Comparative CNS Pharmacology of the Bruton’s Tyrosine Kinase (BTK) Inhibitor Tolebrutinib Versus Other BTK Inhibitor Candidates for Treating Multiple Sclerosis

Abstract Background and objectives Tolebrutinib is a covalent BTK inhibitor designed and selected for potency and CNS exposure to optimize impact on BTK-dependent signaling in CNS-resident cells. We applied a translational approach to evaluate three BTK inhibitors in Phase 3 clinical development in MS with respect to their relative potency to block BTK-dependent signaling and exposure in the CNS Methods We used in vitro kinase and cellular activation assays, alongside pharmacokinetic sampling of cerebrospinal fluid (CSF) in the non-human primate cynomolgus to estimate the ability of these candidates (evobrutinib, fenebrutinib, and tolebrutinib) to block BTK-dependent signaling inside the CNS. Results In vitro kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib, while fenebrutinib, a classical reversible antagonist with a K i value of 4.7 nM and slow off-rate (1.54 x 10-5 s-1), also had an association rate 1760-fold slower (0.00245 μM-1 * s-1). Estimates of cellular potency were largely consistent with the in vitro kinase assays, with an estimated IC50 of 0.7 nM for tolebrutinib against 33.5 nM for evobrutinib and 2.9 nM for fenebrutinib. We then observed that evobrutinib, fenebrutinib, and tolebrutinib achieved similar levels of exposure in non-human primate CSF after oral doses of 10 mg/kg. However, tolebrutinib CSF exposure (4.8 ng/mL) (k p,uu CSF=0.40) exceeded the IC90 (the estimated concentration inhibiting 90% of kinase activity) value, while evobrutinib (3.2 ng/mL) (k p,uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp,uu CSF=0.15) failed to reach the estimated IC90 values. Conclusions Tolebrutinib was the only candidate of the three that attained relevant CSF exposure in non-human primates. Graphical Abstract