John R. Mercer

Biodistribution and Uptake of 3′-Deoxy-3′-Fluorothymidine in ENT1-Knockout Mice and in an ENT1-Knockdown Tumor Model

18F-3′-Deoxy-3′-fluorothymidine (18F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for 18F-FLT uptake in normal tissues and tumors. Methods: ENT1-knockout (ENT1−/−) mice were generated and compared with wild-type (ENT1+/+) mice using small-animal 18F-FLT PET. In addition, ENT1+/+ mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by 18F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography–mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and 18F-FLT uptake using real-time polymerase chain reaction and 3H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by 18F-FLT small-animal PET. Results: Compared with noninjected ENT1+/+ mice, ENT1+/+ mice injected with NBMPR-P and ENT1−/− mice displayed a reduced percentage injected dose per gram (%ID/g) for 18F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for 18F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1−/− mice displayed 1.65-fold greater plasma thymidine levels than did ENT1+/+ mice. Spleen tissue from ENT1+/+ and ENT1−/− mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold 3H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo γ-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for 18F-FLT uptake at 1 h after tracer injection. Conclusion: Loss of ENT1 activity significantly affected 18F-FLT biodistribution in mice and 18F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of 18F-FLT uptake in normal and transformed cells.

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.

Radiopharmacological evaluation of 6-deoxy-6-[18F]fluoro-D-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer.

INTRODUCTION Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. METHODS Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine. RESULTS 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo. CONCLUSION Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.

Preliminary Results of Nanopharmaceuticals Used in the Radioimmunotherapy of Ovarian Cancer

A multistep radioimmunotherapeutic (RIT) approach, exploiting the combination of a bispecific monoclonal antibody (BsMAb) with 90Y labelled biotinylated long-circulating liposomes was tested as a potential adjuvant treatment for epithelial ovarian carcinomatosis. The BsMAb, with anti-CA 125 and anti-biotin epitopes was used with PEGylated liposomes coated with biotin to deliver the cytotoxic radionuclide 90Y to tumor sites. This approach was used to overcome some of the major obstacles associated with conventional strategies, in particular, to increase the amount of radioactivity delivered to the tumor site compared with conventional monoclonal antibody (MAb) radionuclide delivery. Sequential intraperitoneal administration of the targeting and therapeutic moieties provides the basis for enhanced therapeutic ratio, according to our strategy. We report here the results of an in vivo therapy using our RIT approach with the Balb/c nude mouse model xenografted with the NIH:OVCAR-3 (CA 125+) human ovarian cancer cell line. An ongoing tumor growth delay/control study in Balb/c mice xenografted intraperitoneally with the NIH:OVCAR-3 cell line indicates a significant delay in onset of tumor and ascites development in treated vs. control populations.

Inhibition of insulin metabolism by hydroxychloroquine and its enantiomers in cytosolic fraction of liver homogenates from healthy and diabetic rats.

To elucidate the mechanism by which hydroxychloroquine (HCQ) affects glucose metabolism, the effect of this drug and its enantiomers on insulin metabolism was studied using the cytosolic fraction of liver homogenates from healthy and diabetic rats. Eadie-Hofstee plots were monophasic suggesting that only a one-component enzyme system is involved in insulin degradation in the fraction used. Reaction velocity (V) vs substrate concentration plots were consistent with a Vmax model. HCQ caused a significant reduction in Vmax and Vmax/Km values in both healthy (Vmax, 3.63 +/- 0.46 vs 1.97 +/- 0.13, ng/min/mg; protein P < 0.001; and Vmax/Km 0.265 +/- 0.015 vs 0.112 +/- 0.004, ml/min/g protein) and diabetic rats (Vmax, 0.718 +/- 0.06 vs 0.360 +/- 0.024, ng/min/mg protein; and Vmax/Km, 0.05 +/- 0.002 vs 0.023 +/- 0.001, ml/min/g protein). Significant reduction in the V was observed in the presence of racemic (rac)-, R-, or S-HCQ. Ranking of the inhibitory potency was HCQ > S = R except at highest examined concentration (20 mg/mL) which was HCQ > S > R. In conclusion, the effect of HCQ on insulin degradation appears to be, in part, through inhibition of cytosolic insulin metabolizing enzyme. The effect is not stereoselective except at high concentrations. The R- and S-HCQ may have synergistic effects on inhibition of insulin degradation.

