Leonard I. Wiebe

[18F]Fluoroazomycinarabinofuranoside (18FAZA) and [18F]Fluoromisonidazole (18FMISO): a comparative study of their selective uptake in hypoxic cells and PET imaging in experimental rat tumors

The present study compares the uptake of [(18)F]Fluoroazomycinarabinofuranoside ((18)FAZA), a recently developed hypoxia tracer for PET imaging of tissue hypoxia, with an established tracer [(18)F]Fluoromisonidazole ((18)FMISO) both in vitro, using Walker 256 rat carcinosarcoma cells, and in vivo in experimental rat tumors eleven to twelve days after tumor cell implantation. In vitro studies indicated that hypoxia-selective uptake of both (18)FAZA and (18)FMISO in tumor cells, 20 and 100 minutes post-incubation was of the same magnitude (20 min: 1.24 +/- 0.4% ((18)FAZA); 1.19 +/- 0.7% ((18)FMISO); 100 min: 3.6 +/- 1.6% ((18)FAZA); 3.3 +/- 1.7% ((18)FMISO)). PET imaging reflected a similar radiotracer distribution in rat tumors for (18)FAZA and (18)FMISO one h after radiotracer injection. The concentration of (18)FAZA in the tumors as measured by PET, however, was lower in comparison to (18)FMISO (SUV(FAZA) = 0.61 +/- 0.2 vs. SUV(FMISO) = 0.92 +/- 0.3, p < 0.05) although the tumor to muscle ratios for (18)FAZA and (18)FMISO did not differ in the PET images that were obtained after one h (SUV(FAZA) = 2.5 +/- 0.5 vs. SUV(FMISO) = 2.9 +/- 0.7). A comparison of PET data three h post-injection (SUV(FAZA) = 3.0 +/- 0.5 vs. SUV(FMISO) = 4.6 +/- 1.8, p < 0.05) demonstrated a lower (18)FAZA uptake that indicates a lower sensitivity of (18)FAZA in comparison to (18)FMISO in detecting hypoxic regions at a longer time in this animal model. However, these data also show a faster elimination of (18)FAZA from blood, viscera and muscle tissue, via the renal system. This advantage of a faster reduction of unspecific binding, in light of similar or marginally lower tumor uptake, warrants further investigation of (18)FAZA as a marker of regional hypoxia in tumors.

Electrophysiological characterization of a recombinant human Na+-coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

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 Km 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 uridine‐evoked 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 [14C]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 × 1010 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

Targeted Delivery of Macromolecular Drugs: Asialoglycoprotein Receptor (ASGPR) Expression by Selected Hepatoma Cell Lines used in Antiviral Drug Development

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.

Fluoroazomycin arabinoside (FAZA): synthesis, 2H and 3H‐labelling and preliminary biological evaluation of a novel 2‐nitroimidazole marker of tissue hypoxia

1-α-D-(5-Fluoro-5-deoxyarabinofuranosyl)-2-nitroimidazole (fluoroazomycin arabinoside, FAZA) 6, a putative PET imaging agent when labelled with 18F, was synthesized by fluorination of 1-α-D-(2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole 3 with DAST followed by deprotection. The C-5′-deuterated and tritiated analogues were prepared by NaCNBD3 or NaCNBT3 reduction of the protected C-5′-carbonyl intermediate 5, followed by C-5′ fluorination and deprotection, to afford C-5′ deuterated and C-5′ tritiated FAZA, respectively. Preliminary in vivo biodistribution studies in a murine tumour model, and pharmacokinetic studies in rats indicated that 3H-FAZA has biodistribution, tumour uptake and pharmacokinetic properties similar to those of 123I-IAZA, a clinically-proven radiopharmaceutical for SPECT-imaging of hypoxic tissues. Copyright

Asialoglycoprotein receptor-targeted superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide (SPIO) nanoparticles are primarily used as contrast agents in magnetic resonance imaging. SPIO have also been derivatized to add targeting and drug-carrier functionality as drug delivery devices. The preparation and characterization of amino-functionalized SPIO (ASPIO) and lactose-derivatized galactose-terminal-ASPIO are now reported. The target for galactose-terminal-ASPIO is the cell-surface asialoglycoprotein receptor (ASGPR) expressed by hepatocytes. Two batches of ASPIO with average particle sizes of 61 [42]nm and 127 [125]nm [full-width half maximum; FWHM] were prepared. The small ASPIO increased from 61 nm to 278 [309]nm upon lactosylation (Gal-ASPIO-278) and to 302 [280] by N-acetylation (NAcASPIO-302); the larger ASPIO afforded galactosyl-terminal ASPIO of 337 [372]nm and N-acetylated ASPIO of 326 [308]nm. The LD50 of Gal-ASPIO-278 was 1500 microg/mL to HepG2 cells; Gal-ASPIO-278 associated with HepG2 cells in vitro, whereas NAcSPIO-302, prepared from the same ASPIO batch, did not. Gal-ASPIO-278 and NAcASPIO-302 were not bound by ASPGR non-expressing 143B cells. The association of Gal-ASPIO-278 to HepG2 cells was reduced by free galactose, supporting the model of ASGPR-mediated binding. These data underline the potential application of Gal-ASPIO as a targeted ligand for ASPGR-expressing cells in vivo.

Non-invasive scintigraphic monitoring of gene expression in a HSV-1 thymidine kinase gene therapy model.

