Mass spectrometric investigations into the brain delivery of abacavir, stavudine and didanosine in a rodent model.

HIV replication in the brain is unopposed due to reduced antiretroviral drug penetration into the central nervous system (CNS). Prevalence of HIV-associated neurocognitive disorder (HAND) has increased severely in patients living with HIV despite current treatments. The aims of this study were to evaluate the brain bio-distribution of alternative nucleoside reverse transcriptase inhibitors, abacavir, stavudine and didanosine in the CNS and to determine their localization patterns in the brain.Sprague-Dawley rats received 50 mg kg-1 single i.p dose of each drug. Mass spectrometric techniques were then used to investigate the pharmacokinetics and localization patterns of these drugs in the brain using LC-MS/MS and mass spectrometric imaging (MSI), respectively.Abacavir, stavudine and didanosine reached the Brain Cmax with concentration of 831.2, 1300 and 43.37 ngmL-1, respectively. Based on MSI analysis Abacavir and Stavudine were located in brain regions that are strongly implicated in the progression of HAND.Abacavir and Stavudine penetrated into CNS, reaching a Cmax that was above the IC50 for HIV (457.6 and 112.0 ngmL-1, respectively), however, it was noted ddI showed poor entry within the brain, therefore, it is recommended that this drug cannot be considered for treating CNS-HIV.

Discovery of Potent ALK Inhibitors Using Pharmacophore-Informatics Strategy.

Anaplastic lymphoma kinase is a tyrosine kinase receptor protein belonging to insulin receptor superfamily. Gene fusions in anaplastic lymphoma kinase are associated with non-small cell lung cancer development. Hence, they are of immense importance in targeted therapies. Thus, for the treatment of non-small cell lung cancer, effective anaplastic lymphoma kinase inhibitors are of great significance. Therefore, our objective is to find hit compounds that could have better inhibitory activity than the existing anaplastic lymphoma kinase inhibitors. Keeping this in mind, in the present study pharmacophore based virtual screening was performed to identify possible anaplastic lymphoma kinase inhibitors. Initially, a five-point common pharmacophore hypothesis was generated based on twelve anaplastic lymphoma kinase inhibitors using PHASE module of Schrödinger. Subsequently, common pharmacophore hypothesis-based screening was conducted against in-trials subset of ZINC database and a total of 1000 hits were identified. The molecules obtained were further screened by three stages of docking using GLIDE software. The docking results reveal that six hit molecules showed higher glide score in comparison with the reference molecules. Finally, pharmacokinetic properties of the hit molecules were also analysed using QikProp programme. The results indicate that molecules namely videx, dexecadotril, chloramphenicol, naficillin were found to have good pharmacokinetic properties and human oral absorption. Moreover, videx, naficillin and chloramphenicol were found to have significant inhibitory activity for mutant (F1174L) anaplastic lymphoma kinase. It was also found that videx exhibited crucial interactions with the Met1199 residue of the native and mutant anaplastic lymphoma kinase protein. Furthermore, PASS algorithm predicted anti-neoplastic activity for all the four molecules. Thus these hits are found to be promising leads for anaplastic lymphoma kinase inhibitors. We believe that this study will be useful for the discovery and designing of more potent anaplastic lymphoma kinase inhibitors in the near future.

Bioretrosynthetic construction of a didanosine biosynthetic pathway.

Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2',3'-dideoxyinosine). Applying both directed evolution- and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.

Selection and characterization of suitable lipid excipients for use in the manufacture of didanosine-loaded solid lipid nanoparticles and nanostructured lipid carriers.

This research aimed to evaluate the suitability of lipids for the manufacture of solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) loaded with the hydrophilic drug, didanosine (DDI). The crystalline state and polymorphism of lipids with the best-solubulizing potential for DDI was investigated using differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS). DSC and WAXS were also used to determine potential interactions between the bulk lipids and DDI. Precirol® ATO 5 and Transcutol® HP showed the best-solubilizing potential for DDI. Precirol® ATO 5 exists in the ß-modification before heating; however, a mixture of both α- and ß-modifications were detected following heating. Addition of Transcutol® HP to Precirol® ATO 5 changes the polymorphism of the latter from the ß-modification to a form that exhibits coexistence of the α- and ß-modifications. DDI exists in a crystalline state when dispersed at 5% (w/w) in Precirol® ATO 5 or in a Precirol® ATO 5/Transcutol® HP mixture. DSC and WAXS profiles of DDI/bulk lipids mixture obtained before and after exposure to heat revealed no interactions between DDI and the lipids. Precirol® ATO 5 and a mixture of Precirol® ATO 5 and Transcutol® HP may be used to manufacture DDI-loaded SLN and NLC, respectively.

