Peptide nucleic acid as radiopharmaceutical for gene-specific…

Abstracts of the AMI Annual Meeting 2004 107

injected with various dyes show separation of endogenous and exogenous fluorescence. The capability of separating two fluorophores with overlapped emission spectra and the high sensitivity to variations in local chemical properties around a fluorophore will advance molecular imaging research. No. 144 IMAGING OF TUMOR HYPOXIA IN MICE USING 18F-FLUOROAZAMYCIN ARABINOSIDE AND A GERMANIUM ORTHOSILICATE BASED SMALL ANIMAL POSITRON EMISSION TOMOGRAPHY SCANNER D. Binns, D. Dorow, B. Solomon, P. Roselt, C. Cullinane, R. Hicks Peter MacCallum Cancer Centre, East Melbourne, AUSTRALIA. A prototype germanium orthosilicate (GSO) Small Animal positron emission tomography (PET) Scanner (Phillips MOSAIC) was recently installed with the aim of studying specific biological alterations in experimental tumor models in vivo. This 3-D scanner, with a resolution of approximately 2mm, features a 12cm axial field-of-view, which increases count sensitivity and permits dynamic and simultaneous imaging of an entire mouse. Aim: To evaluate preliminary data assessing suitability of this system for tracers with low and/or delayed tumoral uptake, such as those encountered by hypoxic imaging with 18Fluorinated ligands. Method: Twelve mice were implanted with human A431 squamous-cells subcutaneously in the right scapular region. Tumors of between 200 and 600 mm3 developed after 14 days. These mice were injected with 20-40 MBq of 18F-fluoroazamycin arabinoside (FAZA). Following an uptake time of between three and six hours the mice were anesthetized with isofluorane and scanned for 15 minutes. Dynamic and delayed imaging between five and 360 minutes after administration was performed in selected mice to assess optimal uptake time. Results: There was excellent visualization with FAZA in all tumors , despite high uptake in the gut and urinary system. Tumor uptake and soft tissue clearance of FAZA was expectedly delayed, with tumors best visualized at three hours and beyond. Scanner sensitivity was sufficient to allow adequate counts to be acquired in 15 minutes, at times beyond four hours despite very low regional activity. Conclusion: The performance of this high sensitivity scanner should make it an excellent tool in the in vivo assessment of hypoxia in human tumor xenograft models. No. 145 ASSESSMENT OF DNA/RNA PROLIFERATION USING RADIOLABELED GUANINE ANALOGUES BY POSITRON EMISSION TOMOGRAPHY AND SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY K. C. Chao, D. J. Yang, T. Yang, A. Azhdarinia MD Anderson Cancer Center, Houston, TX. Objective. To assess whether 64Cu and 99mTc-ethylenedicysteine-guanine (EC-Guan) can be used to evaluate tumor proliferation and localization by positron emission tomography (PET) and single photon emission computed tomography (SPECT). Method. EC-Guan was prepared by reacting the amino analogue of penciclovir with EC and carbodiimide. After dialysis, EC-Guan was obtained at 67% yield. Labeling EC-Guan with 99mTc was achieved by adding 200 µCi of Na99mTcO4 into a vial containing 5 mg EC-Guan and 100 µg SnCl2. 64Cu-EC-Guan was prepared by adding 64Cu-acetate to 5 mg EC-Guan. Tumor cells were separated using FACS then incubated with 99mTc-EC-Guan and 64Cu-EC-Guan for cell cycle analysis. Tumor (prostate, ovarian, breast, lung) cells that were overexpressing the HSV thymidine kinase (HSVTK) gene, were incubated with 99mTc-EC-Guan (4 µCi/100 µg/well) at 0.5 to two hours. Thymidine incorporation assays were performed in lung cancer cells incubated with EC-Guan at various doses (0.1-1 mg/well). Tissue distribution, autoradi-

