High sensitivity: high-resolution SPECT-CT/MR molecular imaging of angiogenesis in the Vx2 model.

OBJECTIVES: The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality alpha(nu)beta3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis. MATERIALS AND METHODS: In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either alpha(nu)beta3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or alpha(nu)beta3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled alpha(nu)beta3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of alpha(nu)beta3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into alpha(nu)beta3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity - high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping. RESULTS: alpha(nu)beta3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 +/- 0.13, TMR) versus the 11 MBq/kg (7.32 +/- 0.12) and 44 MBq/kg (6.55 +/- 0.07) doses, (P < 0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 +/- 0.09) was less (P < 0.05) than the equivalent dosage of alpha(nu)beta3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc alpha(nu)beta3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P < 0.05) TMR (5.31 +/- 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc alpha(nu)beta3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer. CONCLUSION: Dual modality molecular imaging with alpha(nu)beta3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Preparation and biological evaluation of 111In-, 177Lu- and 90Y-labeled DOTA analogues conjugated to B72.3.

Three 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) analogues were evaluated for relative in vivo stability when radiolabeled with (111)In, (90)Y and (177)Lu and conjugated to the monoclonal antibody B72.3. The DOTA analogues evaluated were "NHS-DOTA" [N-hydroxysuccinimdyl (NHS) group activating one carboxylate], "Arm-DOTA" (also known as MeO-DOTA; with a p-NCS, o-MeO-benzyl moiety on the methylene group of one acetic acid arm) and "Back-DOTA" (with a p-NCS-benzyl moiety on a backbone methylene group of the macrocycle). The B72.3 was conjugated to the DOTA analogues to increase the retention time of the radioloabeled conjugates in vivo in mice. The serum stability of the various radiometalated DOTA conjugates showed them to have good stability out to 168 h (all >95% except (111)In-NHS-DOTA-B72.3, which was 91% stable). Hydroxyapatite stability for the (111)In and (177)Lu DOTA-conjugates was >95% at 168 h, while the (90)Y DOTA-conjugates were somewhat less stable (between 90% and 95% at 168 h). The biodistribution studies of the radiometalated DOTA-conjugates showed that no significant differences were observed for the (111)In and (177)Lu analogues; however, the (90)Y analogues showed lower stabilities, as evidenced by their increased bone uptake relative to the other two [2-20% injected dose per gram (% ID/g) for (90)Y and 2-8% ID/g for (111)In and (177)Lu]. The lower stability of the (90)Y analogues could be due to the higher beta energy of (90)Y and/or to the larger ionic radius of Y(3+). Based on the bone uptake observed, the (177)Lu-NHS-DOTA-B72.3 had slightly lower stability than the (177)Lu-Arm-DOTA-B72.3 and (177)Lu-Back-DOTA-B72.3, but not significantly at all time points. For (90)Y, the analogue showing the lowest stability based on bone uptake was (90)Y-Arm-DOTA-B72.3, perhaps because of the metal's larger ionic radius and potential steric interactions minimizing effective complexation. The (111)In analogues all showed similar biological distributions at the various time points. This study suggests that care must be taken when evaluating (90)Y-labeled antibodies and in using NHS-DOTA-antibody conjugates with (177)Lu. All evaluations should be extended to time points relevant to the half-life of the radiometal and the therapy applications.

Imaging of Vx-2 rabbit tumors with alpha(nu)beta3-integrin-targeted 111In nanoparticles.

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. alpha(nu)beta(3)-Targeted (111)In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx-2 tumor. alpha(nu)beta(3)-Targeted (111)In/NP bearing approximately 10 (111)In/NP vs. approximately 1 (111)In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. alpha(nu)beta(3)-Integrin-targeted NP with approximately 10 (111)In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 +/- 0.2) than approximately 1 (111)In/NP (5.1 +/- 0.1) or nontargeted particles with approximately 10 (111)In/NP (3.7 +/- 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving alpha(nu)beta(3)-integrin-targeted NP was 4-fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of alpha(nu)beta(3)-targeted fluorescent-labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half-life (t(1/2)beta) was estimated to be approximately 5 hr using a 2-compartment model. alpha(nu)beta(3)-Targeted (111)In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary high-resolution imaging modalities, such as MRI.