Evaluation of the in vitro differential protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (NLCs) for potential targeting to the brain.

The preferential in vitro adsorption of apolipoprotein E (Apo E) onto the surface of colloidal drug carriers may be used as a strategy to evaluate the in vivo potential for such systems to transport drugs to the brain. The aim of this research was to investigate the in vitro protein adsorption patterns of didanosine-loaded nanostructured lipid carriers (DDI-NLCs), using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in order to establish the potential for NLCs to deliver DDI to the brain. NLC formulations were manufactured using high-pressure homogenization using a lipid matrix consisting of a mixture of Precirol(®) ATO 5 and Transcutol(®) HP. The 2-D PAGE analysis revealed that NLCs in formulations stabilized using Solutol(®) HS 15 alone or with a ternary surfactant system consisting of Solutol(®) HS 15, Tween(®) 80, and Lutrol(®) F68, preferentially adsorbed proteins, such as Apo E. Particles stabilized with Tween(®) 80 and Lutrol(®) F68 did not adsorb Apo E in these studies, which could be related to the relatively large particle size and hence small surface area observed for these NLCs. These findings have revealed that DDI-loaded NLCs may have the potential to deliver DDI to the brain in vivo and, in addition, to Tween(®) 80, which has already been shown to have the ability to facilitate the targeting of colloidal drug delivery systems to the brain. Solutol(®) HS 15-stabilized nanoparticles may also achieve a similar purpose.

Design and directed evolution of a dideoxy purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) catalyzes the synthesis and phosphorolysis of purine nucleosides, interconverting nucleosides with their corresponding purine base and ribose-1-phosphate. While PNP plays significant roles in human and pathogen physiology, we are interested in developing PNP as a catalyst for the formation of nucleoside analog drugs of clinical relevance. Towards this aim, we describe the engineering of human PNP to accept 2',3'-dideoxyinosine (ddI, Videx((R))) as a substrate for phosphorolysis using a combination of site-directed mutagenesis and directed evolution. In human PNP, we identified a single amino acid, Tyr-88, as a likely modulator of ribose selectivity. RosettaLigand was employed to calculate binding energies for substrate and substrate analog transition state complexes for single mutants of PNP where Tyr-88 was replaced with another amino acid. In parallel, these mutants were generated by site-directed mutagenesis, expressed and purified. A tyrosine to phenylalanine mutant (Y88F) was predicted by Rosetta to improve PNP catalytic activity with respect to ddI. Kinetic characterization of this mutant determined a 9-fold improvement in k(cat) and greater than 2-fold reduction in K(M). Subsequently, we used directed evolution to select for improved variants of PNP-Y88F in Escherichia coli cell extracts resulting in an additional 3-fold improvement over the progenitor strain. The engineered PNP may form the basis for catalysts and pathways for the biosynthesis of ddI.

Nucleoside phosphorylases from clostridium perfringens in the synthesis of 2',3'-dideoxyinosine.

Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2',3'-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2',3'-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.

Didanosine ester prodrugs: synthesis, albumin binding properties and pharmacokinetic studies in rats.

Three half-ester derivatives 10-12 of 5'-O-2',3'-dideoxydidanosine (DDI, 1) have been synthesized. The compounds exhibited excellent correlation between partition coefficients LogP and relative in vitro bovine serum albumin binding. Using high-performance liquid chromatography-mass spectrometry (LC-MS), DDI (1) was quantitatively determined in rat plasma after intravenous injection of the azelaic acid monoester derivative (11) of DDI. The pharmacokinetic data obtained for DDI were consistent with literature. The pharmacokinetic profile of 11 showed no significant difference in AUC(0-360) or curve shape compared to the parent drug DDI (1). The data indicate that the prodrug was converted to DDI within minutes after administration. High relative protein binding in vitro holds a promise for validity of the concept using more stable linker bonds.

Permeation of four oral drugs through human intestinal mucosa.

