Treatment of mouse neoplasms with high doses of tubercidin.

Previous studies from this laboratory demonstrated that a potent inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), protected cultured cells against cytotoxic nucleosides (nebularine, tubercidin, and toyocamycin). NBMPR and its 5'-monophosphate (NBMPR-P) also protected mice against potentially lethal dosage of these agents. This report describes protection of mice from potentially lethal dosages of tubercidin by administration of NBMPR-P and the use of combinations of these agents in treatments of mice bearing transplanted neoplasms. Treatment of mice bearing i.p. implants of the Ehrlich ascites carcinoma, leukemia L1210/TG8, and colon carcinoma 26 with potentially lethal dosages of tubercidin administered together with host-protecting dosages of NBMPR-P resulted in substantial kill of neoplastic cells and long-term survivors. In these experiments, therapeutic effects were achieved at optimal dosages of NBMPR-P, which protected host vital tissues but did not protect neoplastic cells in ascitic fluids (Ehrlich ascites carcinoma cells and leukemia L1210/TG8 cells). However, at supraoptimal dosages of NBMPR-P, the occurrence of therapeutic failures which were neoplastic deaths indicated that NBMPR-P also protected the neoplastic ascites cells against tubercidin cytotoxicity. Thus, the selectivity of tubercidin toxicity toward cells of the Ehrlich ascites carcinoma and leukemia L1210/TG8 was modified by NBMPR-P dosage.

Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer.

To reveal the antiangiogenic capability of cancer chemotherapy, we developed an alternative antiangiogenic schedule for administration of cyclophosphamide. We show here that this antiangiogenic schedule avoided drug resistance and eradicated Lewis lung carcinoma and L1210 leukemia, an outcome not possible with the conventional schedule. When Lewis lung carcinoma and EMT-6 breast cancer were made drug resistant before therapy, the antiangiogenic schedule suppressed tumor growth 3-fold more effectively than the conventional schedule. When another angiogenesis inhibitor, TNP-470, was added to the antiangiogenic schedule of cyclophosphamide, drug-resistant Lewis lung carcinomas were eradicated. Each dose of the antiangiogenic schedule of cyclophosphamide induced the apoptosis of endothelial cells within tumors, and endothelial cell apoptosis preceded the apoptosis of drug-resistant tumor cells. This antiangiogenic effect was more pronounced in p53-null mice in which the apoptosis of p53-null endothelial cells induced by cyclophosphamide was so vigorous that drug-resistant tumors comprising 4.5% of body weight were eradicated. Thus, by using a dosing schedule of cyclophosphamide that provided more sustained apoptosis of endothelial cells within the vascular bed of a tumor, we show that a chemotherapeutic agent can more effectively control tumor growth in mice, regardless of whether the tumor cells are drug resistant.

Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine.

Current therapy for acute myeloid leukemia (AML) primarily includes high-dose cytotoxic chemotherapy with or without allogeneic stem cell transplantation. Targeting unique cellular metabolism of cancer cells is a potentially less toxic approach. Monotherapy with mitochondrial inhibitors like metformin have met with limited success since escape mechanisms such as increased glycolytic ATP production, especially in hyperglycemia, can overcome the metabolic blockade. As an alternative strategy for metformin therapy, we hypothesized that the combination of 6-benzylthioinosine (6-BT), a broad-spectrum metabolic inhibitor, and metformin could block this drug resistance mechanism. Metformin treatment alone resulted in significant suppression of ROS and mitochondrial respiration with increased glycolysis accompanied by modest cytotoxicity (10-25%). In contrast, 6-BT monotherapy resulted in inhibition of glucose uptake, decreased glycolysis, and decreased ATP with minimal changes in ROS and mitochondrial respiration. The combination of 6-BT with metformin resulted in significant cytotoxicity (60-70%) in monocytic AML cell lines and was associated with inhibition of FLT3-ITD activated STAT5 and reduced c-Myc and GLUT-1 expression. Therefore, although the anti-tumor and metabolic effects of metformin have been limited by the metabolic reprogramming within cells, the novel combination of 6-BT and metformin targets this bypass mechanism resulting in reduced glycolysis, STAT5 inhibition, and increased cell death.

ENT1 inhibition attenuates epileptic seizure severity via regulation of glutamatergic neurotransmission.

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.

Efficacy of combination therapy with tubercidin and nitrobenzylthioinosine 5'-monophosphate against chronic and advanced stages of schistosomiasis.

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.

Treatment of schistosomiasis by purine nucleoside analogues in combination with nucleoside transport inhibitors.

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.

