Endoplasmic reticulum transporter OAT2 regulates drug metabolism and interaction.

Xenobiotic metabolic reactions in the hepatocyte endoplasmic reticulum (ER) including UDP-glucuronosyltransferase and carboxylesterase play central roles in the detoxification of medical agents with small- and medium-sized molecules. Although the catalytic sites of these enzymes exist inside of ER, the molecular mechanism for membrane permeation in the ER remains enigmatic. Here, we investigated that organic anion transporter 2 (OAT2) regulates the detoxification reactions of xenobiotic agents including anti-cancer capecitabine and antiviral zidovudine, via the permeation process across the ER membrane in the liver. Pharmacokinetic studies in patients with colorectal cancer revealed that the half-lives of capecitabine in rs2270860 (1324C > T) variants was 1.4 times higher than that in the C/C variants. Moreover, the hydrolysis of capecitabine to 5'-deoxy-5-fluorocytidine in primary cultured human hepatocytes was reduced by OAT2 inhibitor ketoprofen, whereas capecitabine hydrolysis directly assessed in human liver microsomes were not affected. The immunostaining of OAT2 was merged with ER marker calnexin in human liver periportal zone. These results suggested that OAT2 is involved in distribution of capecitabine into ER. Furthermore, we clarified that OAT2 plays an essential role in drug-drug interactions between zidovudine and valproic acid, leading to the alteration in zidovudine exposure to the body. Our findings contribute to mechanistically understanding medical agent detoxification, shedding light on the ER membrane permeation process as xenobiotic metabolic machinery to improve chemical changes in hydrophilic compounds.

Zidovudine Glycosylation by Filamentous Fungi Leads to a Better Redox Stability and Improved Cytotoxicity in B16F10 Murine Melanoma Cells.

BACKGROUND: The strategic development of therapeutic agents, capable of being targeted at their active sites, has been a major goal in treatment of cancer. The delivery of drugs for tumors has as its main challenge the development of safe and effective drugs, since the goal of chemotherapy is to eliminate the tumor completely without affecting healthy cells. The aim of present study was to investigate the antioxidant, anticancer activities of zidovudine and its α-O-glycosylated derivative obtained by biosynthesis of a filamentous fungi, Cunninghamela echinulata. METHODS: An evaluation of the cytotoxic potential of zidovudine and its α-O-glycosylated was performed in fibroblasts and melanoma cells by the tetrazolium reduction method (MTT) and the antioxidant activity of this derivative was observed. RESULTS: The antioxidant activity of zidovudine demonstrated an electrochemical oxidation potential of 0.91V, while the α-O-glycosylated derivative did not exhibit any antioxidant activity. The zidovudine exhibited low cytotoxicity for melanoma and fibroblast cells, while the α-O-glycosylated derivative presented better cytotoxicity on melanoma cells at a concentration of 10mg. mL-1. CONCLUSION: This study demonstrates the specific cytotoxicity of the glycoconjugate and suggests that glycosylation by biosynthesis can be a useful strategy for obtaining new anticancer compounds.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Capillary electrophoresis study of systemin peptides spreading in tomato plant.

Systemin (Sys) is an 18-aa plant peptide hormone involved in the regulation of plant's defensive response. Sys is considered as a fast-spreading systemic wound signal. We developed a simple and rapid CE method to monitor the spreading of Sys peptides through tomato plant. A 1,2,3-triazole-linked AZT-systemin conjugate was designed as a model to study the possibility of translocating small cargo molecules 3'-Azido-2',3'-dideoxythymidine by systemin. The Sys peptides (Sys, N-propiolyl Sys, and AZT-systemin conjugate) were injected into the stem and leaves of mature tomato plant. Its transportation throughout the plant tissue was traced by CE. The peptides were clearly visible in the crude tomato exudates and an optimum separation was achieved in 25 mM phosphate "buffer" at pH 2.5 and a voltage of 20 kV using uncoated fused silica capillary. CE analysis showed that Sys peptides are well separated from tomato plant exudates ingredients and are stable in tomato stem and leaf exudates for up to 24 h. CE study revealed that the Sys peptides are effectively spreading throughout tomato stem and leaves and the peptides could be directly detected in the crude plant matrixes. The translocation was strongly inhibited by sodium azide. The results showed that the established CE method can be used to characterize plant peptides spreading under plant physiological conditions.

