Bruton’s tyrosine kinase inhibitors: a promising emerging treatment option for multiple sclerosis

1. Introduction

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination and neurodegeneration. Although its etiology is not fully understood yet, genetic, epidemiological, and pathological studies support the hypothesis that autoimmunity plays a major role in disease pathogenesis [1]. Classically, CD4⁺ T cells have been considered the primary drivers in MS. However, histopathological studies and the success of clinical trials with B-cell-depleting therapies indicate that the general view of MS as a condition meditated only by CD4⁺ T cells must be reassessed, and the role of CD8⁺ T cells, B cells, mast cells, and myeloid cells emphasized [2].

Over the past two decades, a significant number of disease-modifying treatments (DMT) have been approved for clinical use in patients with relapsing-remitting MS (RRMS). This has dramatically influenced the management of MS patients in daily practice and has positively influenced prognosis of a subset of patients. Nonetheless, DMT are not specific for MS, and given the heterogeneity of the disease, its use may be limited by intolerance, toxicity, or comorbidities that preclude the persistent use of these compounds. Furthermore, novel-targeted therapies should be studied in refractory patients and patients with progressive forms of the disease. Therefore, there remains an unmet need for new MS treatment.

Tyrosine kinases (TYKs) are intracellular enzymes mediating tyrosine phosphorylation of downstream molecules that participate in specific signaling pathways. Reversible phosphorylation is a major mechanism used by all cells, consequently TYKs have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival as well as apoptosis.

TYK comprise two general classes of molecules: receptor TYKs (RTYKs), and non-receptor TYKs (non-RTYKs [3,4]). The binding of a ligand (cytokine or growth factor) to RTYKs facilitates the dimerization of the receptor, its auto-phosphorylation, and the transfer of the phosphate from ATP to the hydroxyl groups of tyrosine residues on the receptor itself or on substrate proteins, both of which promote signal transduction. Likewise, activation of non-RTYKs, which are intracellular TYKs without a direct role in sensing extracellular cues, also occurs following phosphorylation of tyrosine residues on proteins by kinases, which activates downstream intracellular signaling pathways and cellular effector functions [3,4]. Therefore, given their role in multiple signaling processes and disease pathogenesis TYKs have emerged as excellent therapeutic targets for various diseases, resulting in the development of diverse TYKs inhibitors. Indeed, small molecule TYKs inhibitors have expanded the therapeutic armamentarium in oncology, and more recently in different autoimmune diseases [5]. These inhibitors typically, but not always, bind to the nucleotide-binding pocket of the catalytic domain, and can thereby modulate changes in the conformation of the molecule that are necessary for kinase activation [6].

Bruton’s tyrosine kinase (BTK), a member of the Tec family of kinase, is a cytoplasmic non-RTYKs that transmits signals via a variety of cell-surface molecules and is expressed by all cells of the hematopoietic lineage, except T cells, NK cells, and plasma cells [7]. BTK is also highly expressed by lung tissue, mainly localized in the membrane of alveolar epithelial cells [8].BTK is an essential component of different B cell receptor (BCR) signal pathways after antigen engagement, including the PI3K, MAPK, and NF-κB pathways (Figure 1), regulating the survival, activation, proliferation, and differentiation to antibody-producing plasma cells [9,10]. Additionally, BTK plays a critical role signaling through FcRγ-associated receptor in myeloid cells (macrophages and microglial cells), and has high affinity for IgE receptor in mast cells, leading to the secretion of pro-inflammatory cytokines as well as degranulation and histamine release. Moreover, BTK is a non-canonical pathway that is activated downstream of different Toll-like receptors (TLRs [11,12]). Therefore, inhibiting BTK could block the activation of different down-stream cell signaling pathways related to the development of B cell malignancies, and autoimmune diseases. BTK inhibitors can be classified into two types based on their mode of binding to BTK: i) Irreversible inhibitors form a covalent bond with the amino acid residue Cys481 in the ATP binding site of BTK, exerting a powerfully effective clinical benefit; ii) Reversible inhibitors, bind to specific pockets in the targets by weak, reversible forces (hydrogen bonds or hydrophobic interactions), determining an inactive conformation of the kinase. They can be easily removed, and as a result, they lack inhibitory potency and selectivity. Most of the molecules being active in the current clinical trials belong to irreversible BTK inhibitors [13].