ography and planar scintigraphy of 99mTc-EC-Guan and 64Cu-EC-Guan were determined in tumor-bearing rodents at 0.5 to four hours. Results. Radiochemical purity (determined by radio-TLC and HPLC) was >97%. In vitro cell culture assays indicated both 64Cu and 99mTc-EC-Guan were incorporated into cell cycles. S-Phase showed higher uptake than other phases. There was no significant uptake difference between HSVTK overexpressed and normal groups. Biodistribution of 99mTc-EC-Guan in tumor-bearing mice showed increased tumor-to-tissue count density ratios as a function of time. Tumor uptake (%ID/g) of 99mTc-EC-Guan was 0.30.8. Autoradiography of 64Cu-EC-Guan showed that brain tumors could be visualized at one hour. Conclusion. Our results indicated that 64Cu and 99mTc-EC-Guan are novel proliferation-imaging agents. No. 146 IMAGING BRAIN TUMOR INTEGRIN EXPRESSION WITH 64Cu-LABELED PEGYLATED RGD PEPTIDE X. Chen, Y. Hou, R. Park, M. Tohme, J. R. Bading, P. S. Conti University of Southern California, Los Angeles, CA. Cell adhesion molecules αv-integrins that are highly expressed on activated endothelial cells and tumor cells but not on quiescent endothelial cells and normal cells present an excellent target for tumor angiogenesis imaging and anti-angiogenic therapy. We have previously coupled cyclic RGD peptide c(RGDyK) with DOTA (1,4,7,10-tetraazacyclododecaneN,N’,N’’,N’’’-tetraacetic acid) and labeled the RGD-DOTA conjugate with 64Cu for solid tumor targeting with good tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study we introduced a poly(ethylene glycol) (PEG, M.W.=3400) moiety between DOTA and RGD and evaluated this 64CuDOTA-PEG-RGD tracer for microPET imaging of brain tumor models. Compared with 64Cu-DOTA-RGD, this tracer revealed significantly reduced liver uptake and more rapid renal clearance. The high tumor-toorgan ratios for the PEGylated RGD peptide tracer (e.g. at one hour postinjection: tumor-to-blood=20, tumor-to-muscle=12, tumor-to-liver=2.7, and tumor-to-kidney=1.2) were confirmed by microPET and autoradiographic imaging of the subcutaneous U87MG tumor model. Although orthotopic tumor uptake is lower than in subcutaneous model, relatively high tumor/brain ratio was still obtained. Systematic investigation of the effect of size and geometry of PEG on tumor targeting and in vivo kinetics will develop radiotracers suitable for clinical applications. In addition, the same ligand if labeled with therapeutic radionuclides may be applied for angiogenesis targeted internal radiotherapy. [Support by ACS-IRG-580007-42 (X.C.), DOD DAMD17-03-1-0752 (to X.C.), the Wright Foundation (XC), and NIH P20 CA86532 (PSC). Copper-64 was provided by Washington University and funded through NCI R24 CA86307.]. No. 147 PEPTIDE NUCLEIC ACID AS RADIOPHARMACEUTICAL FOR GENE-SPECIFIC RADIOTHERAPY AND IMAGING Y. He1, I. G. Panyutin1, I. V. Panyutin1, E. J. Chuang1, V. V. Demidov2, R. D. Neumann1 1National Institutes of Health, Bethesda, MD, 2Boston University, Boston, MA. Peptide nucleic acids (PNA) are biopolymers with repeating N-(2-amino ethyl) glycine units that replace the sugar-phosphate backbone of DNA. PNA exhibits higher chemical and biological stability as compared to the nuclease-sensitive phosphodiester oligonucleotides, and they bind sequence-specifically to single- and double-stranded DNA by forming stable hybrid duplex or triplex. Thus, PNA may be of a potential interest in the design of gene-targeted radiotherapeutics that could direct radiodamage to specific DNA and/or RNA sites as well as in the development of DNA and RNA imaging probes.

