thioinosine and Disease-Models--Animal

Neuroinflammation and apoptosis are two key factors contributing to early brain injury (EBI) after subarachnoid hemorrhage (SAH) and are strongly associated with a poor prognosis. Recently, equilibrative nucleoside transporter 1 (ENT1) was emerged to accelerate the severity of inflammation and cell apoptosis in several nervous system diseases, including cerebral ischemia, neurodegeneration and epilepsy. However, no study has yet elaborated the expression levels and effects of ENT1 in EBI after SAH.. Sprague-Dawley rats were subjected to SAH by endovascular perforation. Nitrobenzylthioinosine (NBTI) was intranasally administered at 0.5 h after SAH. The protein expression levels of ENT1, NLRP3, Bcl2, Bax, ACS, Caspase-1, IL-1 were detected by western blot. The modified Garcia score and beam balance score were employed to evaluate the neurologic function of rats following SAH. In addition, hematoxylin-eosin, fluoro-jade C and TdT-mediated dUTP nick-end labeling staining were then used to evaluate brain tissue damage and neuronal apoptosis.. Analysis indicated that endogenous levels of ENT1 were significantly upregulated at 24-hour post-SAH, accompanied by NLRP3 inflammasome activation and Bcl2 decline. The administration of NBTI, an inhibitor of ENT1, at a dose of 15 mg/kg, ameliorated neurologic deficits and morphologic lesions at both 24 and 72 h after SAH. Moreover, ENT1 inhibition efficiently mitigated neuronal degeneration and cell apoptosis. In addition, NBTI at 15 mg/kg observably increased Bcl2 content and decreased Bax level. Furthermore, suppression of ENT1 notably reduced the expression levels of NLRP3, apoptosis associated speck like protein containing CARD, caspase-1 and IL-1β.. NBTI relieved SAH-induced EBI partly through ENT1/NLRP3/Bcl2 pathway.

Topics: Animals; Apoptosis; Disease Models, Animal; Equilibrative Nucleoside Transporter 1; Inflammasomes; Neuroinflammatory Diseases; NLR Family, Pyrin Domain-Containing 3 Protein; Proto-Oncogene Proteins c-bcl-2; Rats; Subarachnoid Hemorrhage; Thioinosine

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn't change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50µM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.

Topics: Adenosine; Adenosine A1 Receptor Antagonists; Animals; Disease Models, Animal; Epilepsy; Gene Expression; Hippocampus; Male; Patch-Clamp Techniques; Protein Transport; Pyramidal Cells; Rats; Receptor, Adenosine A1; Thioinosine

Type 1 equilibrative nucleoside transporter (ENT1) promotes glutamate release by inhibition of adenosine signaling. However, whether ENT1 plays a role in epileptic seizure that involves elevated glutamatergic neurotransmission is unknown. Here, we report that both seizure rats and patients show increased expression of ENT1. Intrahippocampal injection of a specific inhibitor of ENT1, nitrobenzylthioinosine (NBTI), attenuates seizure severity and prolongs onset latency. In order to examine whether NBTI would be effective as antiepileptic after peripheral application, we injected NBTI intraperitoneally, and the results were similar to those obtained after intrahippocampal injection. NBTI administration leads to suppressed neuronal firing in seizure rats. In addition, increased mEPSC in seizure are inhibited by NBTI. Finally, NBTI results in deactivation of phosphorylated cAMP-response element-binding protein in the seizure rats. These results indicate that ENT1 plays an important role in the development of seizure. Inhibition of ENT1 might provide a novel therapeutic approach toward the control of epileptic seizure.

Topics: Action Potentials; Adenosine; Adolescent; Adult; Animals; Anterior Temporal Lobectomy; Anticonvulsants; CA1 Region, Hippocampal; Carrier Proteins; Child, Preschool; Convulsants; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Drug Resistance; Epilepsy, Temporal Lobe; Equilibrative Nucleoside Transporter 1; Glutamates; Humans; Male; Middle Aged; Nerve Tissue Proteins; Phosphorylation; Pilocarpine; Protein Processing, Post-Translational; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Seizures; Thioinosine; Young Adult

