Radiosynthesis and evaluation of a high affinity GSK3 ligand [¹¹C]CMP

Objectives: Glycogen synthase kinase-3 (GSK3) plays significant role as a signaling mediator and it deals with more than 100 substrates in most cells. Dysfunction of GSK3 is implicated in the etiology of a variety of psychiatric disorders, neurological disorders, inflammatory diseases and cancer. Non-invasive and in vivo detection of the changes in GSK3 expression using PET imaging can impact the choice of therapy and enable monitoring the progress of treatment. In this presentation, we report the automated radiochemical synthesis and evaluation of a high affinity GSK3 ligand [¹¹C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide ([¹¹C]CMP) (IC50 = 3.4 nM).

Methods: Synthesis of nonradioactive CMP was achieved by the coupling of 2-(cyclopropanecarboxamido) isonicotinic acid and 4-methoxypyridin-3-amine. Desmethyl-CMP was obtained by demethylation of CMP with BBr₃. The radiochemical synthesis of [¹¹C]CMP was optimized and automated on a GE-FX2MeI/FX2M radiochemistry module by alkylating the corresponding desmethyl-CMP precursor with [¹¹C]MeI in DMF in presence of NaOH. The cell uptake of [¹¹C]CMP was determined in glioblastoma U251 culture at 5, 30 and 60 minutes in quadruplicate. Dynamic microPET acquisition of [¹¹C]CMP was performed in anesthetized nude mice (n=3) with Trifoil mPET/CT for 60 minute. Effect of p-GP to brain uptake of [¹¹C]CMP was tested by administering 50 mg/kg (i.v) injection of cyclosporine 60 minute prior to injection of the radiotracer. Results: Synthesis of CMP and desmethyl-CMP were accomplished with 75% and 85% yields respectively. [¹¹C]CMP was produced in high radiochemical purity (>98%) and specific activity (2.2+0.3 Ci/μmol) in 25+5% radiochemical yield, decay corrected to EOS (n >10). The radioligand exhibited modest uptake in U251 cells (15+3.85%) at 30 minute incubation time with ~50% specific binding. PET studies in mice indicated no blood-brain barrier (BBB) penetration of [¹¹C]CMP despite its high affinity and suitable logP (ClogP= 1.1) value for BBB penetration. Administration of cyclosporine prior to 60 minute prior to [¹¹C]CMP administration showed some uptake of radioactivity that is retained in brain.

Conclusions: We report an automated radiolabeling procedure for a high affinity GSK3 ligand [¹¹C]CMP achieving excellent purity and specific activity. [¹¹C]CMP showed moderate uptake and specific binding in glioblastoma cells. Initial mPET imaging in mice showed no BBB penetration; however, a modest effect of cyclosporine on the brain uptake of [¹¹C]CMP indicates failure of BBB penetration is due to efflux transporters such as p-GP. Research support: MH112037, WFSM CTSA TIP (UL1TR001420)