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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 IC 50 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 IC 90 values. Conclusions Tolebrutinib was the only candidate of the three that attained relevant CSF exposure in non-human primates. Graphical Abstract

Neuroinflammation in the central nervous system (CNS), driven largely by resident phagocytes, has been proposed as a significant contributor to disability accumulation in multiple sclerosis (MS) but has not been addressed therapeutically. Bruton’s tyrosine kinase (BTK) is expressed in both B-lymphocytes and innate immune cells, including microglia, where its role is poorly understood. BTK inhibition may provide therapeutic benefit within the CNS by targeting adaptive and innate immunity-mediated disease progression in MS. Using a CNS-penetrant BTK inhibitor (BTKi), we demonstrate robust in vivo effects in mouse models of MS. We further identify a BTK-dependent transcriptional signature in vitro, using the BTKi tolebrutinib, in mouse microglia, human induced pluripotent stem cell (hiPSC)-derived microglia, and a complex hiPSC-derived tri-culture system composed of neurons, astrocytes, and microglia, revealing modulation of neuroinflammatory pathways relevant to MS. Finally, we demonstrate that in MS tissue BTK is expressed in B-cells and microglia, with increased levels in lesions. Our data provide rationale for targeting BTK in the CNS to diminish neuroinflammation and disability accumulation. Bruton’s tyrosine kinase (BTK) is expressed in immune cells and microglia, where its role remains poorly understood. Here, the authors show that BTK modulates microglial neuroinflammatory pathways relevant to multiple sclerosis (MS) and report robust effects of BTK inhibition in human in vitro models and animal models of MS.

Current therapies for multiple sclerosis (MS) reduce both relapses and relapse-associated worsening of disability, which is assumed to be mainly associated with transient infiltration of peripheral immune cells into the central nervous system (CNS). However, approved therapies are less effective at slowing disability accumulation in patients with MS, in part owing to their lack of relevant effects on CNS-compartmentalized inflammation, which has been proposed to drive disability. Bruton tyrosine kinase (BTK) is an intracellular signalling molecule involved in the regulation of maturation, survival, migration and activation of B cells and microglia. As CNS-compartmentalized B cells and microglia are considered central to the immunopathogenesis of progressive MS, treatment with CNS-penetrant BTK inhibitors might curtail disease progression by targeting immune cells on both sides of the blood–brain barrier. Five BTK inhibitors that differ in selectivity, strength of inhibition, binding mechanisms and ability to modulate immune cells within the CNS are currently under investigation in clinical trials as a treatment for MS. This Review describes the role of BTK in various immune cells implicated in MS, provides an overview of preclinical data on BTK inhibitors and discusses the (largely preliminary) data from clinical trials.Bruton tyrosine kinase inhibitors are an emerging treatment for multiple sclerosis. Krämer et al. consider the evidence that central nervous system-penetrant Bruton tyrosine kinase inhibitors might target both peripheral immune cells and compartmentalized inflammation and discuss promising preliminary results of clinical trials of these agents in multiple sclerosis.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Tolebrutinib is an oral, brain‐penetrant, covalent Bruton's tyrosine kinase inhibitor in development to treat multiple sclerosis at 60 mg/day with food. A phase I trial was conducted in healthy volunteers to assess the safety and pharmacokinetics of tolebrutinib at oral doses higher than 60 mg with food and during fasting, and to determine cerebrospinal fluid (CSF) exposure after a single dose of 60 or 120 mg with food. The trial included double‐blind, placebo‐controlled single ascending dose (120, 240, and 300 mg; fed and fasted) and multiple ascending dose (120, 180, and 240 mg) arms. Additional open‐label cohorts received a single 60 mg dose with a high‐fat meal and during fasting using a crossover design or a single 60 or 120 mg dose with food and lumbar puncture to obtain CSF. Tolebrutinib was rapidly absorbed and converted to an active metabolite (designated “M2”), both of which had a terminal half‐life of ~5 h. Tolebrutinib and M2 exposures increased following administration with food versus fasting, and plasma levels were generally dose proportional. For up to 4 h (the last measurement timepoint) after a 60 mg dose, CSF concentrations of tolebrutinib exceeded its in vitro cellular potency (half‐maximal inhibitory concentration [IC50]) for microglia, and tolebrutinib and M2 surpassed their biochemical IC50. Tolebrutinib was well‐tolerated, and treatment‐emergent adverse events were generally mild. Concentration‐QTc modeling showed no effects on QT/QTc intervals for any tolebrutinib dose or fed status. In conclusion, tolebrutinib has an acceptable safety profile at supratherapeutic doses and achieved bioactive CSF exposures at the phase III dose.

