Background: Evobrutinib, a highly selective Brutons tyrosine kinase (BTK) inhibitor, demonstrated a low annualized relapse rate (75mg BID, 0.11; 95%CI 0.040.25) in a phase II, randomized, controlled trial in patients with relapsing multiple sclerosis (RMS; NCT02975349). This was maintained in an open-label extension (OLE) through 108 weeks (0.12; 95%CI 0.060.22, 75mg once-daily for ~48 weeks, then 75mg twice-daily). Evobrutinib concentrations in the brain achieved high BTK occupancy in an EAE model. We investigated evobrutinib distribution into cerebrospinal fluid (CSF) in patients with RMS. Objectives: An exploratory investigation of evobrutinib distribution into cerebrospinal fluid (CSF) relative to plasma concentration in patients with RMS, to further understand the activity of evobrutinib. Methods: Plasma and CSF samples were collected 23 hours post-dose from phase II OLE patients (75mg twice-daily for >1 year, including ?6 days uninterrupted dosing before sampling) and evobrutinib concentrations measured. Plasma protein binding was measured ex vivo in sub-study participant samples to determine free plasma concentrations of evobrutinib. Results: Nine patients from one center were enrolled: 89% female, median age 50.5 years and median disease duration 6.9 years (at phase II study baseline); all had stable RMS. In plasma, mean total evobrutinib (95%CI) was 115.0 ng/mL (71.8184.3) and free evobrutinib was 5.5 ng/mL. Evobrutinib was quantifiable in the CSF of all patients; the geometric mean concentration in CSF was 3.21 ng/mL (2.274.55); 58% of the free plasma concentration. Conclusions: Evobrutinib administered at an efficacious, steady state dose was detected in the CSF of patients with RMS at concentrations consistent with free plasma concentrations. Therefore, evobrutinib may inhibit BTK-expressing cells, including B cells and microglia, in the central nervous system. Role of the Study Sponsor: This study was sponsored by EMD Serono Research and Development Institute, Inc., an affiliate of Merck KGaA, Darmstadt, Germany.