The pharmaceutical industry is in need of rapid and accurate methods to screen new drug leads for intestinal permeability potential in the early stages of drug discovery. Excised human jejunal mucosa was used to investigate the permeability of the small intestine to four oral drugs, using a flow-through diffusion system. The four drugs were selected as representative model compounds of drug classes 1 and 3 according to the biopharmaceutics classification system (BCS). The drugs selected were zidovudine, propranolol HCl, didanosine, and enalapril maleate. Permeability values from our in vitro diffusion model were compared with the BCS permeability classification and in vivo and in vitro gastrointestinal drug permeability. The flux rates of the four drugs were influenced by the length of the experiment. Both class 1 drugs showed a significantly higher mean flux rate between 2 and 6 h across the jejunal mucosa compared to the class 3 drugs. The results are therefore in line with the drugs' BCS classification. The results of this study show that the permeability values of jejunal mucosa obtained with the flow-through diffusion system are good predictors of the selected BCS class 1 and 3 drugs' permeation, and it concurred with other in vitro and in vivo studies.

Influx mechanism of 2',3'-dideoxyinosine and uridine at the blood-placenta barrier.

The blood-placenta barrier (BPB) serves to protect the fetus from exposure to toxins, and to transport various nutrients, including nucleosides, and hormones from mother to fetus. It is known that nucleoside transporters contribute to the transfer of nucleosides and nucleoside analogues. 2',3'-Dideoxyinosine (ddI) has a nucleoside structure, and crosses the BPB. Although ddI is a substrate of several transporters, including equilibrative nucleoside transporters (ENT1 and ENT2), the transport mechanism of ddI in the placenta has not yet been characterized. Therefore, the purpose of this study was to clarify the influx mechanisms of ddI from the maternal to the fetal side, and to examine the interaction between ddI and uridine transport at the BPB. We studied ddI and uridine uptakes using a conditionally immortalized rat syncytiotrophoblast cell line, TR-TBT 18d-1, as a BPB model. The ddI uptake was temperature-dependent, Na(+)-independent and saturable. Kinetic analysis yielded K(m) values for ddI and uridine of 6.51 mM and 23.4 microM, respectively. Uridine uptake was inhibited by ENT1 and ENT2 substrates, and ddI uptake was also inhibited by substrates or inhibitors at concentrations that inhibit ENT2. Uridine uptake in Xenopus laevis oocytes expressing rat ENT2 was inhibited by 5mM ddI, in agreement with the results for TR-TBT 18d-1. Our results indicate that ddI and uridine are both taken up in part via ENT2 in TR-TBT 18d-1 cells, and therefore that ENT2 may contribute to their uptake at the BPB.

Arthrobacter oxydans as a biocatalyst for purine deamination.

Deaminases are enzymes that catalyze the hydrolysis of amino groups of nucleosides or their bases. Because these enzymes play important roles in nucleotide metabolism, they are relevant targets in anticancer and antibacterial therapies. Mammalian deaminases are commercially available but the use of bacterial whole cells, especially as biocatalysts, is continuously growing because of their economical benefits. Moreover, deaminases are useful for the preparative chemoenzymatic transformation of nucleoside and base analogues into a variety of derivatives. The purine deaminase activities of Arthrobacter oxydans, a gram-positive bacterium utilized widely in bioremediation, were studied. The presence of adenosine, adenine and guanine deaminases was demonstrated and some purine bases and nucleosides were analyzed as substrates. Using A. oxydans whole cells as the biocatalyst, different purine compounds such as the anti-HIV, 2',3'-dideoxyinosine (73%, 2 h) were obtained.

Nucleoside reverse transcriptase inhibitors prevent HIV protease inhibitor-induced atherosclerosis by ubiquitination and degradation of protein kinase C.

HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Calpha (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.

Analysis and validation of the phosphorylated metabolites of two anti-human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts: sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath-liquid make-up constituents.

A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.

Pharmacokinetic interaction study of indinavir/ritonavir and the enteric-coated capsule formulation of didanosine in healthy volunteers.