Figure 1. BTK signal transduction pathways. After BCR is activated by phosphorylation, it recruits spleen tyrosine kinase (SYK) to the membrane where it is phosphorylated and subsequently phosphorylates Bruton’s tyrosine kinase (BTK). Phosphorylated BTK is recruited to the plasma membrane via autophosphorylation of Tyr223 in the SRC homology domain, and phosphorylates PLCγ2. Activated PLCγ2 hydrolyzes phosphatidyl inositol 4,5-biphosphate (PIP2), which results in the generation of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 upregulates calcium levels and DAG mediates activation of protein kinase Cβ (PKCβ). Increased of calcium levels, activation of PLCγ2, and PKCβ in turn promote the activation of the NF-κB-, ERK-, and NFAT-dependent pathways, which control the transcriptional expression of genes involved in proliferation, survival and cytokines secretion. In order to facilitate the understanding of the figure, some intermediate pathways have not been drawn. Ag: antigen; BCAP: B cell adaptor for PI3K; BCR: B cell receptor; BLNK: B cell linker; BTK: Bruton’s tyrosine kinase; DAG: diacylglycerol; ERK: extracellular-signal-regulated kinase; Igα/Igβ: signal transduction moiety (CD79); IKK: IκB kinase; IP3: inositol 1,4,5-triphosphate; NFAT: nuclear factor of activated T cells; NF-κB: nuclear factor κB; PKCβ: protein kinase Cβ; PLCγ2: phospholipase C-γ2. SYK: spleen tyrosine kina

The small molecule irreversible BTK inhibitor, ibrutinib is approved for the treatment of B cell malignancies, as well as graft versus host disease. However, ibrutinib inhibits a large number of kinases other than BTK, and the substantial off-target activity of the molecule preclude its evaluation in autoimmune diseases. To improve the overall tolerability of BTK inhibitors while maintaining the efficacy of ibrutinib, new BTK inhibitors have been developed [14].

Although previous studies using CD20-depleting antibodies in experimental autoimmune encephalomyelitis (EAE) have shown contradictory results, increasing or inhibiting monophasic chronic EAE in C57BL/6 mice [15–20], different TKIs and BTK have been explored in this animal model. Imatinib, sorafenib, and GW2580 can each effectively treat EAE. Imatinib and sorafenib abrogated platelet-derived growth factor (PDGF)-induced proliferation of astrocytes, whereas GW2580 and sorafenib suppressed TNF production by macrophages [21]. Tyrphostin AG126 has also proven to be beneficial in EAE inhibiting BTK and TLR-induced pro-inflammatory cytokine expression in microglial cells [22]. Evobrutinib is a new oral, irreversible BTK inhibitor with high kinase selectivity, which may be suitable for the chronic treatment of autoimmune diseases, inhibiting BCR- and FcRγ-mediated signaling. The administration of evobrutinib in EAE started 7 days before immunization inhibited antigen-triggered activation and maturation of B cells, and the release of pro-inflammatory cytokines. Furthermore, evobrutinib impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells. This ultimately reduces CNS infiltration and inflammation, leading to clinical amelioration of EAE [23]. Likewise, SAR442168, a brain penetrant BTK inhibitor that binds to its target covalently, has demonstrated protection from disease induction in a mouse model of EAE [24]. Together these observations have prompted the development of clinical investigations in different phenotypes of MS using BTK inhibitors (Table 1).