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In this study we describe a simple procedure for incorporation of metal radionuclides (In-111) into PNA oligomers, and demonstrate the ability of the radio-labeled PNA bind to their targets and produce sequence-specific DNA breaks in vitro. We utilized the lysine-appended, mixed-base PNA oligomer (H-TAGTTATCTCTATCT-Lysine-NH2) and plasmid pUC19C1. The pUC19-C1 was constructed from the plasmid pUC19 vector by inserting a 29 mer oligo-DNA (3’-TTCCTTT-ATCAATAGAGATAGATTCCTTT-5’) containing complementary sequence for radio-labeled PNA binding site flanked by sequences that are complementary to PNA-opener binding sites. The PNA opener is [7+7]-mers (H-Lys2-TTJJTTT-(eg1)3TTTCCTT-Lys-NH2) (where egl is 8-amino-3,6-dioxaoctanoic acid) that is able to form triplexes with the short flanking sequences around the target site and, thus, assisting the radio-labeled PNA in binding to the target. The binding of radio-labeled PNA to plasmid pUC19-C1 was studied by the gel-shift assay, and the breaks induced by the site-positioned In-111 decay of radio-labeled PNA were analyzed by sequencing PAGE. All data indicate that radio-labeled PNA is a very promising radio-pharmaceutical for gene-specific radiotherapy and imaging.

183.4±6.7nM respectively), confirming the retention of receptor-binding affinity. MicroPET imaging of 68Ga-DOTATOC in AR42J tumor bearing rats at 75 minutes postinjection showed significant uptake and good contrast in the thigh tumor in the Lewis rat model. The AR42J tumor of the Lewis rats showed reduced uptake using microSPECT when coinjection of 0.5mg of DSPE-PEGTOC and 2ug of 68Ga-DOTATOC, suggesting that DSPE-PEGTOC has the ability to compete the specific binding sites of somatostatin receptor in vivo and in vitro. Biodistribution analysis also showed that the tumor uptake of 1.07 %ID/g was determined for 68GaDOTATOC and 0.25 %ID/g for coinjection with 0.5mg of DSPE-PEGTOC and 2ug of 68Ga-DOTATOC in AR42J pancreatic tumor bearing rat at 2h postinjection. We conclude that 68Ga or 123/124I labeled DOTATOC can be used for microPET or microSPECT diagnosis and quantitative imaging-based dosimetry of SSTR positive tumors. We can evaluate new drugs, DSPEPEGTOC and may octreotide-conjugated liposome, which of them do not need to incorporate the radionuclide in the synthetic processes. No. 150

No. 148 MICROWAVE-PROMOTED SYNTHESIS OF [C-11]TOLUENES VIA SUZUKI COUPLING WITH [C-11]METHYL IODIDE E. D. Hostetler, G. E. Terry, H. D. Burns Merck, West Point, PA. A necessity in positron emission tomography (PET) tracer discovery is integration of the radiolabel into an appropriately substituted compound. A methyl substituent on an aromatic ring is a common location for the introduction of a carbon-11 radiolabel into a potential PET tracer. Thus, [C11]toluene derivatives are desirable C-11 labeled analogs. The most common strategy currently utilized for synthesizing [C-11]toluene derivatives is via Stille coupling of the corresponding aryl trialkyltin derivative with [C-11]methyl iodide. However, yields are often low, and most significantly, toxic tin by-products can be difficult to completely remove from the final product, which can be prohibitive for clinical studies. The desire for an alternate synthetic route to the Stille coupling led us to investigate the feasibility of the Suzuki coupling. The Suzuki coupling proved to be an excellent choice, providing high yields of the desired products from the corresponding aryl boronic acids or esters. Heating the reaction using a specially designed microwave cavity proved to be especially effective. We have found the Suzuki coupling to be a robust, non-toxic alternative to the Stille coupling for the synthesis of functionalized [C11]toluene derivatives from [C-11]methyl iodide. The results and scope of this new methodology will be presented in detail. No. 149 EVALUATION OF OCTREOTIDE-CONJUGATED LIPOSOME USING 68Ga OR 123/124I LABELED SOMATOSTATIN ANALOGUE DOTA- D-Phe1-Tyr3-OCTREOTIDE W. C. Hsu, S. L. Chen, S. P. Chiu, W. L. Cheng Institute of Nuclear Energy Research, Lungtan, TAIWAN REPUBLIC OF CHINA. 1,4,7,10-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic-acid-D-Phe1Tyr3-octreotide(DOTATOC) was synthesized by liquid-phase method and labeled with the positron- emitting generator nuclide 68Ga and γ-ray-emitting nuclide 123I by iodogen method in high radiochemical purity and specific activity. These radiolabeled peptides were used to evaluate the SSTR targeting activities of distearoyl phosphatidyl ethanolamine-polyethylene glycol-octreotide (DSPE-PEGTOC), the precursor ligand of a new development octreotide-conjugated liposome, synthesized by a direct synthetic method. In somatostatin receptor binding assays using AR42J cell membranes, the unlabelled DOTATOC and DSPE-PEGTOC showed effective displacement of 125I labeled Tyr1-somatostatin (Ki= 1.14±0.17nM and

