Abstract
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Introduction: A comprehensive summary of PET imaging reveals the superior role for Fibroblast activation protein (FAP) due to the high specificity in the cancer-associated Fibroblasts. Studies have shown that FAPI as potential specific PET-theranostic strategy for glioblastoma with a high uptake rate of FAPI and tumor-to background ratio (TBR) in Isocitrate dehydrogenase (IDH)-wild, grade III or IV glioblastomas. FAPI PET has also been documented for higher tumor gross volumes. These results pinpointed the exploration of FAPI radio conjugates for glioblastoma. Our previous encouraging results with FAPI driven us to explore the 64 Cu labeled Sarchophagine conjugated FAPI radiopharmaceuticals.
Methods: The design of the targeted conjugates depends on the modification of the linker (PEG) length between FAPI structure and Sarchophagine. The targeted urea derived FAPI precursors was achieved by the phosphine mediated azide-amine conjugation using atmospheric carbon dioxide. In this report, we describe three derivatives of homofunctional constructs (FAPI-PEG(n)-urea-sarc, FAPI-PEG(n)-urea-sarc-urea-PEG(n)-FAPI, and FAPI-PEG(n)-urea-sarc-Ph-sarc-urea-PEG(n)-FAPI). Radiolabeling reactions were performed under 37 oC for 1.5 h. In vivo distribution study in small animals was performed using Sedecal PET/CT system. All the synthesized 64 Cu labeled radiopharmaceuticals were intravenously injected into U87 glioblastoma tumor-bearing model and the animal was scanned at 1h, 4h, 24h, and 48h post injection (p.i.). Region of interest was analyzed in Amide software and the organ uptake was calculated after decay correction. In vitro stability of the leading compound that showed the best in vivo distribution profile was performed. To validate the specificity binding, an in vivo blocking experiment was also performed in U87 mouse.
Results: The moderate linker length (PEG4) was found to exhibit highest tumor uptake and best tumor-to-background contrast. The tumor uptake for FAPI-PEG4-urea-sarc was 8.7±2.8 %ID/g at 1h and 7.3±2.6 %ID/g at 4h. The liver uptake was below 1.5 %ID/g at all time points while kidney uptake kept below 2.5 %ID/g at all times. Compared to FAPI-PEG4-urea-sarc, FAPI-PEG4-urea-sarc-urea-PEG4-FAPI has increased and sustained tumor uptake, which was 16.3±1.8 %ID/g at 1h and maintained at 6.2±3.4 %ID/g at 48h. Meanwhile, higher liver and kidney uptake were also observed (5.5±3.2 and 3.8±1.9 %ID/g at 1h, respectively). The potential reason was hypothesized to be that the dimer has a larger size, leading to longer circulation time and slower washout. The dimer may also have increased binding affinity with the target on the tumor site. In monomers, it was noticed that when the linker length reduced from PEG4 to PEG1, the tumor uptake level maintained roughly at the same level, while the liver uptake was raised about 2-fold. Moreover, increasing the linker length from PEG4 to PEG12 made the tumor uptake dropped dramatically while background level at the same range as PEG4. Furthermore, we validated the specificity of the FAPI-targeting effect by comparing the in vivo blocking and unblocking tumor uptake at 1h and 4h. The difference between two groups was statistically significant (2.1±0.4 %ID/g vs 8.7±2.8 %ID/g at 1h, p<0.05; 1.6±0.3 %ID/g vs 7.3±2.6 %ID/g at 4h, p<0.05).
Conclusions: We have succeeded in the synthesis of new FAPI targeting compounds with > 80% radiochemical yield, 97-99% radiochemical purity and high molar activities. Of all the synthesized precursors, FAPI-PEG4-urea-sarc was recognized as the lead agent for its high tumor uptake and tumor-to background ratio. Dimerization further enhanced the tumor uptake and retention. The therapeutic potential of the lead agent is worth being investigated further in the future.
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