Nucleoside trapping during reperfusion prevents ventricular dysfunction, "stunning," in absence of adenosine. Possible separation between ischemic and reperfusion injury.

A previous study has shown that endogenous adenosine trapping during ischemia (by blocking adenine nucleoside transport and inhibiting adenosine breakdown) prevents myocardial stunning. In this study, we tested the hypothesis that delay of administration of inhibitors until reperfusion would similarly prevent myocardial stunning in the absence of entrapped adenosine. In both studies, a selective nucleoside transport blocker, p-nitrobenzyl-thioinosine, was used in combination with a potent adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, to entrap adenosine (preischemic treatment) or inosine (postischemic treatment) in an in vivo canine model of reversible global ischemia. Twenty-five anesthetized adult dogs were instrumented (by sonomicrometry) to monitor left ventricular performance from the relationship between stroke work and end-diastolic length as a sensitive and load-independent index of contractility. Hearts of animals supported by cardiopulmonary bypass were subjected to 30 minutes of normothermic global ischemia and 60 minutes of reperfusion. Saline solution containing the pharmacologic agents were infused into the bypass circuit before ischemia (group 1) or during reperfusion (group 2). Control group (group 3) received saline before and after ischemia. Myocardial biopsy specimens were obtained before, during, and after ischemia, and levels of adenine nucleotides, nucleosides, oxypurines, and the oxidized form of nicotinamide-adenine dinucleotide were determined. Left ventricular contractility fully recovered within 30 minutes of reperfusion in the groups treated with erythro-9-(2-hydroxy-3-nonyl)adenine and p-nitrobenzyl-thioinosine (p < 0.05 versus control group). Myocardial adenosine triphosphate was depleted by 50% in all groups at the end of ischemia. Adenosine triphosphate recovered during reperfusion only in the group that was treated with inhibitors before ischemia (group 1). At the end of ischemia, adenosine levels were low (< 10% of total nucleosides) in the control group (group 3) and in the group treated only after ischemia (group 2). A high level of adenosine (> 90% of total nucleosides) was present in group 1. We infer that selective pharmacologic blockade of nucleoside transport, only after ischemic injury, accelerated functional recovery during reperfusion, even without trapping of endogenous adenosine during ischemia and without adenosine triphosphate recovery during reperfusion. Recovery of myocardial adenosine triphosphate required preischemic treatment and adenosine entrapment during ischemia and reperfusion. Therefore, nucleoside trapping may be used to prevent reperfusion-mediated injury after reversible ischemic injury.

Protection against fludarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine.

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.

Effect of nitrobenzylthioinosinate on the toxicity of tubercidin and ethidium against Trypanosoma gambiense.

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.

Experimental chemotherapy of leishmaniasis with adenosine analogue Formycin A, in combination with inhibitor of nucleoside transport, nitrobenzylthioinosinate.

A single dose of the adenosine analogue Formycin A (FoA) (20 mg/kg), combined with nitrobenzyl mercaptopurine ribonucleoside 5'-monophosphate (NBMPR-P) (10 mg/kg), a prodrug of nitrobenzylthioinosine (NBMPR), was effective in reducing the size of the foot pad lesions from 7.4 +/- 0.2 to 3.9 +/- 0.2 of Syrian golden hamsters infected with Leishmania major. There was a statistical difference (p < 0.01) in the size of the foot pad by the fifth day between the infected groups that received treatment and the controls, as well as between the groups that were treated with combined drugs and FoA only. The initial reduction in size of the foot pad noted in the group that received only FoA was transient. The effect of FoA or FoA combined with NBMPR on the in vitro cultured promastigotes was similar, indicating that the transport inhibitor might be manipulating the availability of FoA in the host's macrophages where the leishmania amastigotes are resident. The results further indicate the need to explore the usefulness of combining cytotoxic nucleoside analogues with host protecting nucleoside transport inhibitors in the treatment of protozoan parasitic infections.

Efficacy of the tubercidin antileishmania action associated with an inhibitor of the nucleoside transport.

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.

7-Deaza-6-benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: activities and selective toxicities.

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-benzylthioinosines act as subversive substrates of T. gondii, but not human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Moreover, 7-deazaadenosine (tubercidin) was shown to be an excellent ligand of T. gondii adenosine kinase. Therefore, we synthesized 7-deaza-6-benzylthioinosine, and analogues with various substitutions at their phenyl ring, to increase the binding affinity of the 6-benzylthioinosines to T. gondii adenosine kinase. Indeed, the 7-deaza-6-benzylthioinosine analogues were better ligands of T. gondii adenosine kinase than the parent compounds, 6-benzylthioinosine and 7-deazainosine. Herein, we report the testing of the metabolism of these newly synthesized 7-deaza-6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the 7-deaza-6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the 7-deaza-6-benzylthioinosine analogues showed a selective antitoxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were 7-deaza-6-(p-methoxybenzylthio)inosine (IC(50)=4.6 microM), 7-deaza-6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=5.0 microM), and 7-deaza-6-(p-cyanobenzylthio)inosine (IC(50)=5.3 microM). These results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that 7-deaza-6-benzylthioinosines are potential antitoxoplasmic agents.