Table 1. Bruton’s Tyrosine Kinase inhibitor emerging drugs for the MS treatment

Compound	Company	Structure	Indication	Stage of development	Mechanism of action
SAR442168 Oral ClinicalTrials.gov Identifier: NCT03889639	Sanofi	•Allocation: Randomized •Control group: placebo •Intervention Model: Crossover Assignment •Masking: Double (Participant, Investigator) •Primary Purpose: Treatment (dose-finding Study) •Primary Objective: new Gd-enhancing lesions on MRI •Secondary Objectives: new T2 lesions and safety	Relapsing MS	Study Type: Interventional Phase: 2b Enrollment: 128 Recruitment Status: completed	Bruton’s Tyrosine Kinase Inhibitor
M2951 Other Name: Evobrutinib Oral ClinicalTrials.gov Identifier: NCT02975349	EMD Serono Collaborator: Merck KGaA,	•Allocation: Randomized •Control group: placebo, dimethyl fumarate •Intervention Model: Parallel Assignment •Masking: Double (Investigator, Outcomes Assessor) •Primary Purpose: Treatment (safety and effectiveness) •Primary Objective: new Gd-enhancing lesions on MRI •Secondary Objectives: ARR, EDSS, new T2 lesions and safety	Relapsing MS	Study Type: Interventional Phase: 2 Enrollment: 267 Recruitment Status: Active, not recruiting	Bruton’s Tyrosine Kinase Inhibitor
SAR442168 Oral ClinicalTrials.gov Identifier: NCT04410991	Sanofi	•Allocation: Randomized •Control group: placebo, teriflunomide •Intervention Model: Parallel Assignment •Masking: Triple (Participant, Investigator, Outcomes Assessor) •Primary Purpose: Treatment (Efficacy and Safety) •Primary Objective: AAR •Secondary Objectives: EDSS, MRI (new or Gd) lesions and safety	Relapsing MS	Study Type: Interventional Phase: 3 Estimated enrollment: 900 Recruitment Status: Active, recruiting	Bruton’s Tyrosine Kinase Inhibitor
SAR442168 Oral ClinicalTrials.gov Identifier: NCT04410978	Sanofi	•Allocation: Randomized •Control group: placebo, teriflunomide •Intervention Model: Parallel Assignment •Masking: Triple (Participant, Investigator, Outcomes Assessor) •Primary Purpose: Treatment (Efficacy and Safety) •Primary Objective: AAR •Secondary Objectives: EDSS, MRI (new or Gd) lesions and safety	Relapsing MS	Study Type: Interventional Phase: 3 Estimated enrollment: 900 Recruitment Status: Active, recruiting	Bruton’s Tyrosine Kinase Inhibitor
SAR442168 Oral ClinicalTrials.gov Identifier: NCT04458051	Sanofi	•Allocation: Randomized •Control group: placebo •Intervention Model: Parallel Assignment •Masking: Triple (Participant, Investigator, Outcomes Assessor) •Primary Purpose: Treatment (Efficacy) •Primary Objective: Confirmed Disability Progression •Secondary Objectives: MRI (new, Gd, brain volume loss) lesions, clinical/cognitive impairment and safety	Primary progressive MS	Study Type: Interventional Phase: 3 Estimated enrollment:990 Recruitment Status: not yet recruiting	Bruton’s Tyrosine Kinase Inhibitor
SAR442168 Oral ClinicalTrials.gov Identifier: NCT04411641	Sanofi	•Allocation: Randomized •Control group: placebo •Intervention Model: Parallel Assignment •Masking: Triple (Participant, Investigator, Outcomes Assessor) •Primary Purpose: Treatment (Efficacy) •Primary Objective: Confirmed Disability Progression •Secondary Objectives: MRI (new, Gd, brain volume loss) lesions, clinical/cognitive impairment and safety	Secondary progressive MS	Study Type: Interventional Phase: 3 Estimated enrollment: 1290 Recruitment Status: recruiting	Bruton’s Tyrosine Kinase Inhibitor
BIIB091 Oral ClinicalTrials.gov Identifier: NCT03943056	Biogen	•Allocation: Randomized •Control group: placebo •Intervention Model: Sequential Assignment •Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor) •Primary Purpose: Treatment (Safety, Tolerability, pharmacokinetic and Pharmacodynamic) •Primary Objective: Safety •Secondary Objectives: pharmacokinetic and Pharmacodynamic	Healthy Volunteer	Study Type: Interventional Phase: 1 Enrollment: 64 Recruitment Status: completed	Bruton’s Tyrosine Kinase Inhibitor