Simultaneous inhibition of the cardiac equilibrative-p-nitrobenzylthioinosine (NBMPR)-sensitive (es) type of the equilibrative nucleoside transport 1 (ENT1) nucleoside transporter, with NBMPR, and adenosine deaminase, with erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), prevents release of myocardial purines and attenuates myocardial stunning and fibrillation in canine models of warm ischemia and reperfusion. It is not known whether prolonged administration of hypothermic cardioplegia influences purine release and EHNA/NBMPR-mediated cardioprotection in acutely ischemic hearts.. Anesthetized dogs (n = 46), which underwent normothermic aortic crossclamping for 20 minutes on-pump, were divided to determine (1) purine release with induction of intermittent antegrade or continuous retrograde hypothermic cardioplegia and reperfusion, (2) the effects of postischemic treatment with 100 μM EHNA and 25 μM NBMPR on purine release and global functional recovery, and (3) whether a hot shot and reperfusion with EHNA/NBMPR inhibits purine release and attenuates ventricular dysfunction of ischemic hearts. Myocardial biopsies and coronary sinus effluents were obtained and analyzed using high-performance liquid chromatography.. Warm ischemia depleted myocardial adenosine triphosphate and elevated purines (ie, inosine > adenosine) as markers of ischemia. Induction of intermittent antegrade or continuous retrograde hypothermic (4°C) cardioplegia releases purines until the heart becomes cold (<20°C). During reperfusion, the levels of hypoxanthine and xanthine (free radical substrates) were >90% of purines in coronary sinus effluent. Reperfusion with EHNA/NBMPR abolished ventricular dysfunction in acutely ischemic hearts with and without a hot shot and hypothermic cardioplegic arrest.. Induction of hypothermic cardioplegia releases purines from ischemic hearts until they become cold, whereas reperfusion induces massive purine release and myocardial stunning. Inhibition of cardiac es-ENT1 nucleoside transporter abolishes postischemic reperfusion injury in warm and cold cardiac surgery.

Topics: Adenine; Adenosine Triphosphate; Animals; Cold Ischemia; Disease Models, Animal; Dogs; Equilibrative Nucleoside Transporter 1; Female; Heart Arrest, Induced; Hypothermia, Induced; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardial Stunning; Myocardium; Recovery of Function; Thioinosine; Time Factors; Ventricular Function, Left; Warm Ischemia

The inhibition of adenosine deaminase with erythro-9 (2-hydroxy-3-nonyl)-adenine (EHNA) and the es-ENT1 transporter with p-nitro-benzylthioinosine (NBMPR), entraps myocardial intracellular adenosine during on-pump warm aortic crossclamping, leading to a complete recovery of cardiac function and adenosine triphosphate (ATP) during reperfusion. The differential role of entrapped intracellular and circulating adenosine in EHNA/NBMPR-mediated protection is unknown. Selective (8-cyclopentyl-1,3-dipropyl-xanthine) or nonselective [8-(p-sulfophenyl)theophyline] A1 receptor antagonists were used to block adenosine A1-receptor contribution in EHNA/NBMPR-mediated cardiac recovery.. Anesthetized dogs (n = 45), instrumented to measure heart performance using sonomicrometry, were subjected to 30 minutes of warm aortic crossclamping and 60 minutes of reperfusion. Three boluses of the vehicle (series A) or 100 μM EHNA and 25 μM NBMPR (series B) were infused into the pump at baseline, before ischemia and before reperfusion. 8-Cyclopentyl-1,3-dipropyl-xanthine (10 μM) or 8-(p-sulfophenyl)theophyline (100 μM) was intra-aortically infused immediately after aortic crossclamping distal to the clamp in series A and series B. The ATP pool and nicotinamide adenine dinucleotide was determined using high-performance liquid chromatography.. Ischemia depleted ATP in all groups by 50%. The adenosine/inosine ratios were more than 10-fold greater in series B than in series A (P < .001). ATP and function recovered in the EHNA/NBMPR-treated group (P < .05 vs control group). 8-Cyclopentyl-1,3-dipropyl-xanthine and 8-(p-sulfophenyl)theophyline partially reduced cardiac function in series A and B to the same degree but did not abolish the EHNA/NBMPR-mediated protection in series B.. In addition to the cardioprotection mediated by activation of the adenosine receptors by extracellular adenosine, EHNA/NBMPR entrapment of intracellular adenosine provided a significant component of myocardial protection despite adenosine A1 receptor blockade.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Chromatography, High Pressure Liquid; Constriction; Disease Models, Animal; Dogs; Ischemic Preconditioning; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardial Stunning; Nucleoside Transport Proteins; Receptor, Adenosine A1; Theophylline; Thioinosine; Xanthines

