purine (MPTF) and 6-mercaptopurine (6-MP), but not 6-azauridine (6-AzaU), prevented salvage of hypoxanthine. In addition, MPTF and 6-MP produced a significant two-cell block, which did not depend upon the presence of hypoxanthine or D-glucose; whereas 6-AzaU was without effect. When embryos were cultured 2 days in the presence or absence of D-glucose, hypoxanthine salvage was significantly reduced in lysates of four-cell embryos exposed to D-glucose. D-glucose had no effect when added directly to the assay mixture. These data demonstrate that the ability of hypoxanthine to block embryo development at the two-cell stage depends on the presence of D-glucose or other glycolyzable sugars and suggest that inhibition of the purine salvage pathway promotes the two-cell block.

Oxygen free radical mediated renal dysfunction.

Postischemic renal dysfunction (PIRD) is characterized by a reduction in glomerular filtration and tubular reabsorption of solute. The relative contribution of oxygen free radicals (OFRs) generated during reperfusion remains unclear. This study characterized the renal response to OFRs--independent of an ischemic insult. Isolated rat kidneys were perfused at 37 degrees C and 90-100 mm Hg with a modified Krebs' buffer. Hypoxanthine (25 mumole) and xanthine oxidase (1 unit) were combined and infused proximal to the kidney. There was a 50% increase in vascular resistance. This was accompanied by a 30% reduction in perfusate flow rate and a 70% reduction in glomerular filtration rate. There was also a significant reduction in urine flow rate and oxygen consumption. The percentage reabsorption of filtered water and sodium by the renal tubules was not diminished, however. This pattern was not observed when the xanthine oxidase was inactivated or when the perfusate was pretreated with superoxide dismutase (250 units/ml) and catalase (500 units/ml). The generation of OFRs, independent of an ischemic insult, causes a decrease in glomerular filtration out of proportion to the decrease in renal flow similar to that observed with PIRD. OFRs may contribute to the hemodynamic and glomerular alterations seen with PIRD. Factors other than OFRs, probably associated with ischemia, must be responsible for the tubular dysfunction.

Reduced urinary serotonin excretion after intake of high doses of hypoxanthine.

Two healthy volunteers were treated with hypoxanthine 3 x 1 g and allopurinol 3 x 100 mg daily for 1 week. During this treatment serum oxypurine concentration and urinary oxypurine excretion increased as expected. No side effects were observed except for some mild daytime drowsiness and lethargy. Measurements of urinary serotonin (5-HT) excretion showed decreases to as much as 60% below initial values. Decreased urinary 5-HT excretion was also found in a patient with incomplete Lesch-Nyhan syndrome during treatment with high doses of hypoxanthine. His neurological symptoms improved slightly. The results suggest that high doses of hypoxanthine exert a nonspecific sedative effect on both patients with Lesch-Nyhan syndrome and healthy controls. The cause is probably a reduced synthesis or release of 5-HT.

[Inhibitory effect of hypoxanthine on monoamine oxidase activity].

Hypoxanthine was demonstrated to have a dose-dependent inhibitory effect on type B monoamine oxidase (MAO-B) activity in liver and brain tissues, and slight inhibitory effect on type A MAO(MAO-A) activity when given orally to mice at doses of 25-500 mg/kg. When mice were given orally hypoxanthine 500 mg/kg, MAO-A and -B activities were all inhibited significantly 16 hours after administration, but the inhibitory action on MAO-A was weaker. Subcutaneous injection of the agent also produced obvious inhibition of MAO activity in the liver but no significant influence on MAO activity in brain tissue was observed. In vitro experiment showed that the action of hypoxanthine on MAO-B was competitive inhibition and that on MAO-A was competitive mixed with noncompetitive inhibition.

Free oxygen radicals decrease electrical resistance of microvascular endothelium in brain.

The effect of free oxygen radicals on the electrical resistance of brain venular endothelium was studied in anesthetized frogs. The technique allowed continuous recording of the electrical resistance of the vascular wall reflecting its ionic permeability. The oxygen radicals were generated by an enzymatic reaction between xanthine oxidase and hypoxanthine supplied to the surface of the exposed brain. Electrical resistance of the venular endothelium decreased within 1-2 s after the reaction was initiated. Hypoxanthine (1 mM) and xanthine oxidase at a concentration of 10, 25, 50, 100, and 250 mU ml-1 lowered resistance to 1.0, 0.9, 0.8, 0.5 and 0.2 X control value, respectively, within a 3 min period of administration. The effect induced by 25 and 50 mU ml-1 of xanthine oxidase was readily reversible, whereas that induced by the two highest concentrations was irreversible within the observation time. The response was totally blocked by allopurinol as well as by superoxide dismutase plus catalase. Pretreatment with methylprednisolone or BW755C (an inhibitor of cyclo- and lipoxygenase) did not inhibit the response, nor did removal of calcium or magnesium from the extracellular medium. Free oxygen radicals are powerful agents that rapidly induce dynamic changes in the electrical resistance of brain vessels, supporting the notion that they may be important mediators of vascular endothelial damage in the brain.

Changes in inspiratory activity after injection of adenosine and hypoxanthine in cats.

