Development of lisinopril radioconjugates for nuclear imaging of the angiotensin converting enzyme

The angiotensin converting enzyme (ACE) is essential in maintaining cardiovascular homeostasis and its dysfunction is associated with various pathological conditions. This study aimed to investigate lisinopril-based radioconjugates for nuclear imaging of ACE.

Lisinopril conjugates were prepared using solid-phase peptide synthesis and labeled with gallium-67. The resulting radioconjugates were assessed for their in vitro stability in saline and in mouse and human blood plasma. ACE-mediated uptake was evaluated in HEK cells transfected with human ACE, and the findings were interpreted using molecular dynamic studies. Tissue distribution profiles of the radioconjugates were evaluated in mice bearing HEK-ACE xenografts by means of nuclear imaging and classic biodistribution studies. The feasibility to detect physiologic ACE was further assessed by ex vivo autoradiography of the lungs and the kidneys collected from mice injected with [⁶⁷Ga]Ga-DOTA-LIS-02.

DOTA-LIS-01, DOTA-LIS-02 and NODAGA-LIS-02 were prepared with > 98% chemical purity. Radiolabeling of the conjugates with gallium-67 was achieved at 80 MBq/nmol with > 99% radiochemical purity. [⁶⁷Ga]Ga-DOTA-LIS-02 and [⁶⁷Ga]Ga-NODAGA-LIS-02 exhibited radiolytic stability for up to 3 h in saline and remained intact in mouse and human blood plasma for 1 h. The uptake of [⁶⁷Ga]Ga-DOTA-LIS-02 and [⁶⁷Ga]Ga-NODAGA-LIS-02 in HEK-ACE cells after 3 h incubation reached ~ 57% and ~ 50%, respectively, while only ~ 21% cell uptake was reached with [⁶⁷Ga]Ga-DOTA-LIS-01. This discrepancy was ascribed to the favorable binding mode of the LIS-02 radioconjugates with a longer linker between DOTA and lisinopril, as demonstrated by molecular dynamic studies. Nuclear images demonstrated uptake of all radioconjugates in HEK-ACE xenografts but not in HEK-ACE2 xenografts. At 1 h after injection of [⁶⁷Ga]Ga-DOTA-LIS-02, the accumulation in HEK-ACE xenografts of mice reached 14 ± 3% IA/g, whereas only 3.6 ± 0.3% IA/g uptake was observed for [⁶⁷Ga]Ga-NODAGA-LIS-02. The ex vivo autoradiograms of kidneys and lungs of mice injected with [⁶⁷Ga]Ga-DOTA-LIS-02 only, or co-injected with excess lisinopril confirmed ACE-specific binding of this radioconjugate.

[⁶⁷Ga]Ga-DOTA-LIS-02 emerged as the most promising candidate to visualize ACE in mice. Further (pre)clinical studies will be necessary to validate the radioconjugate's potential for assessing ACE expression dynamics under pathophysiological conditions.