Gelatin modified lipid nanoparticles for anti- viral drug delivery.

The major challenges to clinical application of zidovudine are its moderate aqueous solubility and relative short half-life and serious side effects due to frequent administrations. We investigated the preparation of zidovudine-loaded nanoparticles based on lipids which were further modified with the polymer gelatin. Formulation and stability of the modified nanoparticles were analysed from the physico-chemical characterizations. The interactions of nanoparticles with blood components were tested by haemolysis and aggregation studies. The drug content and entrapment efficiencies were assessed by UV analysis. The effect of nanoparticles on protein adsorption was assessed by native polyacrylamide gel electrophoresis (PAGE). In vitro release studies showed a sustained release profile of zidovudine. In vitro cytotoxicity and cellular uptake of the zidovudine-loaded nanoparticles were performed in MCF-7 and neuro 2a brain cells. The enhanced cellular internalization of drug loaded modified nanoparticles in both the cell lines were revealed by fluorescence microscopy. Hence the present study focuses on the feasibility of zidovudine-loaded polymer modified lipid nanoparticles as carriers for safe and efficient HIV/AIDS therapy.

Recent progress in prodrug design strategies based on generally applicable modifications.

The development of prodrugs has progressed with the aim of improving drug bioavailability by overcoming various barriers that reduce drug benefits in clinical use, such as stability, duration, water solubility, side effect profile, and taste. Many conventional drugs act as the precursors of an active agent in vivo; for example, the anti-HIV agent azidothymidine (AZT) is converted into its corresponding active triphosphate ester in the body, meaning that AZT is a prodrug in the broadest sense. However prodrug design is generally difficult owing to the lack of general versatility. Thus, these prodrugs, broadly defined, are often discovered by chance or trial-and-error. Recently, many prodrugs that could release the corresponding parent drugs with or without enzymatic action under physiological conditions have been reported. These prodrugs can be easily designed and synthesized because of their generally applicable modifications. This digest paper provides an overview of recent development in prodrug strategies for drugs with a carboxylic acid or hydroxyl/amino group on the basis of a generally applicable modification strategy, such as esterification, amidation, or benzylation.

Structure and conformational analysis of the anti-HIV reverse transcriptase inhibitor AZT using MP2 and DFT methods. Differences with the natural nucleoside thymidine. Simulation of the 1st phosphorylation step with ATP.

A comprehensive quantum-chemical investigation of the conformational landscape of the HIV-1 reverse transcriptase inhibitor AZT (3'-azido-3'-deoxythymidine) nucleoside analogue was carried out. The whole conformational parameters (χ, γ, ß, δ, ϕ, P, νmax) were analysed as well as the NBO charges. The search located at least 55 stable structures, 9 of which were by MP2 within a 1 kcal mol(-1) electronic energy range of the global minimum. Most conformers were anti or high-anti around the glycoside bond and with North sugar ring puckering angles. The distribution of all the conformers according to the ranges of stability of the characteristic torsional angles was established. The results obtained were in accordance with those found in related anti-HIV nucleoside analogues. The best conformer in the anti form corresponded to the calculated values by MP2 of χ = -126.9°, ß = 176.4° and γ = 49.1°. An analysis of the lowest vibrations in conformer C1 was carried out. The first hydration shell was simulated and the structural differences with the natural nucleoside deoxythymidine (dT) were determined. The first phosphorylation step was simulated by interacting ATP with the best hydrated clusters of AZT and dT. The Na cations act as a bridge between the phosphate moieties of ATP making it easy for -P3O3 to receive the H5' proton from AZT or dT. A proton-transfer mechanism is proposed through the water molecules. When the number of the water molecules surrounding AZT is lower than 8, the first phosphorylation step of AZT can be carried out. However, the appropriate orientation of the O5'-H in dT avoids this limitation and it can be performed with large numbers of water molecules.

Tomato thymidine kinase is subject to inefficient TTP feedback regulation.

A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation.

Hepatic microsomal UDP-glucuronosyltransferase (UGT) activities in the microminipig.