Didanosine enteric-coated should be taken on an empty stomach, but the once-daily combination of indinavir/ritonavir can be taken with food. Because these drugs are frequently included in 1 regimen, the food effects on the pharmacokinetics were evaluated. This was a randomized, 4-way crossover study of single doses of didanosine enteric-coated 400 mg and indinavir/ritonavir 1200/400 mg in 8 healthy subjects. The following regimens were given: didanosine enteric-coated 2 hours after breakfast (reference regimen A), indinavir/ritonavir with breakfast (reference regimen B), didanosine enteric-coated + indinavir/ritonavir 2 hours after breakfast (test regimen C), and didanosine enteric-coated + indinavir/ritonavir with breakfast (test regimen D). Breakfast was 550 kcal, 28% fat. Blood samples were drawn before and up to 24 hours after ingestion. Statistical comparisons of test regimens C and D with reference regimens A and B were made using the equivalence approach for indinavir and didanosine area under the curve and C(max) (0.80-1.25). Eight subjects (5 men, 3 women) were enrolled and completed the study. Indinavir area under the curves were bioequivalent in test regimens C and D compared to reference regimen B. A 14% increased C(max) was observed in test regimen C. Didanosine area under the curve in test regimen D was 4% lower and suggestive of bioequivalence compared to reference regimen A. However, test regimen C didanosine area under the curve was 23% lower and bioinequivalent compared to reference regimen A. Didanosine C(max) decreased 42% and 46% in test regimens C and D, respectively, in comparison to reference regimen A. In this study, dosing didanosine enteric-coated 400 mg once daily + indinavir/ritonavir 1200/400 mg once daily with breakfast indicated no decrease in the amount of absorption for either didanosine and indinavir and that this regimen could be administered with food.

Metabolism of tenofovir and didanosine in quiescent or stimulated human peripheral blood mononuclear cells.

OBJECTIVE: As tenofovir disoproxil fumarate substantially increases plasma concentrations of didanosine in patients with human immunodeficiency virus-1 infection, we sought to determine whether tenofovir and didanosine showed a similar intracellular interaction in human peripheral blood mononuclear cells (PBMCs). DESIGN: Comparative in vitro incubation of two antiretrovirals in lymphocytes. SETTING: Clinical research laboratory. MATERIAL: Radiolabeled tenofovir and didanosine in human PBMCs. MEASUREMENTS AND MAIN RESULTS: Phosphorylation of 2 and 20 microM didanosine to dideoxyadenosine triphosphate (ddATP) was determined in quiescent and stimulated PBMCs in the presence or absence of 5 microM tenofovir. Similarly, phosphorylation of 5 microM tenofovir to tenofovir diphosphate (TFVpp) was examined in the presence or absence of 2 and 20 microM didanosine. Intracellular amounts of ddATP and TFVpp were determined by incubating PBMCs with radiolabeled tenofovir or didanosine alone and together for up to 16 hours and then separating the anabolites by high-performance liquid chromatography for quantitation. The presence of tenofovir did not affect the amount of ddATP in quiescent or stimulated PBMCs with 2 or 20 microM didanosine. In addition, didanosine did not alter the amount of TFVpp that formed. The amount of ddATP was modestly (1.5-3-fold) but consistently higher in stimulated than in quiescent PBMCs, but the amount of TFVpp did not differ. CONCLUSION: There is no significant interaction between tenofovir and didanosine in human PBMCs as determined by the extent of formation of the phosphorylated anabolites. This suggests that adjusting didanosine dosage, when given with tenofovir, to achieve similar didanosine plasma concentrations, may be sufficient to accommodate the systemic drug interaction.

Pharmacokinetics of nelfinavir and its active metabolite, hydroxy-tert-butylamide, in infants perinatally infected with human immunodeficiency virus type 1.

BACKGROUND: In children younger than 2 years of age vertically infected with HIV-1, the recommended pediatric dosing regimen for nelfinavir (20 to 30 mg/kg three times a day) provides insufficient drug exposure. This study was conducted to determine the steady state pharmacokinetics of nelfinavir and its active metabolite, M8, in this population. METHODS: Fourteen infants (2.3 to 8.5 months) underwent 18 intensive pharmacokinetic studies of nelfinavir and M8 at steady state. Nelfinavir and M8 concentrations were measured by high performance liquid chromatography coupled with mass spectrometry, and individual pharmacokinetic values were determined. RESULTS: A mean nelfinavir daily dose of 135.7 +/- 18.8 mg/kg (twice or three times a day) resulted in median C(min), C(max), area under the plasma concentration-time curve (AUC(0-24 h)) and CL/ for nelfinavir of 0.627 mg/l, 2.39 mg/l, 30.6 mg*h/l and 4.2 liters/h/kg, respectively. When normalized for a daily dose of nelfinavir of 150 mg/kg/day, 16.7% of C(max) and 27.8% of AUC(0-24 h) values were below the tenth percentile for adult values. CONCLUSIONS: During the first year of life, nelfinavir requirement is much higher than in older children and adults to obtain similar drug exposure. The mechanisms underlying such differences may involve higher first past metabolism and/or drug interactions or might be related to feeding conditions.

