Radiosynthesis and characterization of a carbon-11 PET tracer for receptor-interacting protein kinase 1

Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a K_i of 0.91 nM that can suppress necroptosis in mouse and human cells. To develop a radiotracer for investigating the RIPK1 in vivo, we radiosynthesized [¹¹C]TZ7774 and performed preliminary in vitro and in vivo evaluations in rodents and macaque.

Synthesis of the desmethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [¹¹C]TZ7774 was achieved through TZ7790 reacting with [¹¹C]methyl iodide via N-methylation. Ex vivo biodistribution of [¹¹C]TZ7774 was performed in normal Sprague-Dawley rats. Characterization of [¹¹C]TZ7774 in response to inflammation was performed using ex vivo biodistribution study in normal and LPS treated (10 mg/kg) C57BL/6 mice, and in vitro autoradiography and immunohistochemistry of the spleen. MicroPET brain study of [¹¹C]TZ7774 in the macaque was also performed.

The radiosynthesis of [¹¹C]TZ7774 was achieved with good radiochemical yield (30-40%, decay corrected to the end of bombardment (EOB)), high chemical purity (>90%), high radiochemical purity (>99%), and high molar activity (>207 GBq/μmol, decay corrected to EOB). Biodistribution studies in Sprague-Dawley rats showed [¹¹C]TZ7774 has a high brain uptake of 0.53 (%ID/g) at 5 min post injection; pancreas, spleen, kidney, and liver also showed a relatively high initial uptake of 0.49, 0.41, 0.62, and 0.95 at 5 min respectively. Uptake of [¹¹C]TZ7774 increased in LPS-treated C57BL/6 mice by 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. In vitro autoradiography study also revealed increased uptake of [¹¹C]TZ7774 in the spleen of LPS-treated mice. Further characterization with immunohistochemistry confirmed increased expression of RIPK1 in red and white pulp of the spleen for mice pre-treated with LPS. MicroPET demonstrated that [¹¹C]TZ7774 had good initial brain uptake in macaque with an (SUV) of ∼3.7 at 6-10 min, and quickly washed out from brain. These data confirm successful radiosynthesis of a RIPK1 specific radiotracer [¹¹C]TZ7774. Our preliminary studies showed good response to LPS-induced inflammation in rodents and good uptake in macaque brain. [¹¹C]TZ7774 has a potential to image RIPK1 related necroptosis and inflammatory processes.