RADIOACTIVE ARSENIC ISOTOPES: NEW TOOLS FOR THE IMAGING OF TUMOR TARGETING ANTIBODIES M. Jennewein1, A. Constantinescu1, O. Bergner2, N. Slavine1, S. Seliouline1, M. Lewis1, D. Zhao1, S. O'Kelly3, S. M. Qaim4, E. Tsyganov1, P. P. Antich1, P. E. Thorpe5, R. Schirrmacher6, F. Roesch6, R. P. Mason1 1Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, 2Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, 3Department of Nuclear Engineering, University of Texas at Austin, Austin, TX, 4Institute of Nuclear Chemistry, Research Centre Juelich, Juelich, GERMANY, 5Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, 6Institute of Nuclear Chemistry, Johannes Gutenberg University of Mainz, Mainz, GERMANY. For the long lasting enrichment of antibodies in tumor tissue currently not many suitable isotopes for positron emission tomography (PET) imaging are available. The element arsenic provides several isotopes with an interesting physical half life. The PET isotopes 72As (T1/2 = 26.0 hours) and 74As (T1/2 = 17.77 days) and the therapeutic isotope 77As (T1/2 = 38.83 hours) were used to developing radiochemical separation procedures, a labeling chemistry and for first small animal imaging experiments using a ch3G4 anti-phosphatitylserine antibody as a tumor vasculature targeting agent. Phosphatidylserine (PS), which is the most abundant anionic phospholipid of the plasma membrane, is tightly segregated to the internal leaflet of the plasma membrane in most cell types, including the vascular endothelium. Anionic phospholipids become exposed on tumor vasculature as a result of exposure to stress conditions in the tumor microenvironment. As antibodies to anionic phospholipids have a potential as diagnostic and therapeutic agents for targeting the vasculature of solid tumors they were chosen as a model system to develop new labeling strategies for antibodies. A new 72Se/72As isotope generator and a separation procedure for 74As and 77As from reactor or cyclotron irradiated GeO2 targets, both delivering nca AsI3, were developed. The ch3G4 antibody was chemically modified to introduce free sulfhydryl groups and labeled with radiochemical yields > 90 %. Quantitative imaging was performed using small animal PET and autoradiographic techniques on AT1 prostate tumor bearing male Copenhagen rats. The observed tumor:liver ratios for both imaging and biodistribution were better than 3:1 at 48 hours. No. 151 QUANTITATION OF Cu-64 ACTIVITY DISTRIBUTIONS IN MURINE IMAGES USING A DEDICATED CLINICAL POSITRON EMISSION TOMOGRAPHY/COMPUTED TOMOGRAPHY SCANNER