AAR: Annualized Relapse Rate, Gd: gadolinium, MRI: magnetic resonance imaging, EDSS: expanded disability status scale, MS: multiple sclerosis.

Recently, the results of a phase II trial comparing evobrutinib (25 mg daily, 75 mg daily or 75 mg twice daily) with placebo and using dimetilfumarate (DMF) as a reference in patients with clinical and imaging evidence of active RRMS, and those with secondary progressive MS with superimposed relapses were reported (NCT02975349). Patients with RRMS who received 75 mg of evobrutinib once daily had significantly fewer gadolinium-enhancing lesions 12 and 24 weeks after than those who received placebo. There were no significant differences in the annualized relapse rate or disability progression at any dose [25]. In another phase IIb trial, the BTK inhibitor SAR442168 was evaluated in RRMS patients. A 12-week, randomized, double-blind, placebo-controlled crossover trial (NCT03889639) assessed the response of 4 doses of the study drug (5 mg, 15 mg, 30 mg, and 60 mg), and placebo. After 12 weeks of treatment the data showed an 85% relative reduction in new gadolinium-enhancing lesions in the 60 mg group (primary endpoint), and 89% relative reduction in new or enlarging T2 hyperintense lesions (secondary endpoint) [26]. Interestingly, in secondary progressive MS brain autopsy samples, BTK expression was increased in the microglia in and around lesions. Of note, inhibition of BTK by SAR442168 reduces the expression of RGS1, a molecule implicated in chemokine receptor signaling in both T and B cells [27]. Based on the positive results of the phase II trials, both compounds are now being advanced to phase III evaluations. BIIB091 is another BTK inhibitor which is still in early stages of development, with its ongoing phase I clinical trial (NCT03943056) expected to reach primary completion sometime in 2020/2021 [28].

Specific mutations in BTK resulted in agammaglobulinemia, which is characterized by a paucity of B cells and circulating antibodies, highlighting the need of long-term evaluation, due to an increased risk of infections and/or non-responsiveness to vaccines in patients under treatment with BTK inhibitors. The roles of BTK inhibitors in different studies are diverse and even opposite. For example, the BTK inhibitor ibrutinib impairs polarization toward a pro‐inflammatory profile against Mycobacterium tuberculosis [29]. Similarly, in Staphylococcus aureus‐infected mice, BTK inhibition negatively regulates IL‐1β‐dependent bacterium clearance through impairing NLRP3 inflammasome activation and blocking IL‐1β release [30]. By contrast, in response to Listeria monocytogenes infections, bone marrow-derived macrophages from BTK^−/− mice show enhanced TNF‐α, IL‐6, and IL‐12p40 secretion, which increases the mean survival time of these animals after infection [31]. Likewise, the BTK inhibitor ibrutinib can ameliorate inflammatory myeloid cell responses by activation of macrophages, neutrophils infiltration, and cytokines secretion, to protect mice from pneumococcal pneumonia indicating that BTK inhibition might be beneficial to the host in bacterial infections [32]. Furthermore, opportunistic infections observed during BTK inhibitor therapy cannot be ignored. While the overall event rate remains low, continued vigilance and awareness of these adverse events are warranted. Likewise, data on the effect of BTK inhibitors on vaccine efficacy are limited and mixed [33,34]. Therefore, longer clinical trials are required to obtain more robust data on long-term safety.

