Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of 223Ra-labeled and PSMA-targeted NaA nanozeolites [223RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of 133Ba- and 223Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to 223Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of 223Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.
Rituximab when radiolabelled with (177)Lu or (90)Y has been investigated for the treatment of patients with Non-Hodgkin's Lymphoma. In this study, we optimized the preparation of antibody conjugates with chelating agent in the freeze-dried kit. It shortens procedures needed for the successful radiolabeling with lutetium-177 and yttrium-90 and assures reproducible labelling yields. Various molar ratios of Rituximab:DOTA (from 1:5 to 1:100) were used at the conjugation step and different purification method to remove unbound DOTA were investigated (size-exclusion chromatography, dialysis, ultrafiltration). The final monoclonal antibody concentration was quantified by Bradford method, and the number of DOTA molecules was determined by radiolabeling assay using (64)Cu. The specific activity of (177)Lu-DOTA-Rituximab and (90)Y-DOTA-Rituximab were optimized using various amounts of radiometal. Quality control (SE-HPLC, ITLC) and stability study were performed. An average of 4.2 ± 0.8 p-SCN-Bz-DOTA molecules could be randomly conjugated to a single molecule of Rituximab. The ultrafiltration system was the most efficient for purification and resulted in the highest recovery efficiency (77.2%). At optimized conditions the (177)Lu-DOTARituximab and (90)Y-DOTA-Rituximab were obtained with radiochemical purity >99% and specific activity ca. 600 MBq/mg. The radioimmunoconjugates were stable in human serum and 0.9% NaCl. After 72 h of incubation the radiochemical purity of (177)Lu-DOTA-Rituximab decreased to 94% but it was still more than 88% for (90)Y-DOTA-Rituximab. The radioimmunoconjugate showed stability after six months storage at 2 - 8(0)C, as a lyophilized formulation. Our study shows that Rituximab-DOTA can be efficiently radiolabeled with (177)Lu and (90)Y via p-SCN-Bn-DOTA using a freezedried kit.
Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.
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