Throughout the course of multiple sclerosis, gradually progressive neurologic impairment can occur, which has been called disability accrual. Current disease-modifying therapies for multiple sclerosis have limited effects on disability accrual unrelated to relapses, which is thought to be partially caused by chronic, nonresolving neuroinflammation within the central nervous system. Tolebrutinib is an oral, brain-penetrant Bruton's tyrosine kinase inhibitor that targets myeloid cells (including microglia) and B cells in both the periphery and central nervous system. There are no approved treatments for nonrelapsing secondary progressive multiple sclerosis. In a phase 3, double-blind, placebo-controlled, event-driven trial, we randomly assigned participants with nonrelapsing secondary progressive multiple sclerosis, in a 2:1 ratio, to receive tolebrutinib (60 mg once daily) or matching placebo. The primary end point was confirmed disability progression that was sustained for at least 6 months, assessed in a time-to-event analysis. A total of 1131 participants underwent randomization: 754 were assigned to receive tolebrutinib and 377 to receive placebo. The median follow-up was 133 weeks. A smaller percentage of participants in the tolebrutinib group than in the placebo group had confirmed disability progression sustained for at least 6 months (22.6% vs. 30.7%; hazard ratio, 0.69; 95% confidence interval, 0.55 to 0.88; P = 0.003). Serious adverse events occurred in 15.0% of the participants in the tolebrutinib group and in 10.4% of those in the placebo group. A total of 4.0% of the participants in the tolebrutinib group and 1.6% of those in the placebo group had increases in alanine aminotransferase levels to more than 3 times the upper limit of the normal range. In participants with nonrelapsing secondary progressive multiple sclerosis, the risk of disability progression was lower among those who received treatment with tolebrutinib than among those who received placebo. (Funded by Sanofi; HERCULES ClinicalTrials.gov number, NCT04411641.).

Tolebrutinib is an oral, brain-penetrant, and bioactive Bruton's tyrosine kinase inhibitor that modulates peripheral inflammation and persistent immune activation within the central nervous system, including disease-associated microglia and B cells. More data are needed on its efficacy and safety in treating relapsing multiple sclerosis. In two phase 3, double-blind, double-dummy, event-driven trials (GEMINI 1 and GEMINI 2), participants with relapsing multiple sclerosis were randomly assigned in a 1:1 ratio to receive tolebrutinib (60 mg once daily) or teriflunomide (14 mg once daily), each with matching placebo. The primary end point was the annualized relapse rate. The key secondary end point was confirmed worsening of disability that was sustained for at least 6 months, which was assessed in a time-to-event analysis that was pooled across trials. A total of 974 participants were enrolled in GEMINI 1, and 899 were enrolled in GEMINI 2. The median follow-up was 139 weeks. The annualized relapse rate in the tolebrutinib and teriflunomide groups was 0.13 and 0.12, respectively, in GEMINI 1 (rate ratio, 1.06; 95% confidence interval [CI], 0.81 to 1.39; P = 0.67) and 0.11 and 0.11, respectively, in GEMINI 2 (rate ratio, 1.00; 95% CI, 0.75 to 1.32; P = 0.98). The pooled percentage of participants with confirmed disability worsening sustained for at least 6 months was 8.3% with tolebrutinib and 11.3% with teriflunomide (hazard ratio, 0.71; 95% CI, 0.53 to 0.95; no formal hypothesis testing was conducted owing to the prespecified hierarchical testing plan, and the width of the confidence interval is not adjusted for multiple testing). The percentage of participants who had adverse events was similar in the two treatment groups, although the percentage with minor bleeding was higher in the tolebrutinib group than in the teriflunomide group (petechiae occurred in 4.5% vs. 0.3%, and heavy menses in 2.6% vs. 1.0%). Tolebrutinib was not superior to teriflunomide in decreasing annualized relapse rates among participants with relapsing multiple sclerosis. (Funded by Sanofi; GEMINI 1 and GEMINI 2 ClinicalTrials.gov numbers, NCT04410978 and NCT04410991, respectively.).

Inositol 1,4,5-trisphosphate (IP$_3$)-induced calcium release from intracellular stores is a regulator of cytosolic-free calcium levels. The subsecond kinetics and regulation of IP$_3$-induced calcium-45 release from synaptosome-derived microsomal vesicles were resolved by rapid superfusion. Extravesicular calcium acted as a coagonist, potentiating the transient IP$_3$-induced release of calcium-45. Thus, rapid elevation of cytosolic calcium levels may trigger IP$_3$-induced calcium release in vivo. Extravesicular calcium also produced a more slowly developing, reversible inhibition of IP$_3$-induced calcium-45 release. Sequential positive and negative feedback regulation by calcium of IP$_3$-induced calcium release may contribute to transients and oscillations of cytosolic-free calcium in vivo.

The results of a study suggest that multiple calcium2plus channel types coexist to regulate neurosecretion under normal physiological conditions in the majority of nerve terminals.

Presynaptic calcium channels are crucial elements of neuronal excitation-secretion coupling. In mammalian brain, they have been difficult to characterize because most presynaptic terminals are too small to probe with electrodes, and available pharmacological tools such as dihydropyridines and omega-conotoxin are largely ineffective. Subsecond measurements of synaptosomal glutamate release have now been used to assess presynaptic calcium channel activity in order to study the action of peptide toxins from the venom of the funnel web spider Agelenopsis aperta, which is known to inhibit dihydropyridine and omega-conotoxin-resistant neuronal calcium currents. A presynaptic calcium channel important in glutamate release is shown to be omega-Aga-IVA sensitive and omega-conotoxin resistant