The effects of intra-arterial and intravenous injections of adenosine and hypoxanthine were investigated with special reference to respiratory variables in anesthetized young cats. Studies were made of the effects on inspiratory activity (phrenic nerve activity), heart rate, blood pressure and central venous pressure. To assess the risk of accumulation of adenosine degeneration products after several injections measurements were also made of hypoxanthine, xanthine and urate in plasma at intervals after the injections. It was found that intra-arterial and intravenous injections of adenosine increased central inspiratory activity during the first few breaths after the injection. The blood pressure and heart rate decreased slightly and central venous pressure increased slightly after the injection. Degradation of adenosine and its metabolites takes place rapidly and it is therefore unlikely that metabolites influence the results. It is concluded that adenosine causes brief stimulation of inspiratory activity.

In vivo efficacy of HAT therapy against transplantable murine myelomas resistant to purine analogs.

The therapeutic efficacy of antineoplastic purine analogs can be jeopardized by the emergence of drug-resistant mutant subpopulations of tumor cells. To determine whether such mutant populations might be eradicable in vivo with the type of HAT combination (hypoxanthine + an antifolate + thymidine) known to be selectively cytotoxic to them in vitro, 2 thioguanine-resistant BALB/c murine myeloma lines were transplanted into BALB/c mice to produce tumors capable of progressive growth in the absence of therapy. Treatment of these mice with a modified HAT regimen induced permanent tumor regressions in 37/44 mice; the same treatment was ineffective against tumors produced by a non-mutant myeloma line from which one of the mutant sublines had been derived.

The fate of administered purines in the Dalmatian coach hound.

The fate of radioactively labelled purines was examined in the Dalmatian Coach Hound after oral and intravenous administration. The incomplete intestinal absorption of urate, delayed conversion of urate to allantoin and the increased renal output and clearance of urate found in the Dalmatian Coach Hound when compared with the non-Dalmatian were consistent with a generalized defect in the transport of urate across cell membranes.

Highly selective drug combinations for human colon cancer cells resistant in vitro to 5-fluoro-2'-deoxyuridine.

In the preceding companion paper, we describe a human colon carcinoma cell line that is resistant in vitro to 5-fluoro-2'-deoxyuridine by virtue of impaired nucleoside transport. Two drug combinations, methotrexate: hypoxanthine: thymidine (dThd) and 5,8-dideazaisofolic acid: dThd, selectively kill these resistant cells with no effect on the sensitive cell population. As little as 0.3 microM dThd was sufficient to completely protect sensitive cells from 5-fluoro-2'-deoxyuridine, and 5,8-dideazaisofolic acid at concentrations that produced over 80% lethality in unprotected cells and the same concentration of dThd in combination with 100 microM hypoxanthine fully protected sensitive cells from greater than 99% methotrexate-induced cell lethality. In contrast, when resistant cells were exposed to these drugs, they were not protected by dThd, or by the combination dThd: hypoxanthine, in concentrations up to 300 times higher than those necessary to prevent sensitive cell kill. Thus, it may be possible to protect normal renewal tissues while obtaining selective tumor cell kill with these two drug combinations in patients whose colon carcinoma cells are resistant to 5-fluoro-2'-deoxyuridine by virtue of defective transport.

Combinations of 5-FU, hypoxanthine and allopurinol in chemotherapy for human colon adenocarcinoma xenografts.

A series of four human colon adenocarcinomas, growing as xenografts in immune-deprived mice, have been used to evaluate the efficacy of 5-FU in combination with two purines, hypoxanthine (Hx) and allopurinol (HPP), which have reduced the toxicity of 5-FU in host mice. Tumor-bearing mice were treated at 7-day intervals with 5-FU administered simultaneously with the protecting agents (Hx and HPP). Two tumor lines (HxVRC5 and HxGC3), insensitive to 5-FU alone, failed to show any response to this combination. In 5-FU-sensitive HxELC2 tumors, the combination of 5-FU with Hx and HPP did not increase the therapeutic index, and in HxHC1 xenografts, antagonism to 5-FU cytotoxicity was observed. Tumor response in relation to the pathways of 5-FU metabolism is discussed.

Autoradiographic studies on the effect of allopurinol on 14C-hypoxanthine metabolism in the squirrel monkey.

The Effect of orally given allopurinol in the distribution of intravenously administered 14C-hypoxanthine radioactivity was studied in squirrel monkeys 8 hr after administration of the label by the whole body autoradiography. Although the distribution of radioactivity in the normal and allopurinol-treated animals was essentially similar to each other, more intense radioactivity was noted in the latter monkey; salvage of 14C-hypoxanthine was enhanced. Similarly to our previous observation in mice, significant radioactivity in monkeys was seen in tissues undergoing rapid nucleic acid synthesis except for slight species differences in some organs. 14C-Allantoin alone as the urinary metabolite of the hypoxanthine in the normal monkey whereas significant amounts of 14C-hypoxanthine and 14C-xanthine as well were detected in the urine of the drug-treated animal.

Thymidine and hypoxanthine requirements for the proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts in the presence of methotrexate.

Cultured normal and Rous sarcoma virus-infected chicken fibroblasts do not differ in the concentrations of thymidine or of hypoxanthine that they require to proliferate in the presence of a methotrexate block. For maximal proliferation, thymidine is required at 10(-6) M, while hypoxanthine is required at 10(-5) M. The normal and Rous-infected fibroblasts show very similar, if not identical, decreases in proliferation rates at suboptimal concentrations of thymidine or hypoxanthine. These results suggest that conversion of fibroblasts to the neoplastic state does not alter their capacity to salvage thymidine or purines from the extracellular fluid or to metabolize these compounds.