In vivo transduction of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene and subsequent administration of antiviral drugs such as ganciclovir has emerged as a promising gene therapy protocol for proliferative disorders. However, the evaluation of HSV-1 TK gene expression in transduced tissue has relied on invasive techniques for detection. We now report that HSV-1 TK expression can be detected non-invasively using scintigraphy. The radioiodinated nucleoside analogue, (E)-5-(2-iodovinyl)-2‘-fluoro-2’-deoxyuridine (IVFRU), becomes metabolically trapped in tumour cells transduced with the HSV-1 TK gene on a retroviral vector. Selective phosphorylation of radiolabelled IVFRU by HSV-1 TK results in elevated radioactivity in HSV-1 TK-expressing cells in vitro and in vivo relative to cells lacking the HSV-1 TK gene. Due to low non-target tissue uptake, unambiguous imaging of HSV-1 TK-expressing tumours in mice is possible with labelled IVFRU. We have monitored the process of tumour regression non-invasively during ganciclovir treatment using labelled IVFRU and scintigraphy.

In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses.

A group of myristic acid analogs, designed as alternative substrates for N-myristoyltransferase (NMT), were evaluated against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in vitro. Antiviral potency was increased when S or O was substituted for -CH2- in myristic acid and selectivity was affected by the presence and position of the heteroatoms and phenyl groups. A correlation was established among anti-HIV activity, Log P and Log D7.4 and between anti-HIV activity and carbonyl-heteroatom interatomic distances in the myristoyl analogs. 12-Thioethyldodecanoic acid 6 was moderately active (EC50 = 9.37 microM) against HIV-infected T4-lymphocytes (CEM-SS cell line), and it exhibited in vitro activity (EC50 = 17.8 microM) against HBV-producing 2.2.15 cell cultures derived from a human hepatoblastoma cell line (Hep G2). 12-Methoxydodecanoic acid 1 exhibited in vitro activity (EC50 = 20-30 microM) against hepatitis B in the HBV DNA-transfected 2.2.15 cell line. At a concentration of 10 microg/ml, none of the fatty acids significantly inhibited the replication of DHBV in infected hepatocytes.

Varicella-zoster virus thymidine kinase gene and antiherpetic pyrimidine nucleoside analogues in a combined gene/chemotherapy treatment for cancer

Ten pyrimidine nucleoside analogues, including (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5′ phosphotransferase, EC 2.7.2.21) gene. (E)-5-(2-bromovinyl)-1-β- D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2′-deoxy-2′-fluoro-1-β- D-arabinofurano- syluracil (IVFAU) and (E)-5-(2-bromovinyl)-2′-deoxy-4′-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. Inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-β- D-arabinofuranosylpurine (araM), and at least five- to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.

Synthesis and In vivo studies of the radiosensitizer 4-[82Br]bromomisonidazole

[82Br]Misonidazole ( [82Br]MISO) was investigated as a potential agent for scintillation imaging and indexing of hypoxic areas in tumor masses. [82Br]MISO was prepared by irradiating samples of Br-MISO in a SLOWPOKE reactor for 2 h at a thermal neutron flux of 10(12) n cm-2 s-1. Radiochemical yields ranged from 11 to 16%. [82Br]MISO had a plasma clearance half-life of approximately 40 min in BALB/c mice bearing EMT-6 tumors. The major metabolic product was identified as [82Br]Desmisonidazole. [82Br]MISO showed relatively high tumor uptake and persistently high plasma levels.

Synthesis of hypoxia imaging agent 1-(5-deoxy-5-fluoro-α-D-arabinofuranosyl)-2-nitroimidazole using microfluidic technology.

INTRODUCTION Microfluidic technology allows fast reactions in a simple experimental setup, while using very low volumes and amounts of starting material. Consequently, microfluidic technology is an ideal tool for radiolabeling reactions involving short-lived positron emitters. Optimization of the complex array of different reaction conditions requires knowledge of the different reaction parameters linked to the microfluidic system as well as their influence on the radiochemical yields. 1-(5-Deoxy-5-fluoro-α-d-arabinofuranosyl)-2-nitroimidazole ([(18)F]FAZA) is a frequently used radiotracer for PET imaging of tumor hypoxia. The present study describes the radiosynthesis of [(18)F]FAZA by means of microfluidic technology and subsequent small animal PET imaging in EMT-6 tumor-bearing mice. METHODS Radiosyntheses were performed using the NanoTek Microfluidic Synthesis System (Advion BioSciences, Inc.). Optimal reaction conditions were studied through screening different reaction parameters like temperature, flow rate, residency time, concentration of the labeling precursor (1-(2,3-di-O-acetyl-5-O-tosyl-α-d-arabinofuranosyl)-2-nitroimidazole) and the applied volume ratio between the labeling precursor and [(18)F]fluoride. RESULTS Optimized reaction conditions at low radioactivity levels (1 to 50 MBq) afforded 63% (decay-corrected) of HPLC-purified [(18)F]FAZA within 25 min. Higher radioactivity levels (0.4 to 2.1 GBq) gave HPLC-purified [(18)F]FAZA in radiochemical yields of 40% (decay-corrected) within 60 min at a specific activity in the range of 70 to 150 GBq/μmol. Small animal PET studies in EMT-6 tumor-bearing mice showed radioactivity accumulation in the tumor (SUV(20min) 0.74 ± 0.08) resulting in an increasing tumor-to-muscle ratio over time. CONCLUSIONS Microfluidic technology is an ideal method for the rapid and efficient radiosynthesis of [(18)F]FAZA for preclinical radiopharmacological studies. Careful analysis of various reaction parameters is an important requirement for the understanding of the influence of different reaction parameters on the radiochemical yield using microfluidic technology. Exploration of microfluidic technology for the radiosynthesis of other PET radiotracers in clinically relevant radioactivity levels is currently in progress.