Developing DLBCL PET Probe: Radiofluorination and Preclinical PET Studies

Introduction: Positron emission tomography (PET) has become an essential tool for non-invasive and specific disease diagnosis, utilizing positron-emitting radioisotopes such as fluorine-18 known for its advantageous nuclear properties. Diffused Large B-Cell Lymphoma (DLBCL), a prevalent non-Hodgkin’s lymphoma, poses challenges in accurate diagnosis using PET imaging, primarily due to limitations associated with [¹⁸F]FDG. The current research aims to develop a PET probe with enhanced specificity for DLBCL, focusing on superior biological activities to improve the diagnostic properties.

Methods: The radiopharmaceutical probe (OICR-01) utilized in this study was supplied by the Ontario Institute for Cancer Research (OICR) and subjected to radiofluorination through nucleophilic substitution, as depicted in the provided scheme. The detailed quality control assessment was conducted, encompassing examinations of formulation and serum stability, purity, lipophilicity, and stickiness. The biological activity of the radiolabeled compounds ([¹⁸F]F-OICR) was evaluated in vitro, including a saturation binding assay with Karpas422 cells to determine binding affinity. Subsequently, preliminary imaging and biodistribution studies, performed at one hour time point, were undertaken in healthy NCr nude mice for preliminary in vivo analysis. These multifaceted analyses aim to provide a robust understanding of the radiopharmaceutical properties and performance, which is crucial for its potential application in diagnostic imaging.

Results: The radiolabeling of OICR-01 was consistently reproducible across multiple attempts, resulting in 11.5 ± 4.0% radiochemical yield (RCY) (n = 4). The final [¹⁸F]F-OICR, exhibited radiochemical purity (RCP) exceeding 99%. The molar activity (MA) was calculated at approximately 0.89 ± 0.04 mCi/μmol.

[¹⁸F]F-OICR exhibited stability in the formulation buffer for up to 4 hours and in mouse serum for up to 2 hours. The hydrophobic character of [¹⁸F]F-OICR, as indicated by a Log P test, elucidated the substantial accumulation observed in the gallbladder during PET imaging studies 60 minutes post-injection (P.I.).

The Micro-PET studies in NCr mice revealed renal and hepatobiliary excretion of [¹⁸F]F-OICR, with gallbladder accumulation consistent with its apparent lipophilicity. Notably, blood uptake remained minimal, approaching background levels, and blood clearance was rapid, suggesting potential tumor uptake in xenografted mouse models using high-affinity molecules. Additionally, limited uptake in the bone emphasized the in vivo stability of [¹⁸F]F-OICR-01. However, subsequent saturation assays revealed concerns regarding the potential limitations of the MA value (0.89 mCi/μmol) in terms of binding efficiency to Karpas422 cells. At a concentration of 10 μCi/mL [¹⁸F]F-OICR exhibited minimal binding, less than 0.5%, prompting scrutiny into the validity of tumor cell uptake at improved MA value.

Conclusions: The DLBCL probe named OICR-01 was successfully radiofluorinated with a RCY of 11.5 ± 4.0 and RCP of >99%. The micro-PET study revealed renal and hepatobiliary excretion, along with swift blood clearance and minimal blood uptake, indicating the prospect of tumor uptake in xenograft mouse models. Nonetheless, subsequent saturation assays have highlighted potential limitations in the binding efficiency with Karpas422 cells due to inherent self-blocking at the calculated MA of 0.89 mCi/μmol. This prompts further consideration for improving the MA value to enhance the efficiency of tumor cell uptake.

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