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Alimentary Pharmacology & Therapeutics Feb 2017Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations.
AIM
To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment.
METHODS
The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5.
RESULTS
Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 10 erythrocytes was defined. Analysis of patients on stable thiopurine dose (n = 174) showed that those exceeding the 6-MMPR threshold were at increased risk of hepatotoxicity: OR = 3.8 (95% CI: 1.8-8.0). Age, male gender and BMI were significant determinants. A predictive algorithm was developed based on these determinants and the 6-MMPR threshold to assess hepatotoxicity risk [AUC = 0.83 (95% CI: 0.75-0.91)]. 6-MMPR concentrations above the threshold also correlated with gastrointestinal complaints: OR = 2.4 (95% CI: 1.4-4.3), and general malaise: OR = 2.0 (95% CI: 1.1-3.7).
CONCLUSIONS
In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.
Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Chemical and Drug Induced Liver Injury; Cohort Studies; Early Diagnosis; Female; Genotype; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Prognosis; Risk Factors; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult
PubMed: 27943397
DOI: 10.1111/apt.13879 -
The Journal of Biological Chemistry Nov 1997Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we...
Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues.
Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.
Topics: Affinity Labels; Amino Acid Sequence; Animals; Carrier Proteins; Cloning, Molecular; Dilazep; Dipyridamole; Electrophoresis, Polyacrylamide Gel; Equilibrative Nucleoside Transport Proteins; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Nucleoside Transport Proteins; Peptide Mapping; Protein Structure, Secondary; Rats; Sequence Alignment; Thioinosine; Vasodilator Agents; Xenopus
PubMed: 9353301
DOI: 10.1074/jbc.272.45.28423 -
The Journal of Biological Chemistry Mar 2000We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although...
Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine.
We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.
Topics: Biological Transport; Carrier Proteins; Cell Line; Cytidine; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Genetic Complementation Test; Guanosine; Humans; Inosine; Kinetics; Membrane Proteins; Nucleosides; Thioinosine
PubMed: 10722669
DOI: 10.1074/jbc.275.12.8375 -
Diabetes, Obesity & Metabolism Dec 2018Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate...
AIM
Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin.
METHODS
Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells.
RESULTS
AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes.
CONCLUSIONS
We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.
Topics: AMP-Activated Protein Kinases; Aminoimidazole Carboxamide; Animals; Biological Transport; Equilibrative Nucleoside Transporter 1; Female; Glucose; Hepatocytes; Hypoglycemic Agents; Liver; Metformin; Mice; Phosphorylation; Ribonucleotides; Signal Transduction; Thioinosine
PubMed: 29962100
DOI: 10.1111/dom.13455 -
Chemico-biological Interactions Sep 2017To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
AIM
To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
METHODS
Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10 M azathioprine and 1.1 × 10 M mercaptopurine, concentrations corresponding to the IC values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA).
RESULTS
Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037).
CONCLUSION
Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.
Topics: Azathioprine; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Hepatocytes; Humans; Mercaptopurine; Purines; Tandem Mass Spectrometry
PubMed: 28811125
DOI: 10.1016/j.cbi.2017.08.009 -
Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.Pharmacology Research & Perspectives Aug 2021Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to...
Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.
Topics: Animals; Antimetabolites, Antineoplastic; Biological Transport; Cells, Cultured; Clofarabine; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Humans; Lamivudine; Male; Rats, Sprague-Dawley; Telomerase; Testis; Thioinosine; Thionucleotides; Rats
PubMed: 34288585
DOI: 10.1002/prp2.831 -
The Journal of Physiology Nov 19881. Uptake and metabolism of adenosine were investigated from both maternal (M) and fetal (F) circulations of the isolated, dually perfused guinea-pig placenta by using a...
1. Uptake and metabolism of adenosine were investigated from both maternal (M) and fetal (F) circulations of the isolated, dually perfused guinea-pig placenta by using a single-circulation paired-tracer [( 14C]sucrose as extracellular reference, and [3H]adenosine) dilution technique. 2. Maximal [3H]adenosine uptakes (percentage of dose) from adenosine-free perfusates were 75 +/- 1 and 87 +/- 2% (mean +/- S.E. of mean) at maternal and fetal blood-tissue interfaces respectively. Rapid backflux (percentage of influx) of tritium (labelled adenosine and/or adenosine derivatives) from the placental tissue into the ipsilateral circulation was higher at the fetal (24 +/- 2%) than at the maternal side (11 +/- 2%). 3. Tritium uptakes were reduced to 50 +/- 4 (M) and 60 +/- 6% (F) when the perfusion medium contained 100 microM-unlabelled adenosine; backflux was highly stimulated (44% M and 84% F). Neither uptake nor backflux were affected by inosine, uridine, adenine or hypoxanthine present in the perfusion medium (1 mM). 4. Tissue sequestration of tritium (5-6 min) was approximately 60% of the injected dose when perfusates were adenosine-free and 20% or less in the presence of 100 microM-adenosine. 