2. Expert opinion

During the last two decades, an increasing number of molecules have been developed for the treatment of MS. However, given the heterogeneity of the disease and the existence of refractory patients to current treatment schemes, innovation of new therapeutic alternatives is required. On the other hand, because several sources of evidence indicate that both B cells and cells of the myeloid lineage are important drivers in the development of MS, there has been an abundant interest in developing BTK inhibitors. It is believed that BTK inhibitors can provide attractive therapeutic benefit for MS and other autoimmune diseases, as well as for hematological malignancies. BTK inhibitors would have some potential advantages over biological compounds. They are less likely to trigger antibody responses against the drug and consequent allergic reactions. However, we do not know whether BTK inhibitors will offer benefit to the one achieved by biological compounds, only head-to-head studies will be able to provide this answer accurately. Although ibrutinib provides a break-through for therapies of B cell malignancies, the possibility of resistance to treatment has emerged as a consequence of Cys481 mutations. On the other hand, BTK inhibitors for autoimmune diseases may present other limitations. First, although kinome-wide platforms have comprehensively identified the relevant kinases that are targeted by TKIs, recent studies have discovered non-kinase targets of TKIs. These non-kinase targets can contribute to the desired or undesired activities of inhibitors, or act as silent bystanders [35,36]. Although ibrutinib provides a break-through for therapies of B cell malignancies, the possibility of resistance to treatment has emerged as a consequence of Cys481 mutations. Second, the therapeutic effect (or lack thereof) in the animal model cannot always be reliable to be extrapolated to human MS patients. Third, BTK inhibitors might additionally inhibit other tyrosine kinases with structurally related cysteines (such as Cys773 in EGFR family kinases), that are not involved in MS, and thus be more likely to cause unrelated tissue damage that limits their therapeutic use. Therefore, given these limitations, the development of second-generation BTK inhibitors is necessary. Many reversible BTK inhibitors have been investigated for long-term administration, exclusively for the treatment of autoimmune diseases. However, there are still doubts as to their efficacy. The emergence of second-generation of BTK inhibitors has opened new expectations, and results of two-phase II trials in active MS using evobrutinib and SAR442168 have been encouraging. However, longer and larger trials are required to determine the efficacy and risks of these two molecules. Because BTK inhibitors have many uncertain or opposite characteristics in pathogenic microorganism infections, future studies should focus on elucidating the definite role of BTK in infectious diseases, as well as in responsiveness to vaccines.

The authors believe that more researches on BTK inhibitor’s family and other TKIs will lead to more refined therapies, driving the field toward personalized therapeutic approaches for MS. However, with hundreds of potential kinases to target, modern techniques that allow determination of cellular targets and selectivity data (e.g. KINOMEscan, recombinant enzyme panels, chemical proteomics, and in cell chemical proteomics), are critical for the development of the field.

Declaration of interest

E Carnero Contentti has received reimbursement for developing educational presentations, educational and research grants, consultation fees, and/or travel stipends from Biogen Argentina, Genzyme Argentina, Merck Argentina, Roche Argentina, Raffo, and Novartis Argentina. JC is a board member of Merck-Serono Argentina, Novartis Argentina, Genzyme LATAM, Genzyme global, Biogen-Idec LATAM, and Merck-Serono LATAM. He is part of the Steering Committee for the clinical trials of Ofatumumab (Novartis Global). J Correale has received reimbursement for developing educational presentations for Merck-Serono Argentina, Merck-Serono LATAM, Biogen-Idec Argentina, Genzyme Argentina, Novartis Argentina, Novartis LATAM, Novartis Global, and Roche Argentina, as well as professional travel/accommodation stipends. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgment

The authors are grateful to  Ms. Adriana Zufriategui for the design and drawing of the figure.