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Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na + down its electrochemical gradient. The two-microelectrode voltage-clamp technique was used to measure steady-state and presteady-state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na + currents included the anticancer drugs 5-fluorouridine, 5-fluoro-2 -deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent K m values for 5-fluorouridine, 5-fluoro-2 -deoxyuridine and zidovudine were 18, 15 and 450 µM, respectively. hCNT1 was Na + specific, and the kinetics of steady-state uridineevoked Na + currents were consistent with an ordered simultaneous transport model in which Na + binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na + -nucleoside coupling stoichiometry, determined directly by comparing the uridine-induced inward charge movement to [ 14 C]uridine uptake was 1 : 1. hCNT1 presteady-state currents were used to determine the fraction of the membrane field sensed by Na + (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 10 10 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

This study suggests that (18)F-FAZA may be a very useful radiopharmaceutical to image hypoxia in the tumour types selected. Especially the high uptake by gliomas was encouraging. Given the good imaging properties, including acceptable T/B ratios in the tumour categories studied, (18)F-FAZA could be considered as a very promising agent for assessing the hypoxic fraction of these tumour types.

3'-Azido-2',3'-dideoxythymidine (AZT, 1, zidovudine, RetrovirTM) is used to treat patients with human immunodeficiency virus (HIV) infection. AZT, after conversion to AZT-5'-triphosphate (AZT-TP) by cellular enzymes, inhibits HIV-reverse transcriptase (HIV-RT). The major clinical limitations of AZT are due to clinical toxicities that include bone marrow suppression, hepatic abnormalities and myopathy, absolute dependence on host cell kinase-mediated activation which leads to low activity, limited brain uptake, a short half-life of about one hour in plasma that dictates frequent administration to maintain therapeutic drug levels, low potential for metabolic activation and/or high susceptibility to catabolism, and the rapid development of resistance by HIV-1. These limitations have prompted the development of strategies for designing prodrugs of AZT. A variety of 5'-O-substituted prodrugs of AZT constitute the subject of this review. The drug-design rationale on which these approaches are based is that the ester conjugate will be converted by hydrolysis and/or enzymatic cleavage to AZT or its 5′-monophosphate (AZT-MP). Most prodrug derivatives of AZT have been prepared by derivatization of AZT at its 5'-O position to provide two prominent classes of compounds that encompass: A) 5'-O-carboxylic esters derived from 1) cyclic 5'-O-carboxylic acids such as steroidal 17b-carboxylic acids, 1-adamantanecarboxylic acid, bicyclam carboxylic acid derivatives, O-acetylsalicylic acid, and carbohydrate derivatives, 2) amino acids, 3) 1, 4-dihydro-1-methyl-3-pyridinylcarboxylic acid, 4) aliphatic fatty acid analogs such as myristic acid containing a heteroatom, or without a heteroatom such as stearic acid, and 5) long chain polyunsaturated fatty acid analogs such as retinoic acid, and B) masked phosphates such as 1) phosphodiesters that include monoalkyl or monoaryl phosphate, carbohydrate, ether lipid, ester lipid, and foscarnet derivatives, 2) a variety of phosphotriesters that include dialkylphosphotriesters, diarylphosphotriesters, glycolate and lactate phosphotriesters, phosphotriester approaches using simultaneous enzymatic and chemical hydrolysis of bis(4-acyloxybenzyl) esters, bis(S-acyl-2-thioethyl) (SATE) esters, cyclosaligenyl prodrugs, glycosyl phosphotriesters, and steroidal phosphotriesters, 3) phosphoramidate derivatives, 4) dinucleoside phosphate derivatives that possess a second anti-HIV moiety such as AZT-P-ddA, AZT-P-ddI, AZTP2AZT, AZTP2ACV), and 5) 5'-hydrogen phosphonate and 5'-methylene phosphonate derivatives of AZT. In these prodrugs, the conjugating moiety is linked to AZT via a 5'-O-ester or 5'-O-phosphate group. 5'-O-Substituted AZT prodrugs have been designed with the objectives of improving anti-HIV activity, enhancing blood-brain barrier penetration, modifying pharmacokinetic properties to increase plasma half-life and improving drug delivery with respect to site-specific targeting or drug localization. Bypassing the first phosphorylation step, regulating transport and conferring sustained rel...

The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.