No / The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.

Positron emission tomography (PET) tracer 3-fluoro-3-deoxythymidine (FLT) is used for imaging tumor proliferation. Prior to this work, human equilibrative nucleoside transporter 1 (hENT1) was the only known human nucleoside transporter (hNT) capable of FLT transport. The aim of this research was to determine if other hNTs, including hENT2, human concentrative nucleoside transporter 1 (hCNT1), hCNT2 and hCNT3, were capable/important of/for FLT transport in mammalian cells. Transport assays performed in Xenopus laevis oocytes producing recombinant hNTs demonstrated that hENT1/2 and hCNT1/3 were capable of FLT transport. FLT uptake assays with or without hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) in various cultured cancer cell lines demonstrated that hENT1 was responsible for the majority of mediated FLT uptake in all tested cell lines, suggesting that hENT1 was important for FLT uptake. The in vivo role of hENT1 in FLT uptake was determined by performing [18F]FLT PET on wild-type and ENT1 knockout mice. One hour after [18F]FLT injection, ENT1 knockout mice displayed significantly reduced [18F]FLT accumulation in the blood, heart, brain, kidney, liver, and lungs compared to wild-type mice. Interestingly, ENT1 knockout mice displayed increased [18F]FLT accumulation in the bone marrow and spleen which both have high CNT expression, suggesting that loss of ENT1 significantly alters FLT biodistribution in mice. hENT1 is a predictive marker of gemcitabine response in pancreatic cancers. Since FLT uptake and gemcitabine toxicity are dependent on hENT1, FLT uptake may predict gemcitabine response in pancreatic cancers. To test this hypothesis, six different pancreatic cancer cell lines were analyzed for FLT uptake and gemcitabine toxicity. hENT1/2 inhibition in cells decreased FLT uptake and gemcitabine sensitivity. In five of six cell lines, a positive correlation was observed between FLT uptake and gemcitabine toxicity, suggesting that FLT PET may be clinically useful for predicting gemcitabine response in pancreatic cancers. The results from this research suggest that hNTs, especially hENT1, are important for FLT uptake in mammalian cells and that FLT uptake can predict gemcitabine response in most cultured pancreatic cancer cells. The results warrant FLT PET clinical trials in pancreatic cancer patients to determine the potential of FLT PET in predicting gemcitabine response.

No description available.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...

The nasal route has primarily been used to deliver drugs for the treatment of local diseases such as nasal infections, nasal congestion and allergies. The nasal route can also be used as a non-invasive alternative route to deliver drugs systemically when a rapid onset of action and/or avoidance of hepatic metabolism are desired. Moreover, there is a growing interest in the use of this route for direct transport of drugs from the nose to the brain. Most of the drugs that have been studied for nasal delivery are either small molecules which are lipophilic enough to passively diffuse through the nasal epithelia or macromolecules where bioavailabilities less than 1% are clinically effective and acceptable. This study focused on identifying carrier proteins or transporters in the nasal mucosa that could improve the absorption of specific drug substrates across the nasal respiratory and olfactory epithelia. The presence of drug transporters in the nasal mucosa of humans and commonly used animal models were investigated. DNA microarray results for nasal samples from humans and two commonly used models, mice and rats, were obtained from GenBank and were analyzed in collaboration with the University of Iowa Center for Bioinformatics and Computational Biology. While cow tissues are frequently used in in-vitro nasal permeability analyses, there is limited information available in GenBank for this species. Both DNA microarray analysis and RT-PCR were performed on bovine nasal explants to determine transporter expression. Good agreement between the microarray and RT-PCR results was observed. While human and three animal species commonly used as models in nasal drug delivery research (mouse, rat, and cow) show similar patterns of expression for several transporters, interspecies differences in the level of expression were observed. Therefore, the expression level of transporters remains a factor to consider when translating results obtained using animal models to humans. The nucleoside transporter family was selected for further evaluation of the potential to improve the nasal absorption of substrates. Nucleoside transporters are integral proteins responsible for mediating and facilitating the flux of nucleosides across cellular membranes; they are also known to be responsible for the uptake of nucleoside analog drugs such as anti-cancer and anti-viral agents. RT-PCR and Western blotting were used to verify the presence of two transporter subtypes, ENT1 and CNT3, in the bovine nasal respiratory and olfactory mucosa. The expression level of both transporters in the respiratory mucosa was comparable to that in the olfactory mucosa. Using immunohistochemistry, ENT1 and CNT3 were found to be localized primarily at the apical surface of the nasal epithelial cells. This indicates that the nasal epithelium likely absorbs exogenous nucleosides for intracellular uses such as nucleic acid synthesis and regulating other cellular activities. The contribution of the nucleoside transporters to the permeation of a nucleoside analogue drug, alovudine, across the nasal epithelia was also studied. The transport of alovudine showed a non-linear increase with increasing donor concentration over the range of 50 to 3000 µM which suggests that nucleoside transporters play a role in its uptake. Polarized transport was not observed suggesting that the facilitative nature of ENT1 plays a major role in alovudine transport. S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an ENT1 inhibitor, incompletely decreased alovudine permeability across the nasal mucosa. This demonstrates that at least one transporter, ENT, plays a significant role in the uptake of this nucleoside drug across the nasal mucosa.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Šárka Strachoňová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Studium of gene regulation of nucleoside transporters in BeWo cell line Nucleoside transporters (NTs) localized in syncytiotrophoblast control placental uptake of nucleosides. Dysregulation of NTs can disrupt nucleoside homeostasis with a negative consequences on placental and fetal development and can lead to a change in placental pharmacokinetics of nucleoside-derived drugs. Therefore, understanding the expression and function of NTs is necessary for effective and safe pharmacotherapy during pregnancy. The aim of this diploma thesis was to study the adenylate cyclase (AC) activated regulatory pathways of gene expression of concentrative nukleoside transporter 2 (CNT2). For this purpose, qRT-PCR and in vitro accumulation assays using the model substrate [3 H]-adenosine were employed. The human placental choriocarcinoma-derived BeWo cell line has been exposed to an AC activator, forskolin (50 µM), and/or inhibitors of AC/cAMP/PKA, AC/cAMP/MAPK (MEK1/2, p38 MAPK) signaling pathways, PKA inhibitor, KT 5720 (5 μM), an inhibitor of MEK1/2, U0126 (10 μM) and an inhibitor of p38 MAPK, SB202190 (10 μM). The...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Křečková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Role of drug transporters in placental transfer of entecavir Entecavir (ETV), an analogue of guanosine, is a highly efficient anti-hepatitis B antiviral drug. It is the first-line therapy for both adults and children. Its use in pregnancy is limited due to a number of factors, including lack of data on placental pharmacokinetics. The placental transition of drugs is frequently controlled by drug transporters. ATP-binding (ABC) transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) or multidrug resistance-associated protein 2 (MRP2) localized in the apical membrane of syncytiotrophoblast and pumping their substrates in the feto-maternal direction belong to most significant determinants of placental pharmacokinetics. Moreover placental transport of nucleoside-derived drugs can be affected by the activity of nucleoside transporters (NTs); equilibrative nucleoside transporters (ENTs) mediate facilitated diffussion, while the concentrative nucleoside transporters (CNTs) control active influx of their substrates. The aim of the diploma thesis was to describe the role of P-gp, BCRP, MRP2 and NTs (ENTs and...