Cardioprotective effects of novel tetrahydroisoquinoline analogs of nitrobenzylmercaptopurine riboside in an isolated perfused rat heart model of acute myocardial infarction.

We have investigated the cardioprotective effects of novel tetrahydroisoquinoline nitrobenzylmercaptopurine riboside (NBMPR) analog nucleoside transport (NT) inhibitors, compounds 2 and 4, in isolated perfused rat hearts. Langendorff-perfused heart preparations were subjected to 10 min of treatment with compound 2, compound 4, or vehicle (control) followed by 30 min of global ischemia and 120 min of reperfusion. For determination of infarct size, reperfusion time was 180 min. At 1 microM, compounds 2 and 4 provided excellent cardioprotection, with left ventricular developed pressure (LVDP) recovery and end-diastolic pressure (EDP) increase of 82.9 +/- 4.0% (P<0.001) and 14.1 +/- 2.0 mmHg (P<0.03) for compound 2-treated hearts and 79.2 +/- 5.9% (P<0.002) and 7.5 +/- 2.7 mmHg (P<0.01) for compound 4-treated hearts compared with 41.6 +/- 5.2% and 42.5 +/- 6.5 mmHg for control hearts. LVDP recovery and EDP increase were 64.1 +/- 4.2% and 29.1 +/- 2.5 mmHg for hearts treated with 1 microM NBMPR. Compound 4 was the best cardioprotective agent, affording significant cardioprotection, even at 0.1 microM, with LVDP recovery and EDP increase of 76.0 +/- 4.9% (P<0.003) and 14.1 +/- 1.0 mmHg (P<0.03). At 1 microM, compound 4 and NBMPR reduced infarct size, with infarct area-to-total risk area ratios of 29.13 +/- 3.17 (P<0.001) for compound 4 and 37.5 +/- 3.42 (P<0.01) for NBMPR vs. 51.08 +/- 5.06% for control hearts. Infarct size was more effectively reduced by compound 4 than by NBMPR (P<0.02). These new tetrahydroisoquinoline NBMPR analogs are not only potent cardioprotective agents but are, also, more effective than NBMPR in this model.

Therapy in childhood acute nonlymphocytic leukemia (ANLL). Evolution of current concepts of chemotherapy.

Chemotherapy remains the major treatment modality in childhood acute nonlymphocytic leukemia (ANLL). Current remission induction rates range from 60% to 80%; but even with the improved rate of response to therapy, the median duration of remission has seldom exceeded 1 year. On the other hand, an increasing number of children with ANLL who were treated with intensive induction and maintenance chemotherapy regimens for a prescribed period followed by discontinuation of therapy are remaining in remission. The evolution of present-day chemotherapy approaches to childhood ANLL are reviewed in this article.

[Butoctamide enhancement of the antitumor activity of 6-mercaptopurine on Ehrlich solid tumors in mice (author's transl)].

Effects of butoctamide (N-(2-ethylhexyl)-3-hydroxybutyramide, L-2) on the antitumor activity of 6-mercaptopurine (6-MP) against Ehrlich solid tumors in mice were investigated. No change was observed in tumor growth after either oral or intraperitoneal administration of butoctamide (100 mg/kg/day X 7). This drug increased the activity of a low dose of 6-MP (2.5 approximately 10 mg/kg/day. i.p., X 7), but did not change the activity of a high dose of 6-MP (40 approximately 80 mg/kg/day, i.p., X 7). The antitumor activity of thioinosine (6-MP riboside) was similarly increased by administration of butoctamide (100 mg/kg/day, i.p., X 7). On the other hand, concomitant administration of butoctamide with cyclophosphamide, methotrexate, mitomycin C or adriamycin had no effect on the activity of these anticancer drugs. In butoctamide (100 mg/kg/day, i.p., X 7)-treated mice, the antitumor activities of a single administration of 6-MP and cyclophosphamide were not increased. Butoctamide stimulated the hypoxanthine-guanine phosphoribosyltransferase activity and inhibited the xanthine oxidase activity of mouse liver, to a certain degree as compared to controls. Butoctamide may promote conversion from 6 MP to thioinosinic acid monophosphate to a biologically active state, rather than to thiouric acid or hypoxanthine which would be inactive.

