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

Abstract

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 (IC50, Kd, Bmax, autoradiography) and in vivo (biodistribution, and blocking experiments) evaluations in rodents and human striatum tissues. Procedures [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, Kd, and Bmax 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 [3H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with Kd of 26 ± 3 and 43 ± 8, 48 ± 2 nM, respectively. In rat, the density of LRRK2 binding sites (Bmax) 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 Bmax 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. 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.

Reduced LRRK2 in association with retromer dysfunction in post-mortem brain tissue from LRRK2 mutation carriers

“…It may be important to determine if LRRK2 mutation carriers have an earlier increase in LRRK2 protein levels, similar to the early increase reported for idiopathic Parkinson’s disease brain ( Dzamko et al , 2017 ). Novel radioligands for LRRK2 may make such studies possible in the future ( Malik et al , 2017 ). Moreover, it will be important to determine the relationship between LRRK2 protein and LRRK2 kinase activity, studies that could potentially be achieved by measuring phosphorylation levels of recently described LRRK2 substrates, such as the Rab GTPases ( Steger et al , 2016 ), when suitable techniques become available.…”

The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson’s Disease

“…Similarly, Wang M. et al (2017) described the radiolabeling of [ 11 C]-HG-10-102-01 but as with the Roche/Genentech probes, no in vitro or in vivo PET characterization of this molecule was described. Malik et al (2017) reported that they had successfully radiolabeled [ 3 H]-LRRK2-IN-1, however, its use as a CNS PET tracer is limited by poor off-target selectivity and limited brain penetration of the base molecule. Most recently, Chen et al, 2019 reported on the development of [ 11 C]-GNE-1023 and reported excellent in vitro specific binding of [ 11 C]-GNE-1023 to LRRK2 in rat and NHP brain sections ( Chen et al, 2019 ).…”