Targeting of the Cholecystokinin-2 Receptor with the Minigastrin Analog<sup>177</sup>Lu-DOTA-PP-F11N: Does the Use of Protease Inhibitors Further Improve In Vivo Distribution? (2018)

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog <sup>177</sup>Lu-DOTA-PP-F11N (<sup>177</sup>Lu-DOTA-(dGlu)<sub>6</sub>-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH<sub>2</sub>) performs better than reference analogs with varying in vivo stability, namely <sup>177</sup>Lu-DOTA-MG11 (<sup>177</sup>Lu-DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH<sub>2</sub>) and <sup>177</sup>Lu-DOTA-PP-F11 (<sup>177</sup>Lu-DOTA-(dGlu)<sub>6</sub>-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH<sub>2</sub>), and whether the use of protease inhibitors further improves CCKR2 targeting. First human data on <sup>177</sup>Lu-DOTA-PP-F11N are also reported. <b>Methods:</b> In vitro stability of all analogs was assessed against a panel of extra- and intracellular endoproteases, whereas their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon and thiorphan was assessed 4 h after injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of <sup>177</sup>Lu-DOTA-PP-F11N (without and with phosphoramidon) and NanoSPECT/CT were performed. SPECT/CT images of <sup>177</sup>Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. <b>Results:</b><sup>nat</sup>Lu-DOTA-PP-F11N is less of a substrate for neprilysins than the other analogs, whereas intracellular cysteine proteases, such as cathepsin-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors than in A431-CCK2R(+), apparently because of the higher number of binding sites on MZ-CRC-1 cells. <sup>177</sup>Lu-DOTA-PP-F11N had the same biodistribution as <sup>177</sup>Lu-DOTA-PP-F11; however, uptake in the MZ-CRC-1 tumors was almost double (20.7 ± 1.71 vs. 11.2 ± 2.94 %IA [percentage injected activity]/g, <i>P</i> = 0.0002). Coadministration of phosphoramidon or thiorphan increases <sup>177</sup>Lu-DOTA-MG11 uptake significantly in the CCK2R(+) tumors and stomach. Less profound was the effect on <sup>177</sup>Lu-DOTA-PP-F11, whereas no influence or even reduction was observed for <sup>177</sup>Lu-DOTA-PP-F11N (20.7 ± 1.71 vs. 15.6 ± 3.80 [with phosphoramidon] %IA/g, <i>P</i> < 0.05 in MZ-CRC-1 tumors). The first clinical data show high <sup>177</sup>Lu-DOTA-PP-F11N accumulation in tumors, stomach, kidneys, and colon. <b>Conclusion:</b> The performance of <sup>177</sup>Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of <sup>177</sup>Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight the stomach as a potential dose-limiting organ besides the kidneys.