Synthesis and In Vitro and In Vivo Evaluation of [H]LRRK2-IN-1 as a Novel Radioligand for LRRK2.

Purpose: LRRK2 (leucine-rich repeat kinase 2) has recently been proven to be a promising drug target for Parkinson's disease (PD) due to an apparent enhanced activity caused by mutations associated with familial PD. To date, there have been no reports in which a LRRK2 inhibitor has been radiolabeled and used for in in vitro or in vivo studies of LRRK2. In the present study, we radiolabeled the LRRK2 ligand, LRRK-IN-1, for the purposes of performing in vitro (IC, K , B , autoradiography) and in vivo (biodistribution, and blocking experiments) evaluations in rodents and human striatum tissues.

Procedures: [H]LRRK2-IN-1 was prepared with high radiochemical purity (>99 %) and a specific activity of 41 Ci/mmol via tritium/hydrogen (T/H) exchange using Crabtree's catalyst. For IC, K , and B determination, LRRK2-IN-1 was used as a competing drug for nonspecific binding assessment. The specific binding of the tracer was further evaluated via an in vivo blocking study in mice with a potent LRRK2 inhibitor, Pf-06447475.

Results: In vitro binding studies demonstrated a saturable binding site for [H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with K of 26 ± 3 and 43 ± 8, 48 ± 2 nM, respectively. In rat, the density of LRRK2 binding sites (B ) was higher in kidney (6.4 ± 0.04 pmol/mg) than in brain (2.5 ± 0.03 pmol/mg), however, in human brain striatum, the B was 0.73 ± 0.01 pmol/mg protein. Autoradiography imaging in striatum of rat and human brain tissues gave results consistent with binding studies. In in vivo biodistribution and blocking studies in mice, co-administration with Pf-06447475 (10 mg/kg) reduced the uptake of [H]LRRK2-IN-1 (%ID/g) by 50-60% in the kidney or brain.

Conclusion: The high LRRK2 brain density observed in our study suggests the feasibility for positron emission tomography imaging of LRRK2 (a potential target) with radioligands of higher affinity and specificity.