5. Cellular uptake of [3H]adenosine at both sides of the placenta was markedly reduced by the nucleoside transport inhibitors dipyridamole (DIP, 10 microM) and nitrobenzylthioinosine (NBMPR, 5 microM). 6. Thin-layer chromatographic separation of [3H]inosine, [3H]hypoxanthine and [3H]phosphorylated derivatives in venous effluents following a bolus arterial injection of [3H]adenosine showed a greater fraction of metabolites at the fetal side (about 0.75) than at the maternal side (about 0.50). The percentage of [3H]inosine increased when perfusates contained 100 microM-adenosine and the effect was more marked in the fetal circulation. In the presence of DIP and NBMPR the fractional recovery of 3H-labelled metabolites was greatly reduced. 7. During steady-state perfusion of [3H]adenosine (100 microM) a maintained (5-60 min) tritium uptake of about 55% was observed and all the effluent activity was 3H-labelled metabolites [( 3H]adenosine was only 2.8 +/- 0.2%). Under these conditions high-performance liquid chromatography (HPLC) showed that effluents contained xanthine and urate at 16 +/- 1 and 23 +/- 2 microM respectively. 8. Transplacental transfer (6 min) of tritiated compounds (of which only 10-20% was [3H]adenosine) was often less than that of the extracellular marker [14C]sucrose in both maternal-to-fetal and fetal-to-maternal directions.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Adenosine; Animals; Biological Transport; Dipyridamole; Female; Guinea Pigs; Hypoxanthine; Hypoxanthines; Inosine; Maternal-Fetal Exchange; Placenta; Pregnancy; Thioinosine; Tritium; Uric Acid
PubMed: 3255799
DOI: 10.1113/jphysiol.1988.sp017345 -
Digestive Diseases and Sciences Mar 2023The thiopurine medications are well established in the treatment of inflammatory bowel disease (IBD). There is significant variation in levels of toxic and therapeutic... (Observational Study)
Observational Study
BACKGROUND
The thiopurine medications are well established in the treatment of inflammatory bowel disease (IBD). There is significant variation in levels of toxic and therapeutic metabolites. Current data from small or short-term studies support therapeutic drug monitoring (TDM) in assessing azathioprine (AZA) and 6-mercaptopurine (6MP). TDM of thiopurines involves measurement and interpretation of metabolites 6-TGN and 6-MMPR.
AIMS
This study aimed to assess long-cterm outcomes of patients on thiopurines following therapeutic drug monitoring.
METHODS
A multicenter retrospective observational study of outcomes post thiopurine TDM was conducted. Demographics, disease characteristics, physician global assessment, IBD therapy at baseline TDM and again at 12 months were collected. Clinical outcomes were analyzed according to TDM result, and indication for TDM including proactive and other indications.
RESULTS
The study included 541 patients. Only 39% of patients had appropriate dosing of thiopurines. AZA/6MP TDM informed a management change in 61.9%, and enabled 88.8% of the cohort to continue AZA/6MP following TDM. At 12 months following TDM the majority (74.1%) of the cohort remained on AZA/6MP. Clinical remission was higher at 12-months following thiopurines TDM (68%) compared to baseline (37%), including proactive TDM. Post TDM, 13.0% of patients were identified as shunters and commenced on thiopurine-allopurinol co-therapy.
CONCLUSION
Thiopurine TDM resulted in a change in management for the majority of patients. Post TDM significantly more patients were in remission. TDM allowed the identification of non-adherence and shunters who, without intervention, would not reach therapeutic drug levels. Proactive TDM allowed identification and management of inappropriate dosing, and was associated with increased levels of clinical remission.
Topics: Humans; Azathioprine; Mercaptopurine; Inflammatory Bowel Diseases; Retrospective Studies; Methylthioinosine; Immunosuppressive Agents
PubMed: 35687221
DOI: 10.1007/s10620-022-07556-y -
Antimicrobial Agents and Chemotherapy Nov 2021Fluoroquinolones-the only clinically used DNA gyrase inhibitors-are effective against tuberculosis (TB) but are in limited clinical use for nontuberculous mycobacteria...
Fluoroquinolones-the only clinically used DNA gyrase inhibitors-are effective against tuberculosis (TB) but are in limited clinical use for nontuberculous mycobacteria (NTM) lung infections due to intrinsic drug resistance. We sought to test alternative DNA gyrase inhibitors for anti-NTM activity. Mycobacterium tuberculosis gyrase inhibitors (MGIs), a subclass of novel bacterial topoisomerase inhibitors (NBTIs), were recently shown to be active against the tubercle bacillus. Here, we show that the MGI EC/11716 not only has potent anti-tubercular activity but is active against M. abscessus and M. avium . Focusing on M. abscessus, which causes the most difficult to cure NTM disease, we show that EC/11716 is bactericidal, active against drug-tolerant biofilms, and efficacious in a murine model of M. abscessus lung infection. Based on resistant mutant selection experiments, we report a low frequency of resistance to EC/11716 and confirm DNA gyrase as its target. Our findings demonstrate the potential of NBTIs as anti-M. abscessus and possibly broad-spectrum anti-mycobacterial agents.
Topics: Animals; Mice; Microbial Sensitivity Tests; Mycobacterium Infections, Nontuberculous; Mycobacterium abscessus; Mycobacterium tuberculosis; Nontuberculous Mycobacteria; Thioinosine; Topoisomerase II Inhibitors
PubMed: 34606340
DOI: 10.1128/AAC.01514-21 -
Purinergic Signalling Apr 2024Evaluation of kinetic parameters of drug-target binding, k, k, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving...
Evaluation of kinetic parameters of drug-target binding, k, k, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.
Topics: Humans; Biological Transport; Thioinosine; Equilibrative Nucleoside Transporter 1
PubMed: 37423967
DOI: 10.1007/s11302-023-09948-9