Synthesis and In Vitro and In Vivo Evaluation of [³H]LRRK2-IN-1 as a Novel Radioligand for LRRK2

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 (IC₅₀, K _d , B _max, autoradiography) and in vivo (biodistribution, and blocking experiments) evaluations in rodents and human striatum tissues.

[³H]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 IC⁵⁰, K _d , and B _max 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.

In vitro binding studies demonstrated a saturable binding site for [³H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with K _d of 26 ± 3 and 43 ± 8, 48 ± 2 nM, respectively. In rat, the density of LRRK2 binding sites (B _max) 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 _max 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 [³H]LRRK2-IN-1 (%ID/g) by 50-60% in the kidney or brain.

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.