Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors.

Evaluation of kinetic parameters of drug-target binding, kon, koff, 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.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1).

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.

Three Faces of Mercaptopurine Cytotoxicity In Vitro: Methylation, Nucleotide Homeostasis, and Deoxythioguanosine in DNA.

Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.

The glutathione transferase Mu null genotype leads to lower 6-MMPR levels in patients treated with azathioprine but not with mercaptopurine.

The conversion of azathioprine (AZA) to mercaptopurine (MP) is mediated by glutathione transferase Mu1 (GSTM1), alpha1 (GSTA1) and alpha2 (GSTA2). We designed a case-control study with data from the TOPIC trial to explore the effects of genetic variation on steady state 6-methylmercaptopurine ribonucleotide (6-MMPR) and 6-thioguanine nucleotide (6-TGN) metabolite levels. We included 199 patients with inflammatory bowel disease (126 on AZA and 73 on MP). GSTM1-null genotype carriers on AZA had two-fold lower 6-MMPR levels than AZA users carrying one or two copies of GSTM1 (2239 (1006-4587) versus 4371 (1897-7369) pmol/8 × 108 RBCs; P<0.01). In patients on MP (control group) 6-MMPR levels were comparable (6195 (1551-10712) versus 6544 (1717-11600) pmol/8 × 108 RBCs; P=0.84). The 6-TGN levels were not affected by the GSTM1 genotype. The presence of genetic variants in GSTA1 and GSTA2 was not related to the 6-MMPR and 6-TGN levels.

Enhancement of inosine-mediated A2AR signaling through positive allosteric modulation.

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15h) than adenosine (half-life <10s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A2A receptor (A2AR). Relative to adenosine inosine exhibits a lower affinity towards the A2AR. Therefore, it is generally believed that inosine is incapable of activating the A2AR through direct engagement, but indirectly activates the A2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.

ITPA Activity in Adults and Children Treated With or Without Azathioprine: Relationship Between TPMT Activity, Thiopurine Metabolites, and Co-medications.

BACKGROUND: The implication of inosine triphosphate pyrophosphatase (ITPA) on thiopurine drug response variability has been investigated but little data are available on its role on thiopurine metabolites. The ability of ITPA to modify the thiopurine metabolite levels is currently used to optimize azathioprine (AZA) therapy in relation to thiopurine S-methyltransferase (TPMT) activity, the aim of this study is to investigate ITPA phenotype in a large population and to evaluate the relation between ITPA and TPMT activities and thiopurine metabolites. METHODS: ITPA activity was determined in 183 adults and 138 children with or without AZA therapy. 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotides (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in red blood cells. Using the Gaussian mixture model, distribution of ITPA activity was evaluated. Intraindividual variability and influence of age, sex, AZA treatment and associated co-medications on ITPA activity were also assessed. RESULTS: This retrospective study shows a quadrimodal distribution in ITPA activity. No influence of age, sex, AZA therapy, and co-medications was found. In adults, ITPA activity was not significantly associated with 6-TGN or 6-MeMPN concentrations, whereas a weak negative correlation was observed with 6-MeMPN levels in pediatric populations (rs = -0.261; P = 0.024). A weak positive correlation was observed between ITPA and TPMT activities in children (rs = 0.289; P = 0.001). CONCLUSIONS: ITPA activity was poorly influenced by nongenetic parameters and has no influence on 6-TGN and 6-MeMPN concentrations in adults and only a weak correlation with 6-MeMPN and TPMT activity in children. These results demonstrate that ITPA is not a rate-limiting enzyme in the formation of 6-TGN but suggest that a decrease in ITPA activity in children may be a risk factor for accumulation of 6-MeMPN in cells.

Early prediction of thiopurine-induced hepatotoxicity in inflammatory bowel disease.

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 × 108 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.

Extended exposure to substrate regulates the human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 µM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.

N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function.

Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic and anti-viral drugs. Previous work, and in silico prediction, suggest that hENT1 is glycosylated at Asn(48) in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without peptide-N-glycosidase F (PNGase-F), followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N-linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9 pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45 pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01 pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06 pmol/mg protein; mock-transfected 74.31±19.65 pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function and oligomerization of hENT1.

Review article: recent advances in pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in inflammatory bowel disease.

BACKGROUND: Azathioprine and mercaptopurine have a pivotal role in the treatment of inflammatory bowel disease (IBD). However, because of their complex metabolism and potential toxicities, optimal use of biomarkers to predict adverse effects and therapeutic response is paramount. AIM: To provide a comprehensive review focused on pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in IBD. METHODS: A literature search up to July 2015 was performed in PubMed using a combination of relevant MeSH terms. RESULTS: Pre-treatment thiopurine S-methyltransferase typing plus measurement of 6-tioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels during treatment have emerged with key roles in facilitating safe and effective thiopurine therapy. Optimal use of these tools has been shown to reduce the risk of adverse effects by 3-7%, and to improve efficacy by 15-30%. For the introduction of aldehyde oxidase (AOX) into clinical practice, the association between AOX activity and AZA dose requirements should be positively confirmed. Inosine triphosphatase assessment associated with adverse effects also shows promise. Nucleoside diphosphate-linked moiety X-type motif 15 variants have been shown to predict myelotoxicity on thiopurines in East Asian patients. However, the impact of assessments of xanthine oxidase, glutathione S-transferase, hypoxanthine guanine phosphoribosyltransferase and inosine monophosphate dehydrogenase appears too low to favour incorporation into clinical practice. CONCLUSIONS: Measurement of thiopurine-related enzymes and metabolites reduces the risk of adverse effects and improves efficacy, and should be considered part of standard management. However, this approach will not predict or avoid all adverse effects, and careful clinical and laboratory monitoring of patients receiving thiopurines remains essential.

Routinely Established Skewed Thiopurine Metabolism Leads to a Strikingly High Rate of Early Therapeutic Failure in Patients With Inflammatory Bowel Disease.

BACKGROUND: The conventional thiopurines azathioprine and mercaptopurine are considered maintenance immunosuppressive drugs of choice in the treatment of inflammatory bowel disease (IBD). Unfortunately, treatment is often discontinued because of adverse events (AEs) or refractoriness, retrospectively associated with the high levels of the thiopurine metabolites 6-methylmercaptopurine ribonucleotides (6-MMPR). Patients with a clinically "skewed" thiopurine metabolism may be particularly at risk for therapy failure. We determined the predictive value of this pharmacological phenomenon in patients with IBD during regular thiopurine therapy. METHODS: Clinical effectiveness and tolerability of weight-based thiopurine therapy were determined in all patients with IBD displaying a skewed metabolism [ratio 6-MMPR/6-thioguanine nucleotide (6-TGN) >20]. All samples were routinely assessed between 2008 and 2012, as part of standard clinical follow-up after initiation of conventional thiopurine therapy. RESULTS: Forty-one (84%) of 49 included patients with IBD discontinued thiopurines (55% female, 53% with Crohn disease) with a median duration of 14 weeks (range, 7-155). The majority of patients with a skewed metabolism discontinued thiopurines because of adverse events (55%) or refractoriness (12%). The most commonly observed adverse event was hepatotoxicity (18 patients, 37%). Median 6-TGN level was 159 pmol/8 × 10 RBC (range, 46-419), median 6-MMPR level was 11,020 pmol/8 × 10 RBC (range, 3610-43,670), and the median 6-MMPR/6-TGN ratio was 72 (range, 29-367). Thiopurine therapy failure was associated with a ratio above 50 (P < 0.03). Hepatotoxicity occurred more frequently in patients with an extremely skewed metabolism (6-MMPR/6-TGN ratio >100) (P < 0.01). CONCLUSIONS: This study demonstrates that a routinely established skewed metabolism is a major risk factor for future thiopurine failure in patients with IBD. These observations imply that routine thiopurine metabolite measurements may be used as a prognostic tool to identify those patients with an aberrant-skewed metabolism at an early stage, possibly benefitting from therapy adjustments.

