Interindividual variability in TPMT enzyme activity: 10 years of experience with thiopurine pharmacogenetics and therapeutic drug monitoring.

BACKGROUND & AIMS: TPMT activity and metabolite determination (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) remain controversial during thiopurine management. This study assessed associations between patient characteristics and TPMT activity, and their impact on metabolite levels. PATIENTS & METHODS: A retrospective review of the laboratory database from a French university hospital identified 7360 patients referred for TPMT phenotype/genotype determination, and/or for 6-TGN/6-MMPN monitoring. RESULTS: Four TPMT phenotypes were identified according to TPMT activity distribution: low, intermediate, normal/high and very high. Based on 6775 assays, 6-TGN concentrations were 1.6-fold higher in TPMT-deficient patients compared with TPMT-normal patients. Azathioprine dose and TPMT genotype were significant predictors of metabolite levels. Furthermore, 6-MMPN and 6-MMPN: 6-TGN ratios were, respectively, 1.6- and 2.2-fold higher in females than in males, despite similar TPMT, 6-TGN and azathioprine doses. An unfavorable ratio (≥20) was associated with a slightly higher TPMT activity. CONCLUSION: These results illustrate the usefulness of pharmacogenomics and metabolite measurement to improve the identification of noncompliance and patients at high risk for toxicity or therapeutic resistance. Original submitted 13 November 2013; Revision submitted 30 January 2014.

Hot shot induction and reperfusion with a specific blocker of the es-ENT1 nucleoside transporter before and after hypothermic cardioplegia abolishes myocardial stunning in acutely ischemic hearts despite metabolic derangement: hot shot drug delivery before hypothermic cardioplegia.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Prevention of early loss of transplanted islets in the liver of mice by adenosine.

BACKGROUND: The low efficiency of islet transplantation necessitating sequential transplantations with the use of 2 to 3 donors for a recipient has been a major obstacle facing clinical islet transplantation. We determined whether adenosine has any beneficial effects on preventing early loss of transplanted islets in the liver, thereby facilitating successful islet transplantation from one donor to one recipient in mice. METHODS: Two hundred islets, the number of islets from a single mouse pancreas, were grafted into the liver of streptozotocin-induced diabetic C57BL/6 mice. Adenosine was administered once at the time of islet transplantation. Mononuclear cells in the liver of mice receiving islets were isolated and examined by flow cytometry. RESULTS: A single injection of adenosine at the time of transplantation ameliorated hyperglycemia of diabetic mice receiving 200 syngenic islets with suppression of interferon (IFN)-gamma production of hepatic NKT cells and neutrophils, while that of control did not. The IFN-gamma production of NKT cells and neutrophils in the liver of mice treated with alpha-galactosylceramide, a synthetic ligand of NKT cells was suppressed by adenosine. The beneficial effect of adenosine was also observed for BALB/c islet allografts when alloimmune rejection was prevented by anti-CD4 antibody. CONCLUSIONS: Adenosine suppresses the NKT cell-mediated IFN-gamma production of neutrophils in the liver of mice receiving islets, thus leading to prevention of early loss of transplanted syngenic and allogenic islets. The findings indicate that adenosine may improve efficiency of clinical islet transplantation.

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate.

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

Trimetrexate inhibits progression of the murine 32Dp210 model of chronic myeloid leukemia in animals expressing drug-resistant dihydrofolate reductase.

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.

Effects of nitrobenzylthioinosine on neuronal injury, adenosine levels, and adenosine receptor activity in rat forebrain ischemia.

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.

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.

Myocardial infarction and purine transport inhibition in anaesthetised ferrets.

The potential cytoprotective effect of the purine transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI) in a model of myocardial ischaemia and reperfusion was investigated in the anaesthetised ferret. The left anterior descending coronary artery (LAD) was occluded for 90 min, producing ischaemia in 53 +/- 3% of the left ventricular free wall, followed by 240 min reperfusion. NBTI (0.5 mg kg-1, i.v.) was given prior to ischaemia or prior to reperfusion. In addition the effect of purine transport inhibition was investigated in animals subjected to ischaemia without reperfusion. NBTI reduced infarct size from 84.0 +/- 1.7 to 71.4 +/- 3.7% of the area at risk (P < 0.05) when given prior to occlusion of the LAD. NBTI was ineffective however when given 15 min prior to reperfusion. NBTI had no effect upon infarct size produced by ischaemia without reperfusion. The effect of NBTI was independent of significant changes in myocardial blood flow during ischaemia and reperfusion or upon neutrophil infiltration.

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.

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.

[Combination therapy of CPT-11, a camptothecin derivative, with various antitumor drugs against L 1210 leukemia].

Antitumor effect of CPT-11 in combination with cyclophosphamide (CY), nimustin hydrochloride (AC-NU), thio-TEPA (TESPA), methotrexate (MTX), 5-fluorouracil (5-FU), cytosine arabinoside (ara-C), thioinosine (6-MPR), adriamycin (ADM), bleomycin (BLM), mitomycin C (MMC), actinomycin D (ACT-D), vincristine sulfate (VCR), etoposide (VP-16) or cisplatin (CDDP) against L 1210 murine leukemia was investigated. The combination treatment of CPT-11 with CY, ACNU, ADM, CDDP, TESPA and ACT-D showed synergistic effects and significantly prolonged the survival time of L 1210-inoculated mice compared with CPT-11 alone or antitumor drug alone. Although the combination with 5-FU, 6-MPR, VP-16, MMC or VCR had synergistic effect for some schedules exceptionally with ara-C, MTX or BLM had slight synergistic effect against L 1210.

Combination therapy of Schistosoma japonicum by tubercidin and nitrobenzylthioinosine 5'-monophosphate.

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.

Antitumor activity of N-phosphonacetyl-L-aspartic acid in combination with nitrobenzylthioinosine.

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.

Combination therapy of schistosomiasis by tubercidin and nitrobenzylthioinosine 5'-monophosphate.

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.

Therapy of mouse leukemia L1210 with combinations of nebularine and nitrobenzylthioinosine 5'-monophosphate.

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.

Effects of levamisole administration on treatment with 6-mercaptopurine riboside of mice bearing L1210 leukemia cells.

DBA/3 mice and BALB/c X DBA/2 F1 mice were inoculated i.p. with 1 X 10(5) L1210 leukemia cells on Day 0. Beginning with Day 1 following inoculation, the animals were given injections of 6-mercaptopurine riboside (200 mg/kg i.p.) once daily for 5 consecutive days in combination with a single s.c. injection of levamisole (LMS) (10 mg/kg). In DBA/2 mice, a marked decrease in the total peritoneal cell count was achieved when LMS was given on any day from Day -2 to 3 as compared with therapy using 6-mercaptopurine riboside alone, although the survival time was not prolonged in these mice. When LMS was given on Day 1 or 3, a decrease was observed in the in vitro growth rate of the L1210 cells obtained on Day 11 from the peritoneal cavity. In BALB/c X DBA/2 F1 mice, no significant changes were observed in either the total peritoneal cell count or the in vitro growth rate when LMS was injected on Day 3 or daily from Day 3 to 5. Daily injection of LMS from Day 1 to 3 resulted in a conspicuous inhibition of the in vitro growth rate, although no significant changes were observed in the total peritoneal cell count. It seems reasonable to conclude from these results that LMS has a potentiating effect on the antitumor activity of 6-mercaptopurine riboside.