Acute lymphocytic leukemia in a child with congenital xanthine oxidase deficiency: implications for therapy.

Xanthine oxidase deficiency was identified in a 16-year-old girl with acute lymphocytic leukemia (ALL). Despite the enzyme deficiency, the patient tolerated high doses of 6-mercaptopurine (6-MP), a drug which is normally inactivated by the action of xanthine oxidase. This rate clinical situation may be analogous in the practice to that of the patient receiving 6-MP who is also given allopurinol, which inhibits xanthine oxidase activity. The implications of these observations on the determination of appropriate drug dosages for patients receiving these two agents are discussed.

Comparative pharmacokinetics of oral 6-mercaptopurine and intravenous 6-mercaptopurine riboside in children.

BACKGROUND: The poor absorption of orally administered 6-mercaptopurine (6MP) causes a wide variation in its cytotoxic efficacy. An i.v. dosage form would eliminate this problem. Our objective was to compare the pharmacokinetics of 6MP administered orally with those of an i.v. dosage form 6-mercaptopurine riboside (6MPR), in children with acute lymphoblastic leukemia or malignant lymphoma. METHODS: A total of 10 children were treated with oral 6MP, 50 mg/m(2) per day, while five children were treated with 6MPR, 50 mg/m(2) per day, administered by rapid i.v. injection. The plasma concentrations of 6MP and of 6MPR were measured on day 0, while the concentrations of 6-thioguanine nucleotides (6TGN) in red blood cells (RBC) were measured on day 2. The area under the plasma concentration-time curve (AUC1-5) was calculated from 1 to 5 h after drug administration. RESULTS: With the intravenously administered 6MPR, the AUC1-5 ranged from 124 to 186 (1.5-fold range, median 145) microM min; only two samples were obtained for the RBC concentration of 6TGN, and were 121 and 273 pmol per 25 mg hemoglobin. With the orally administered 6MP, the AUC1-5 ranged from 23 to 65 microM min (2.8-fold range, median 56); the RBC concentration of 6TGN ranged from 18 to 152 pmol per 25 mg hemoglobin (median 75). CONCLUSION: The i.v. administration of 6MPR showed less interindividual variation in the AUC1-5 coupled with a higher RBC level of 6TGN as compared with those by oral 6MP. We conclude that the i.v. administration of 6MPR achieves stable blood levels of active drug in children undergoing cancer chemotherapy.

Protection of the stunned myocardium. Selective nucleoside transport blocker administered after 20 minutes of ischemia augments recovery of ventricular function.

BACKGROUND: Metabolic interventions capable of preventing ventricular dysfunction "stunning" or accelerating its functional recovery have potential clinical importance. Myocardial protection of the stunned myocardium has not been documented when drugs were administered only during postischemic reperfusion. The role of ATP depletion and release of purines in myocardial injury was assessed using the selective nucleoside transport blocker p-nitrobenzylthioinosine (NBMPR) in a combination with specific adenosine deaminase inhibitor erythro-9-[hydroxy-3-nonyl]adenine (EHNA) administered during reperfusion after reversible ischemic injury. METHODS AND RESULTS: Sixteen anesthetized dogs were instrumented with minor axis sonocrystals and intraventricular Millar. Ventricular performance was determined, off bypass, from the slope of the relationship between stroke-work and end-diastolic length as a sensitive and load-independent index of contractility within physiological range. Hearts were subjected to 20 minutes' warm global ischemia and reperfused with warm blood treated with either saline (control group, n = 8) or saline containing 100 mumol/L EHNA and 25 mumol/L NBMPR (EHNA/NBMPR-treated group, n = 8). Myocardial biopsies were collected and analyzed for ATP and metabolites using high-performance liquid chromatography. Warm ischemia induced significant depletion of ATP (P < .05 versus preischemia) and accumulation of inosine at the end of ischemia (> 90% of total nucleosides) in both groups. Complete functional recovery was observed in the EHNA/NBMPR-treated group (P < .05 versus control group). CONCLUSIONS: Selective entrapment of adenine nucleosides during postischemic reperfusion attenuated ventricular dysfunction (stunning) after brief global ischemia. It is concluded that nucleoside transport plays an important role in myocardial stunning, and its blockade augmented myocardial protection against reperfusion injury. Selective entrapment of endogenous inosine, generated during ischemia, represents an attractive therapeutic approach to the alleviation of postischemic dysfunction mediated by reperfusion in a wide spectrum of ischemic syndromes, including percutaneous transluminal coronary angioplasty and coronary artery bypass graft surgery.