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.

The effect of ITPA polymorphisms on the enzyme kinetic properties of human erythrocyte inosine triphosphatase toward its substrates ITP and 6-Thio-ITP.

The role of inosine triphosphatase (ITPase) in adverse drug reactions associated with thiopurine therapy is still under heavy debate. Surprisingly, little is known about the way thiopurines are handled by ITPase. We studied the effect of ITPA polymorphisms on the handling of inosine triphosphate (ITP) and thioinosine triphosphate (TITP) to gain more insight into this phenomenon. Human erythrocyte ITPase activity was measured by incubation with ITP using established protocols, and the generated inosine monophosphate (IMP) was measured using ion-pair RP-HPLC. Molecular analysis of the ITPA gene was performed to establish the genotype. Kinetic parameters were established for the two common polymorphisms for both ITP and TITP as substrates using the above mentioned protocol. Both ITP and TITP are substrates for ITPase and their enzyme activities are comparable. Substrate binding is not altered in the different ITPA polymorphisms. It is shown that the velocity of pyrophosphohydrolysis is compromised when the c.94C > A polymorphism is present, both in the heterozygous and in the homozygous state. TITP is handled by ITPase in a similar way as for ITP, which implies that TITP will accumulate in the erythrocytes of patients with an ITPase deficiency, resulting in adverse drug reactions (ADRs) on thiopurine therapy. In carriers of ITPA polymorphisms, the matter is more complex and the development of ADR may depend on additional epigenetic factors rather than on the accumulation of thiopurinenucleotides.

Antimutagenic, antigenotoxic and antioxidant activities of phenolic-enriched extracts from Teucrium ramosissimum: combination with their phytochemical composition.

The evaluation of the mutagenic and antimutagenic actions of extracts obtained from aerial part of Teucrium ramosissimum was assayed using the Salmonella typhimurium assay system. The effect of the same extracts on genotoxicity and SOS response induced by aflatoxin B(1) as well as nitrofurantoin was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The different extracts showed no mutagenicity when tested with Salmonella typhimurium strains TA100, TA98 and TA1535 either with or without S9 mix. In contrast, our results prove that T. ramosissimum extracts possess antimutagenic effects against sodium azide, aflatoxin B1, benzo[a]pyrene and 4-nitro-o-phenylenediamine. Moreover, the T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixtures. However, all the extracts significantly decreased the genotoxicity induced by aflatoxin B(1) and nitrofurantoin. The result obtained by the Ames test confirms those of SOS chromotest. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) and the non enzymatic (NBT/riboflavine, DPPH and ABTS assays) systems. All extracts exhibited high antioxidant activity except the chloroform and the methanol extracts in DPPH and NBT/riboflavine assays respectively. Our results underline the potential of T. ramosissimum to avoid mutations and also its antioxidant potential.

The role of inosine-5'-monophosphate dehydrogenase in thiopurine metabolism in patients with inflammatory bowel disease.

BACKGROUND: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated. METHODS: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤ 20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities. RESULTS: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤ 20 (P < 0.001). Metabolite ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001). CONCLUSIONS: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.

Immunomodulatory effects of proanthocyanidin A-1 derived in vitro from Rhododendron spiciferum.

The objective of this study was to evaluate the immunomodulatory effects of proanthocyanidin A-1 (PAA-1) from leaves of Rhododendron spiciferum (Ericaceae). In vitro tests showed that PAA-1 stimulated cell proliferation of splenocytes and peritoneal macrophages significantly enhanced the cytotoxicity of natural killer (NK) cells and increased CD4(+) and CD8(+) cell populations. PAA-1 also regulated the expression of Th1- and Th2-related cytokines. Moreover, this study showed that PAA-1 exhibited a significant effect on NBT dye reduction and lysosomal enzyme activity responses in macrophages, indicating effective phagocytic activation. These results revealed that PAA-1 exhibits immunomodulatory activity without a clear dose response.

Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells.

The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.