An evaluation of iodine-123 iodoazomycinarabinoside as a marker of localized tissue hypoxia in patients with diabetes mellitus

Peripheral vascular disease is a serious and common complication in patients with diabetes mellitus (DM). Evaluation is, conventionally, by transcutaneous oxygen tension measurements (TcpO2), although this technique has some limitations in the evaluation of tissue viability. We have evaluated a new, radiolabelled, in vivo marker of tissue hypoxia, iodoazomycinarabino-side (IAZA), by comparing TcpO2 measurements with patterns of iodine-123 IAZA uptake in ten patients (19 lower limbs) with DM and peripheral vascular disease using conventional gamma camera imaging techniques. Normal uptake patterns were seen in limbs in which normal TcpO2 measurements were obtained. Diffusely increased uptake of [123I]IAZA was seen in limbs with reduced TcpO2 Focally increased uptake was seen in ulcers or in areas of atrophic skin change. A semiquantitative measure showed an inverse correlation between [123I]IAZA and TcpO2 values. These data suggest that tissue hypoxia can be imaged in this population of patients and that severity of disease can be assessed. A longitudinal prospective trial is now being developed.

Synthesis and application of 4-[18F]fluorobenzylamine: A versatile building block for the preparation of PET radiotracers

A novel synthesis of 4-[(18)F]fluorobenzylamine ([(18)F]FBA) by means of transition metal-assisted sodium borohydride reduction of 4-[(18)F]fluorobenzonitrile ([(18)F]FBN) is described. This approach could successfully be extended to borohydride exchange resin (BER) enabling a viable option for use in automated syntheses. [(18)F]FBA was used for the synthesis of 4-[(18)F]fluorobenzylamine-based thiol group-reactive prosthetic groups 4-[(18)F]fluorobenzyl-2-bromoacetamide ([(18)F]FBBA) and 4-[(18)F]fluorobenzylamidopropionyl maleimide ([(18)F]FBAPM). [(18)F]FBBA and [(18)F]FBAPM were obtained in radiochemical yields of 75% and 55%, respectively. Feasibility of using [(18)F]FBAPM as novel prosthetic group for peptide and protein labelling was demonstrated with cysteine-containing tripeptide glutathione (GSH). [(18)F]FBBA was used for labelling of a fully phosphorothioated 20mer oligodesoxynucleotide (ODN).

Radiolabeling of phosphatidylserine-binding peptides with prosthetic groups N-[6-(4-[18F]fluorobenzylidene)aminooxyhexyl]maleimide ([18F]FBAM) and N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB).

The widely used (18)F-prosthetic group N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) and the recently developed N-[6-(4-[(18)F]fluorobenzylidene)aminooxyhexyl]maleimide ([(18)F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reactions conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [(18)F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [(18)F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [(18)F]FBAM. Results indicate that for the peptides in this study, [(18)F]FBAM is a more useful prosthetic group compared to [(18)F]SFB due to its excellent chemoselectivity and high radiochemical yield.

Entropic Fragment-Based Approach to Aptamer Design: Entropic Fragment-Based Approach

Aptamers are short RNA/DNA sequences that are identified through the process of systematic evolution of ligands by exponential enrichment and that bind to diverse biomolecular targets. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology. They are recognized as having potential therapeutic and diagnostic clinical applications. The success of the systematic evolution of ligands by exponential enrichment process requires that the RNA/DNA pools used in the process have a sufficient level of sequence diversity and structural complexity. While the systematic evolution of ligands by exponential enrichment technology is well developed, it remains a challenge in the efficient identification of correct aptamers. In this article, we propose a novel information‐driven approach to a theoretical design of aptamer templates based solely on the knowledge regarding the biomolecular target structures. We have investigated both theoretically and experimentally the applicability of the proposed approach by considering two specific targets: the serum protein thrombin and the cell membrane phospholipid phosphatidylserine. Both of these case studies support our method and indicate a promising advancement in theoretical aptamer design. In unfavorable cases where the designed sequences show weak binding affinity, these template sequences can be still modified to enhance their affinities without going through the systematic evolution of ligands by exponential enrichment process.

Detecting functional changes with [(18)F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy.

BackgroundThe multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [18F]FAZA.MethodsThe present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells.ResultsDuring therapy with sunitinib (40 mg/kg/day), uptake of [18F]FAZA into Caki-1 mice decreased by 46±5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22±5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumors hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [18F]FAZA uptake increased to 126±6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [18F]FAZA uptake into Caki-1 cells in vitro.Conclusion[18F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumors hypoxia status.