The microminipig, a small minipig, was bred as a novel experimental animal for nonclinical pharmacology/toxicology studies by Fuji Micra Inc. (Shizuoka, Japan). Species differences in drug metabolism between humans and the microminipig need to be elucidated in more detail in order to discuss the results of nonclinical studies. Glucuronidation catalysed by UDP-glucuronosyltransferase (UGT) is an important pathway in the metabolism of a wide variety of compounds. The aim of the present study was to identify the characteristics of hepatic UGT activity in the microminipig and compare them with those in humans and other experimental animals. This study examined in vitro UGT activities using liver microsomes from microminipigs (8 months old and 1 day old), humans, mice, rats, dogs, monkeys and minipigs. The glucuronides of estradiol, imipramine, serotonin, propofol, 3'-azido-3'-deoxythymidine (AZT) and morphine, selective substrates of human UGT1A1, 1A4, 1A6, 1A9 and 2B7 (AZT and morphine), respectively, were measured using LC-MS/MS. Estradiol-3-glucuronidation activity was higher in the microminipig than in humans and the other animals. High levels of estradiol-3-glucuronidation were observed in the microsomes of 1-day-old microminipigs. Imipramine-N-glucuronidation, a distinctive conjugation by human UGT1A4, was catalysed by microminipig liver microsomes, but not by dog liver microsomes. Although AZT-glucuronidation activity was low in the microminipig compared with humans, morphine-3-glucuronidation activity in the microminipig was higher than that in humans. The UGT activities in the microminipig were similar to those in the minipig. The results of the present study have provided useful information for selecting an appropriate animal model for nonclinical studies.

Differential gene expression in human hepatocyte cell lines exposed to the antiretroviral agent zidovudine.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the most widely used nucleoside reverse transcriptase inhibitor for the treatment of AIDS patients and prevention of mother-to-child transmission of HIV-1. Previously, we demonstrated that AZT had significantly greater growth inhibitory effects upon the human liver carcinoma cell line HepG2 as compared to the immortalized human liver cell line THLE2. We have now used gene expression profiling to determine the molecular pathways associated with toxicity in both cell lines. HepG2 cells were incubated with 0, 2, 20, or 100 µM AZT for 2 weeks; THLE2 cells were treated with 0, 50, 500, or 2,500 µM AZT, concentrations that were equi-toxic to those used in the HepG2 cells. After the treatment, total RNA was isolated and subjected to microarray analysis. Global analysis of gene expression, with a false discovery rate ≤0.01 and a fold change ≥1.5, indicated that 6- to 70-fold more genes were differentially expressed in a significant concentration-dependent manner in HepG2 cells when compared to THLE2 cells. Comparative analysis indicated that 7 % of these genes were common to both cell lines. Among the common differentially expressed genes, 70 % changed in the same direction, most of which were associated with cell death and survival, cell cycle, cell growth and proliferation, and DNA replication, recombination, and repair. As determined by the uptake of [methyl-(3)H]AZT, the intracellular levels of total AZT were approximately twofold higher in THLE2 cells than in HepG2 cells. The expression of thymidine kinase 1 (TK1) and UDP-glucuronosyltransferase 2B7 (UGT2B7) genes that regulate the metabolic activation and deactivation of AZT, respectively, was increased in HepG2 cells but decreased in THLE2 cells after treatment with AZT. This differential response in AZT metabolism was confirmed by real-time PCR, western blotting, and/or enzymatic assays. These data indicate that molecular pathways involved with cell death and survival, cell cycle, cell growth and proliferation, and DNA replication, recombination, and repair are involved in the toxicities associated with AZT in both human cell lines, and that the difference in expression of TK1 and UGT2B7 in response to AZT treatment in HepG2 cells and THLE2 cells might explain why HepG2 cells are more sensitive than THLE2 cells to the toxicity of AZT.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

Using human recombinant UDP-glucuronosyltransferase isoforms and a relative activity factor approach to model total body clearance of laropiprant (MK-0524) in humans.