Adenosine is an endogenous nucleoside that signals through G-protein coupled receptors. Extracellular adenosine is required for receptor activation and two pathways have been identified for formation and cellular release of adenosine. The CLASSICAL pathway relies on intracellular formation of adenosine from adenine nucleotides and cellular efflux of adenosine via equilibrative nucleoside transporters (ENTs). The ALTERNATE pathway involves cellular release of adenine nucleotides, hydrolysis via ecto-5'-nucleotidases and extracellular formation of adenosine.. A rat model of cerebral ischemia and primary cultures of rat forebrain astrocytes and neurons were used.. Using a rat model of cerebral ischemia, the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) significantly increased post-ischemic forebrain adenosine levels and significantly decreased hippocampal neuron injury relative to saline-treatment. NBMPR-induced increases in adenosine receptor activation were not detected, suggesting that altering the intracellular:extracellular distribution of adenosine can affect ischemic outcome. Using primary cultures of rat forebrain astrocytes and neurons, adenosine release was evoked by ischemic-like conditions. Dipyridamole, an inhibitor of ENTs, was more effective at inhibiting adenosine release from neurons than from astrocytes. In contrast, alpha , beta-methylene ADP, an inhibitor of ecto-5'-nucleotidase, was effective at inhibiting adenosine release from astrocytes, but not from neurons. Thus, during ischemic-like conditions, neurons released adenosine via the CLASSICAL pathway, while astrocytes released adenosine via the ALTERNATE pathway.. These cell type differences in pathways for adenosine formation during ischemia may allow transport inhibitors to block simultaneously adenosine release from neurons and adenosine uptake into astrocytes. In principle, this could improve neuronal ATP levels without decreasing adenosine receptor activation.

Topics: Adenosine; Affinity Labels; Animals; Astrocytes; Brain Ischemia; Cells, Cultured; Deoxyglucose; Dipyridamole; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Glucose; Hypoxia; Inosine; Models, Biological; Neurons; Oligomycins; Phosphodiesterase Inhibitors; Prosencephalon; Purines; Rats; Thioinosine; Tritium

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

This study assessed the cardioprotective effects of inhibitors of adenosine metabolism in an isolated perfused rat heart model. Specifically, we studied the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine and the selective nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine, in terms of their potential to enhance protection when added to Bretschneider's cardioplegic solution.. Rat hearts were infused for 5 min with Krebs-Henseleit buffer solution (group 1), Bretschneider's cardioplegic solution (group 2), Bretschneider's cardioplegic solution with the addition of 25 microM erythro-9-(2-hydroxy-3-nonyl)-adenine and 5 microM S-(p-nitrobenzyl)-6-thioinosine (group 3), and Bretschneider's cardioplegic solution with the addtion of 25 microM erythro-9-(2-hydroxy-3-nonyl)-adenine only (group 4). After cardioplegic arrest and 45 min of ischemic storage at 25 degrees C, the functional recovery of the hearts was tested during 15 min of Langendorff reperfusion and then 45 min of working heart reperfusion.. In relation to the cardioprotective effects of Bretschneider's cardioplegic solution alone, we observed an improved recovery of hemodynamic function of the hearts with the addition of both erythro-9-(2-hydroxy-3-nonyl)-adenine and S-(p-nitrobenzyl)-6-thioinosine. However, the myocardial adenosine triphosphate (ATP) concentration remained unchanged. Bradycardia observed under the addition of erythro-9-(2-hydroxy-3-nonyl)-adenine alone was prevented by the addition of S-(p-nitrobenzyl)-6-thioinosine.. A combination of both substances may be tested further for cardiac preservation, as it might improve the recovery from ischemia at moderate temperatures.

Topics: Adenine; Adenosine Deaminase; Affinity Labels; Animals; Cardioplegic Solutions; Disease Models, Animal; Enzyme Inhibitors; Glucose; In Vitro Techniques; Male; Mannitol; Myocardial Reperfusion Injury; Myocardium; Potassium Chloride; Procaine; Rats; Rats, Sprague-Dawley; Thioinosine

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