Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Assessment of nucleosides as putative tumor biomarkers in prostate cancer screening by CE-UV.

Cancer is responsible for millions of deaths worldwide, but most base diseases may be cured if detected early. Screening tests may be used to identify early-stage malignant neoplasms. However, the major screening tool for prostate cancer, the prostate-specific antigen test, has unsuitable sensitivity. Since cancer cells may affect the pattern of consumption and excretion of nucleosides, such biomolecules are putative biomarkers that can be used for diagnosis and treatment evaluation. Using a previously validated method for the analysis of nucleosides in blood serum by capillary electrophoresis with UV-vis spectroscopy detection, we investigated 60 samples from healthy individuals and 42 samples from prostate cancer patients. The concentrations of nucleosides in both groups were compared and a multivariate partial least squares-discriminant analysis classification model was optimized for prediction of prostate cancer. The validation of the model with an independent sample set resulted in the correct classification of 82.4% of the samples, with sensitivity of 90.5% and specificity of 76.7%. A significant downregulation of 5-methyluridine and inosine was observed, which can be indicative of the carcinogenic process. Therefore, such analytes are potential candidates for prostate cancer screening. Graphical Abstract Separation of the studied nucleosides and the internal standard 8-Bromoguanosine by CE-UV (a); classification of the external validation samples (30 from healthy volunteers and 21 from prostate cancer patients) by the developed Partial Least Square - Discriminant Analysis (PLS-DA) model with accuracy of 82.4% (b); Receiver Operating Characteristics (ROC) curve (c); and Variable Importance in the Projection (VIP) values for the studied nucleosides (d). A significant down-regulation of 5- methyluridine (5mU) and inosine (I) was observed, which can be indicative of the presence of prostate tumors.

Studies on the adenosine deaminase-catalyzed conversion of adenosine and nucleoside prodrugs by different capillary electrophoresis modes.

Four kinds of fast and efficient capillary electrophoresis modes, i.e., immobilized enzymatic reactor (IER), electrophoretically mediated microanalysis (EMMA), capillary zone electrophoresis (CZE), and micellar electrokinetic chromatography (MEKC), were first developed to study the adenosine deaminase (ADA)-catalyzed conversion of adenosine and nucleoside prodrugs, which is critical for releasing prodrugs into the intracellular compartment for phosphorylation. The enzyme-activated prodrug approach is a strategy that has been successfully employed to improve physicochemical and pharmacokinetic properties of potential therapeutic agents, especially in the search for antiviral nucleoside analogues. Adenosine, amino-ddG, and amino-D4G could be converted by ADA to different extents under our experimental conditions. Steady-state parameters K(m), V(max), and k(cat) were also determined. The substrate efficiencies (k(cat)/K(m)) of adenosine, amino-ddG, and amino-D4G were 0.19±0.01, 0.047±0.005, and 0.017±0.010 µM(-1) s(-1), respectively. The enzymatic reaction could be performed at a nanoliter scale and all manipulation steps were combined into a fully automated assay in on-line modes, which opened the possibilities of high-throughput screening of large libraries of synthetic nucleoside analogues for biological activity and a relative mechanism study of nucleoside and its analogues.

Screening of catalytically active microorganisms for the synthesis of 6-modified purine nucleosides.

Modified nucleosides can be prepared by microbial transglycosylation from cheaper nucleoside precursors using free or immobilised whole cells. An efficient screening method to find transglycosylation activity in microorganisms was developed for the synthesis of 6-modified purine nucleosides, such as 6-chloro-, 6-methoxy-, 6-iodo- and 6-mercaptopurine ribonucleoside. Out of 100 microorganisms screened, Bacillus stearothermophilus ATCC 12980 was the best for this purpose.

Nucleoside analogues in the treatment of haematological malignancies.

The nucleoside analogues are a group of antimetabolite cytotoxics which generally have to be metabolised to the equivalent nucleotide before incorporation into DNA. Cytarabine is a well established component of the treatment of acute leukaemias and has its principal action on dividing cells. New formulations include a liposome encapsulated product for intrathecal use and oral cytarabine ocfosfate which may be suitable for long-term outpatient use. Pentostatin acts by causing accumulation of deoxynucleotides and, although active against hairy cell leukaemia, is associated with a poor tolerance profile. Cladribine and fludarabine have substantial activity in the treatment of chronic lymphocytic leukaemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL). Fludarabine is the more thoroughly investigated of the two and is currently being developed in combination therapies for CLL and NHL and also in a combination with cytarabine for acute myeloid leukaemia. Fludarabine's immunosuppressive activity is being exploited in the conditioning of patients for non-myeloablative stem cell transplantation. Gemcitabine is an established agent in the treatment of a number of solid tumours but also has activity in haematological malignancies which might be exploited by the use of extended infusion schedules. Newer agents including nelarabine, clofarabine and troxacitabine are undergoing clinical evaluation and show promising activity.

Biochemical assessment of the effects of acivicin and dipyridamole given as a continuous 72-hour intravenous infusion.

Since this Phase I trial was based on a strategy of biochemical modulation, namely, the inhibition of nucleoside uptake by dipyridamole, a biochemical assessment of the actions of acivicin and dipyridamole was undertaken in order to aid our interpretation of the clinical findings. The primary biochemical objectives of this trial were: (a) to determine whether plasma levels of dipyridamole sufficient to inhibit nucleoside uptake could be achieved with a 72-h continuous i.v. infusion; (b) to monitor the effects of acivicin on two key enzymatic targets, CTP synthetase and GMP synthetase; and (c) to evaluate changes in cellular ribonucleoside triphosphate pools during therapy. Since peripheral blood mononuclear cells have relevant biochemical targets and can be serially obtained during the course of therapy, the biochemical effects of acivicin and dipyridamole were determined in these cells. At the maximally tolerated dose of dipyridamole (23.1 mg/kg/72 h), the steady-state concentrations of total and free dipyridamole averaged 11.9 microM and 27.8 nM, respectively. These levels were sufficient to inhibit cytidine (1 microM) uptake by greater than 50% in the lymphocytes of five of six patients so treated. Using lymphocytes obtained from 14 normal volunteers the concentration of free dipyridamole needed to inhibit the uptake of 1 microM cytidine by 50% averaged 13.8 +/- 1.1 nM. The plasma levels of alpha 1-acid glycoprotein, which tightly binds dipyridamole, ranged from 60 to 300 mg/dl in the patients in this study. As a consequence there were wide variations in the percentage of dipyridamole present as the unbound, pharmacologically active form and in the rates of dipyridamole clearance. The decreased rate of dipyridamole clearance seen in patients with high levels of alpha 1-acid glycoprotein resulted in higher plasma concentrations of total dipyridamole and compensated for the reduced fraction of free drug. Therefore, the plasma concentration of free dipyridamole varied much less than the total drug concentration in these patients. CTP synthetase and GMP synthetase activities were measured in patients' peripheral mononuclear cells prior to and at various times during therapy. CTP synthetase activity was inhibited in a time-dependent fashion by greater than 75% in seven of 13 evaluable courses; GMP synthetase was similarly inhibited in only three of ten cases. Ribonucleoside triphosphate pools were also measured in the patient's lymphocytes. CTP pool reductions of 30 to 50% were seen in nine of 19 courses, but in only four cases was the inhibition greater than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)