A major pathway of elimination of the prostaglandin D2 receptor 1 antagonist laropiprant in humans is by uridine diphosphate-glucuronosyltransferase (UGT)-mediated biotransformation. In this study, liver and kidney relative activity factors were developed for UGT1A1, 1A9 and 2B7 to allow for in vitro-in vivo extrapolation of intrinsic clearance data to whole organ clearance using recombinant human UGT isoforms applying this to laropiprant as a model substrate. The total body metabolic clearance of laropiprant determined using this approach (5.0 L/hr) agreed well with the value determined in vivo following intravenous administration to healthy human volunteers (5.1 L/hr). The results suggest that approximately 36%, 36% and 28% of the hepatic metabolic clearance of laropiprant was mediated by UGT1A1, 1A9 and 2B7, respectively. Likewise, 80% and 20% of the renal metabolic clearance was mediated by UGT1A9 and 2B7, respectively. Furthermore, the data suggested that the contribution of the kidney to the overall total metabolic clearance was minor relative to the liver (≈ 12%).

Toxicology and carcinogenesis studies of mixtures of 3'-azido-3'-deoxythymidine (AZT), lamivudine (3TC), nevirapine (NVP), and nelfinavir mesylate (NFV) (Cas Nos. 30516-87-1, 134678-17-4, 129618-40-2, 159989-65-8) in B6C3F1 Mice (transplacental exposure studies).

BACKGROUND: Antiretroviral drugs are used to treat patients positive for the human immunovirus HIV-1, and increasingly treatments include a combination of such drugs. The noninfected children of women who are pregnant and receiving such treatment may also be exposed to the drugs by transplacental exposure. We studied the long-term effects of such transplacental exposure in mice by exposing pregnant mice to combinations of four such antiretroviral drugs for seven days and then observing their pups for two years following birth. The four drugs studied were 3'-azido-3'-deoxythymidine (AZT), lamivudine (3TC), nevirapine (NVP), and nelfinavir mesylate (NFV). METHODS: Four different sets of exposure studies were performed: exposure to AZT; to AZT plus 3TC; to AZT, 3TC, and NVP; or to AZT, 3TC, and NFV. In each of these studies, groups of pregnant females were given one of three concentrations of the drug combinations seven times though a tube directly into their stomachs, and after birth their pups were maintained with no further exposure for two years. The offspring of another group of pregnant females not treated with the drugs served as controls. At the end of the study, tissues from more than 40 sites were examined for every animal. RESULTS: Survival of pups whose mothers were exposed to AZT or AZT plus 3TC was similar to their controls, while the survival rates for offspring of mice exposed to AZT, 3TC, and NVP or AZT, 3TC, and NFP were lower than for controls. In most cases the body weights of pups from mothers exposed were slightly less than those of the controls. There were slight increases in the incidences of thyroid gland tumors and skin tumors in the female pups of mothers exposed to AZT alone and of lung tumors in female pups of mothers exposed to AZT plus 3TC. For offspring of mothers exposed to AZT, 3TC, and NVP there were increased incidences of skin tumors in both male and female pups, and more so in the males. CONCLUSIONS: We conclude that exposure to the combination of AZT, 3TC, and NVP during pregnancy caused an increase in skin tumors in the male offspring and possibly also to the female offspring. Exposure to AZT alone during pregnancy may have been related to thyroid gland or skin tumors in female offspring, and exposure to AZT plus 3TC may have been related to lung tumors in female offspring.

A pyrophosphatase activity associated with purified HIV-1 particles.

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

Gossypol exhibits a strong influence towards UDP-glucuronosyltransferase (UGT) 1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances.

Gossypol, the polyphenolic constituent isolated from cottonseeds, has been used as a male antifertility drug for a long time, and has been demonstrated to exhibit excellent anti-tumor activity towards multiple cancer types. The toxic effects of gossypol limit its clinical utilization, and enzyme inhibition is an important facet of this. In the present study, in vitro human liver microsomal incubation system supplemented with UDPGA was used to investigate the inhibition of gossypol towards UGT1A1, 1A9 and 2B7-mediated metabolism of xenobiotics and endogenous substances. Estradiol, the probe substrate of UGT1A1, was selected as representative endogenous substance. Propofol (a probe substrate of UGT1A9) and 3'-azido-3'-deoxythimidine (AZT, a probe substrate of UGT2B7) were employed as representative xenobiotics. The results showed that gossypol noncompetitively inhibits UGT-mediated estradiol-3-glucuronidation and propofol O-glucuronidation, and the inhibition kinetic parameters (K(i)) were calculated to be 34.2 and 16.4 µM, respectively. Gossypol was demonstrated to exhibit competitive inhibition towards UGT-mediated AZT glucuronidation, and the inhibition kinetic parameter (K(i)) was determined to be 14.0 µM. All these results indicated that gossypol might induce metabolic disorders of endogenous substances and alteration of metabolic behaviour of co-administered xenobiotics through inhibition of UGTs' activity.

