Comparison of In Vitro Mouse Serum Stability of ⁶⁸Ga-Labeled PSMA Targeting Conjugates of Acyclic and Macrocyclic Chelators

Objectives: The objective of the present work was (1) to measure and compare in vitro stability of ⁶⁸Ga-labeled PSMA targeting conjugates of acyclic and macrocyclic chelators in mouse serum at 37^oC, and (2) to correlate stability data with in-vivo tumor uptake in 22Rv1 xenografted mice¹. The knowledge of mouse serum stability is critical for the understanding of the efficacy of a potential imaging pharmaceutical.

Methods: Glu-Urea-Lys (GUL) derivatives of acyclic and macrocyclic chelators have been reported as prostate-specific membrane antigen (PSMA) agents. In the present work, GUL was conjugated with two chelators, NOTA and DOTA, via a thiourea linker and designated as NOTA-GUL and DOTA-GUL, respectively. The well-known radioligand PSMA-11, NOTA-GUL, and DOTA-GUL were labeled with ⁶⁸Ga (37-100 MBq) using a standard radiolabeling technique developed in our laboratories. ⁶⁸GaCl₃ was produced using a Gallia Pharm generator from Eckert & Ziegler. The ⁶⁸Ga-labeled PSMA conjugates were analyzed and mouse serum stability was monitored by using a reversed-phase High-Performance Liquid Chromatography (RP-HPLC) method. In a typical stability study, a ⁶⁸Ga-labeled conjugate sample was incubated with mouse serum for 4 hours at 37 °C and the sample was injected onto a RP-HPLC column at a predefined time intervals. Progress of the degradation of the ⁶⁸Ga-labeled PSMA conjugate in mouse serum was monitored by monitoring area of the main peak with time. Control experiments were performed in each case by monitoring degradation of ⁶⁸Ga-labeled NOTA-GUL, DOTA-GUL, and PSMA-11 in Phosphate Buffer Saline (PBS).

Results: Efficiency of ⁶⁸Ga-labeling of NOTA-GUL, DOTA-GUL, and PSMA-11 was achieved >96% with > 99% purity. No degradation of ⁶⁸Ga-labeled PSMA-11 and NOTA-GUL was observed after 4 h incubation in mouse serum. On the contrary, degradation (mainly demetalation) of ⁶⁸Ga-labelded DOTA-GUL was seen even after 1 h incubation and reaching up to 8% after 4 h incubation. Consistent with the human serum stability study,¹ mouse serum stability of ⁶⁸Ga-labeled PSMA conjugates followed the order: PSMA-11~NOTA-GUL >DOTA-GUL. A similar trend was observed in the in-vivo tumor uptake (%ID/g) in 22Rv1 xenografted mice 1 h post-injection,i.e 6.5, 5.4, and 4.66 for PSMA-11, NOTA-GUL, and DOTA-GUL, respectively. Interestingly, in the present work ⁶⁸Ga-labeled DOTA-GUL is less inert than the ⁶⁸Ga-labeled PSMA-11 in mouse serum.

Conclusions: In summary, the in-vitro mouse serum stability of the three of ⁶⁸Ga-Labeled PSMA targeting conjugates of acyclic and macrocyclic chelators follow the order PSMA-11~ NOTA-GUL >DOTA-GUL which reflects in the in-vivo tumor uptake in preclinical mice model.¹ Ref. Moon S-H, Hong MK, Kim YJ et al. Bioorg Med Chem 2018; 26: 2501-2507.