Diadenosine pentaphosphate vasodilates the forearm vascular bed: inhibition by theophylline and augmentation by dipyridamole.

BACKGROUND: In rats, diadenosine pentaphosphate (AP(5)A) has been implicated in the pathogenesis of essential hypertension. This study describes for the first time the vasomotor action of AP(5)A in humans by means of the "perfused forearm technique." RESULTS: AP(5)A evoked a dose-dependent forearm vasodilator response equal to that of adenosine but less than that of adenosine triphosphate (ATP) at equimolar doses. The P(1)-purinoceptor antagonist theophylline (0.28 micromol/min per deciliter) reduced the percentage decrease in forearm vascular resistance (FVR) to AP(5)A (0.6, 6, and 20 nmol/min/dL): -8% +/- 6%, -50% +/- 6%, and -68% +/- 4% during saline solution versus -7% +/- 4%, -33% +/- 5%, and -45% +/- 6% during theophylline (mean +/- standard error [SE]; ANOVA for repeated measures; P <.05 for the interaction between purine dose and theophylline; n = 10). An inhibitor of equilibrative nucleoside transport, dipyridamole (7.4 nmol/min per deciliter), augmented the AP(5)A (0.6 and 6 nmol/min per deciliter)-induced reduction in FVR as follows: -34% +/- 6% and -67% +/- 5% during saline versus -49% +/- 5% and -80% +/- 3% during dipyridamole (P <.05 for the effect of dipyridamole; n = 6). The bivalent cation chelator ethylenediaminetetra-acetic acid (EDTA) inhibited the rapid degradation of AP(5)A in vitro. In vivo, the highest tolerated intra-arterial EDTA dose was not sufficient to inhibit AP(5)A metabolism. CONCLUSION: Intra-arterial AP(5)A caused a dose-dependent reduction in FVR. This is, at least in part, mediated by its degradation product adenosine. The data do not support an in vivo vasoconstrictor action of AP(5)A, and as such AP(5)A does not seem likely to contribute to the pathogenesis of primary hypertension in humans.

Differential kinetics of transport of 2',3'-dideoxyinosine and adenosine via concentrative Na+ nucleoside transporter CNT2 cloned from rat blood-brain barrier.

The concentrative Na+ nucleoside transporter type 2 (CNT2), cloned from a rat blood-brain barrier cDNA library, yields very high flux ratios for purine nucleosides after expression in frog oocytes. This high activity of the rat CNT2 produced from the blood-brain barrier-derived cDNA, designated clone A-11, enabled a kinetic analysis of 2',3'-dideoxyinosine transport via the rat CNT2. CNT2 transported both adenosine and 2',3'-dideoxyinosine. The 2',3'-dideoxyinosine transport parameters included a Km of 29.2 +/- 8.3 microM, a V(max) of 0.40 +/- 0.11 pmol/oocyte/min, and a constant of nonsaturable transport (KD) of 15.7 +/- 0.6 nl/oocyte/min. The 2',3'-dideoxyinosine Vmax was 27-fold lower than the adenosine Vmax and the 2',3'-dideoxyinosine KD was >15-fold greater than the KD of adenosine transport. Adenosine inhibited both the saturable component of 2',3'-dideoxyinosine transport with a K(I) of 14.8 +/- 1.6 microM, and inhibited the nonsaturable component of 2',3'-dideoxyinosine transport. Both the saturable and nonsaturable components of 2',3'-dideoxyinosine transport were sodium-dependent with a sodium K0.5 of 8.7 +/- 0.9 mM, and a Hill coefficient of 1.00 +/- 0.10. The transport of 2',3'-dideoxyinosine was strongly inhibited by thymidine, whereas thymidine was a weak inhibitor of adenosine transport via rat CNT2. Thymidine was transported by rat CNT2 with a Km = 130 +/- 44 microM and a Vmax = 1.7 +/- 0.5 pmol/oocyte/min. These studies provide evidence for asymmetric transport sites on rat CNT2, where 2',3'-dideoxyinosine and thymidine compete selectively at a low Vmax site on the transporter, whereas adenosine is transported at a high Vmax site.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.

In the present study, one has determined the relative role of plasma membrane equilibrative (Na+-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na+-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2'3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine) and ddI (2'3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.