<Sup>177</Sup>Lu and <Sup>225</Sup>Ac dose escalation using a novel class of PSMA radioligands with low salivary gland and kidney retention in a preclinical model of human prostate cancer

Introduction: Radioligands targeting prostate-specific membrane antigen (PSMA) have gained popularity for the diagnosis and targeted therapy of prostate cancer. Many of these ligands utilize the Lys-urea-Glu pharmacophore, which might contribute to the off-target binding in salivary gland and high kidney retention in clinical trials. In our recent pre-clinical work, we found that by replacing the Glu residue with aminoadipic acid (Aad), the resulting peptide HTK03149 showed significantly reduced salivary gland and kidney retention in a mouse model of human prostate cancer. To further improve the tumor retention, we incorporated an albumin binder to yield HTK03170. The aim of this study was to evaluate the efficacy of ¹⁷⁷Lu- and ²²⁵Ac-labeled HTK03149 and HTK03170 in dose escalation studies in a preclinical model of human prostate cancer.

Methods: The DOTA-conjugated peptides were prepared by standard Fmoc chemistry on solid phase. HTK03149 was synthesized following the procedures for HTK03141 (Kuo HT, et al. J Nucl Med 2021; 62: 521) with Glu replaced by Aad. HTK03170 was synthesized by incorporating 4-(4-methoxyphenyl)butyric acid as an albumin binder to HTK03149. In vitro competitive binding assays were performed using LNCaP cells. Both peptides were radiolabeled with ¹⁷⁷Lu and evaluated by SPECT/CT imaging and biodistribution studies at 1, 4, 24, 72, and 120 h post-injection (p.i.) in LNCaP tumor-bearing NRG mice (n ≥ 5/group). Furthermore, dose escalation studies were conducted in LNCaP tumor-bearing mice (n ≥ 7/group) treated with a single dose of vehicle control, non-radioactive lutetium (^natLu)-conjugated peptides, [¹⁷⁷Lu]Lu-HTK03149 (9.3 to 148 MBq), [¹⁷⁷]Lu-HTK03170 (9.3 to 37 MBq), [¹⁷⁷Lu]Lu-PSMA-617 (37 MBq), [²²⁵Ac]Ac-HTK03149 (20 and 40 kBq) or [²²⁵Ac]Ac-HTK03170 (7.5 to 60 kBq). The mice were monitored for ~180 days unless predefined endpoints were reached, including body weight loss > 15% and tumor volume > 1,000 mm³.

Results: HTK03149, HTK03170 and their ^natLu-conjugated standards were prepared in high chemical purity. Binding affinity for ^natLu-HTK03149 and ^natLu-HTK03170 was 1.57 ± 0.60 nM and 1.22 ± 0.43 nM, respectively. SPECT/CT imaging and biodistribution studies using [¹⁷⁷Lu]Lu-HTK03149 showed sustained tumor uptake [13.8-20.9 (min-max values) percentage injected dose per gram of tissue, %ID/g], and low salivary gland uptake (peak uptake at 1 h p.i. at 0.23 ± 0.06 %ID/g) and low kidney retention (peak uptake at 1 h p.i. at 7.67 ± 1.35 %ID/g, and cleared to < 1%ID/g at 24 h p.i.). With the albumin binder, [¹⁷⁷Lu]Lu-HTK03170 showed substantially increased tumor uptake from 27.2 ± 7.55%ID/g at 1 h p.i. to 71.91 ± 19.39 %ID/g at 120 h p.i. [¹⁷⁷Lu]Lu-HTK03149 and [¹⁷⁷Lu]Lu-HTK03170 treatments were well-tolerated in the dose escalation studies. Complete remission was observed for all the mice treated with ¹⁷⁷Lu-labeled peptides except for one mouse in the 9.3 MBq group of HTK03149. Median survival for [¹⁷⁷Lu]Lu-HTK03149 at 9.3, 18.5, 37, 74, and 148 MBq groups was 52, 63, 88, 142, and > 180 days, respectively. In comparison, median survival for [¹⁷⁷Lu]Lu-PSMA-617 at 37 MBq was 62 days. [¹⁷⁷Lu]Lu-HTK03170 showed much higher efficacy with the majority of the mice in the 37 and 74 MBq groups survived the duration of the study (median survival > 180 days). Dose escalation studies with ²²⁵Ac-labeled HTK03149 and HTK03170 showed similar results, i.e. the treatment was well-tolerated and complete remission was observed in all the mice, and median survival was > 180 days in the higher dose groups, i.e. 40 kBq group for [²²⁵Ac]Ac-HTK03149, as well as 30 and 60 kBq groups for [²²⁵Ac]Ac-HTK03170.

Conclusions: In the preclinical mouse model of human prostate cancer xenograft, ¹⁷⁷Lu-labeled HTK03149 and HTK03170 showed favorable pharmacokinetics with sustained tumor uptake and low salivary gland and kidney retention in SPECT imaging and biodistribution studies, and treatments with ¹⁷⁷Lu- and ²²⁵Ac-labeled HTK03149 and HTK03170 were well-tolerated and efficacious.

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