Predictive Markers of LRRK2 Inhibition in Biofluids

Abstract

Background: Hyper-activated LRRK2 is linked to Parkinson’s disease susceptibility and progression. Quantitative measures of LRRK2 inhibition, especially in the brain, may be critical in the development of successful LRRK2- targeting therapeutics. Methods: Three structurally distinct, brain-penetrant, and selective LRRK2 small-molecule kinase inhibitors (PFE360, MLi2, and RA283) were orally administered to groups of cynomolgus macaques at different doses. Biofluid markers with proposed predictive value for assessing LRRK2 inhibition were measured from samples of blood, urine, and cerebral-spinal fluid (CSF). Findings: LRRK2 kinase inhibition led to consistent reduced pS935-LRRK2 and pRab10 proteins normalized to β- actin in blood mononuclear cells, reduced exosome-total LRRK2 protein normalized to TSG101 and didocosahexaenoyl (22:6) bis (monoacylglycerol) phosphate normalized to creatinine in urine, and reduced exosometotal LRRK2 and autophosphorylated pS1292-LRRK2 protein normalized to flotillin in CSF. Incomplete LRRK2 kinase inhibition reduced LRRK2 protein secretion in exosomes, whereas high drug exposures reduced both exosome and cellular levels of LRRK2 protein. Interpretation: Orthogonal pairs of markers for LRRK2 inhibition were identified in urine and CSF that can be used in combination with blood markers to non-invasively monitor the potency of LRRK2-targeting therapeutics in the brain and periphery. Funding Statement: Research support was provided by NIH U01 NS097028, R01 NS064934, and experiments were further supported through internal research programs at Atuka Inc, Sanofi, and Nextcea Inc. Declaration of Interests: S.W., K.K., and A.B.W. declare no competing conflicts of interest. L.D., S.B., and N.S. are employees of Sanofi and have shares in Sanofi. J.B.K. and J.M.B. are employees of Atuka and have shares in Atuka Inc. F.H. and E.T. are employed by Nexctea which holds patent rights to the di-22:6-BMP biomarker for neurological diseases involving lysosomal dysfunction (US 8,313,949, Japan 5,702,363, and Europe EP2419742). Ethics Approval Statement: All procedures were performed in full compliance with standards for the care and use of laboratory animals, according to French and European Community (Directive 2010/63/EU) legislation, and approved by the local Animal Ethics Committee and the French Ministry for Research, under authorization H94-002-4 arrete 2015-160.