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position -7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the -7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the -7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1.

The first step for the intracellular retention of several anticancer or antiviral nucleoside analogues is the addition of a phosphate group catalysed by a deoxyribonucleoside kinase such as thymidine kinase 1 (TK1). Recently, human TK1 (HuTK1) has been crystallized and characterized using different ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT) compared with the natural substrate thymidine. The crystal structure of the T163S-mutated HuTK1 reveals a less ordered conformation of the ligand thymidine triphosphate compared with the wild-type structure but the cause of the changed specificity towards AZT is not obvious. Based on its highly increased AZT activity relative to thymidine activity this TK1 mutant could be suitable for suicide gene therapy.

Mechanism of nucleoside uptake in rat placenta and induction of placental CNT2 in experimental diabetes.

The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [(3)H]adenosine and [(3)H]zidovudine from maternal blood was much higher than that of [(14)C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [(3)H]adenosine with K(m) values of 6.1 and 26 µM, respectively. [(3)H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [(3)H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 µM NBMPR on [(3)H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)-induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZ-induced diabetic rats. Consistently, Na(+)-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus.

Structures of reverse transcriptase pre- and post-excision complexes shed new light on HIV-1 AZT resistance.

HIV-1 resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) results from mutations in reverse transcriptase that increase the ability of the enzyme to excise AZT-monophosphate after it has been incorporated. Crystal structures of complexes of wild type and mutant reverse transcriptase with double-stranded DNA with or without the excision product, AZT adenosine dinucleoside tetraphosphate (AZTppppA), have recently been reported. The excision-enhancing mutations dramatically change the way the enzyme interacts with the excision product.

Determinants of individual variation in intracellular accumulation of anti-HIV nucleoside analog metabolites.

Individual variation in response to antiretroviral therapy is well-known, but it is not clear if demographic characteristics such as gender, age, and ethnicity are responsible for the variation. To optimize anti-HIV therapy and guide antiretroviral drug discovery, determinants that cause variable responses to therapy need to be evaluated. We investigated the determinants of intracellular concentrations of nucleoside analogs using peripheral blood mononuclear cells from 40 healthy donors. We observed individual differences in the concentrations of the intracellular nucleoside analogs; the mean concentrations of the triphosphate metabolite of ethynylstavudine (4'-Ed4T), zidovudine (AZT), and lamivudine (3TC) were 0.71 pmol/10(6) cells (minimum and maximum, 0.10 and 3.00 pmol/10(6) cells, respectively), 0.88 pmol/10(6) cells (minimum and maximum, 0.10 and 15.18 pmol/10(6) cells, respectively), and 1.70 pmol/10(6) cells (minimum and maximum, 0.20 and 7.73 pmol/10(6) cells, respectively). Gender and ethnicity had no effect on the concentration of 4'-Ed4T and 3TC metabolites. There was a trend for moderation of the concentrations of AZT metabolites by gender (P = 0.17 for gender·metabolite concentration). We observed variability in the activity and expression of cellular kinases. There was no statistically significant correlation between thymidine kinase 1 (TK-1) activity or expression and thymidine analog metabolite concentrations. The correlation between the activity of deoxycytidine kinase (dCK) and the 3TC monophosphate metabolite concentration showed a trend toward significance (P = 0.1). We observed an inverse correlation between the multidrug-resistant protein 2 (MRP2) expression index and the concentrations of AZT monophosphate, AZT triphosphate, and total AZT metabolites. Our findings suggest that the observed variation in clinical response to nucleoside analogs may be due partly to the individual differences in the intracellular concentrations, which in turn may be affected by the cellular kinases involved in the phosphorylation pathway and ATP-binding cassette (ABC) transport proteins.