The dual role of Bruton's Tyrosine Kinase Inhibitors in multiple sclerosis: from clinical efficacy to molecular modulation of EBV-Host interactions

Background Multiple sclerosis (MS) is an immune-mediated central nervous system disorder characterized by recurrent local neurological deficits and progressively accumulating neurodegenerative damage. As the disease progresses, patients experience varying degrees of motor, sensory, and cognitive dysfunction. Although current treatments have made progress in alleviating symptoms, reducing relapses, and slowing disease progression, they remain ineffective in controlling the accumulation of neurological damage, particularly in chronic progressive MS, where further control of neurodegenerative damage remains a therapeutic challenge. In recent years, the relationship between Epstein-Barr virus (EBV) and MS has garnered significant attention. EBV is a widely prevalent human virus, with approximately 95% of the global population being infected at some point in their lives. Studies suggest that EBV infection may be a critical trigger for MS development, particularly the immune dysregulation following EBV infection that may drive the immune pathogenesis of MS. EBV activates immune cells such as B cells and T cells, affecting immune regulation and promoting immune system attacks on the nervous system, leading to demyelination. Therefore, modulating EBV-related immune responses may provide a new therapeutic strategy for MS. Bruton’s tyrosine kinase (BTK) is a key tyrosine kinase widely present in immune cells such as B cells, T cells, dendritic cells, and microglia. It plays a central role in regulating multiple immune signaling pathways, particularly in B cell activation, antibody production, and T cell co-activation. In recent years, Bruton’s tyrosine kinase inhibitors (BTKis) have emerged as novel immunomodulatory drugs, demonstrating significant therapeutic potential in various immune-mediated diseases, especially in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Due to the central role of BTK in immune responses, BTKis are also considered potential candidates for the treatment of MS. Several BTKis, including Evobrutinib, Tolebrutinib, Fenebrutinib, and Orelabrutinib, have been developed for MS treatment. These drugs not only inhibit immune cell activation and reduce central nervous system inflammation but also possess good blood-brain barrier permeability, allowing them to directly target the central nervous system, thus providing new therapeutic targets for MS. The regulation of EBV-related immune responses by BTKis is of particular interest. Studies have shown that EBV activates immune responses through proteins such as LMP1 (latent membrane protein 1) and EBNA2 (EBV nuclear antigen 2), which may promote the development of MS. Therefore, BTKis may slow or halt the progression of MS by modulating EBV-related immune responses. This study systematically reviews and conducts a meta-analysis to evaluate the efficacy and safety of Evobrutinib, Tolebrutinib, Fenebrutinib, and Orelabrutinib in MS treatment. We also investigate two main objectives: 1) whether BTKi compounds affect molecules important in the EBV lifecycle in B cells, either directly through their viral origin or indirectly through exploitation by the virus for survival; and 2) whether there are differences in the therapeutic efficacy of various BTKi compounds. This research not only advances our understanding of how BTKis modulate EBV-related immune responses in MS but also provides insights into potential personalized treatment strategies, particularly for EBV-driven immunopathology, where BTKis may offer an effective treatment option. Methods In this study, we evaluated the efficacy and safety of BTKis in MS treatment through systematic review and meta-analysis. We conducted a literature search in the PubMed, Embase, Web of Science, and Cochrane Library databases from the inception of the databases to January 2025. According to the inclusion criteria, only randomized controlled trials (RCTs) involving MS patients with both treatment and control groups using BTKis and placebo were included. Studies with incomplete data or missing necessary information were excluded, resulting in 9 eligible studies. In statistical analysis, both random-effects and fixed-effects models were applied using R Studio and Stata 15.1 software. The primary effect size was the standard mean difference (SMD) risk ratio (RR), and 95% confidence intervals (CIs) were calculated. All studies were grouped according to the different dosages of BTKis (ranging from 5mg to 200mg) for subgroup analysis. Subgroup analyses were conducted based on drug concentration, treatment duration, and drug type to explore their potential effects on efficacy. The focus of data analysis was to assess the impact of BTKis on MS patients’ imaging endpoints, including new T1 Gd+ lesions, new or enlarged T2 lesions, relapse rates, and side effects. Additionally, in vitro experiments were conducted using peripheral blood mononuclear cells (PBMCs) from MS patients to isolate B cells for further processing. IC50 analysis was used to assess the inhibitory effects of the four BTKis on cell proliferation at different concentrations to determine the minimum effective concentration. Western blot (WB) and quantitative PCR (qPCR) analyses were used to evaluate the effects of Evobrutinib, Tolebrutinib, Fenebrutinib, and Orelabrutinib on the expression of MS-related proteins (such as BTK, LMP1, EBNA2, NF-κB, STAT3, ADAR1) and mRNA (such as EBNA2, LMP1, CD40, AICDA). Results The meta-analysis results showed that BTKi treatment significantly reduced the number of new T1 Gd+ lesions, with an SMD of 0.340, 95% CI of 0.108 to 0.573, and a p-value of less than 0.05. Additionally, BTKi treatment significantly decreased the number of new or enlarged T2 lesions, with an SMD of 0.349, 95% CI of 0.192 to 0.506, and a p-value of less than 0.001. These results indicate that BTKi treatment effectively reduces active lesions in the central nervous system of MS patients and slows disease progression. Furthermore, BTKi treatment significantly reduced relapse rates, with an effect size (ES) of 0.134, 95% CI of 0.096 to 0.173, and a p-value of less than 0.001, further validating its potential in improving clinical function. Regarding side effects, no significant differences were observed between the BTKi treatment and control groups (RR = 0.99, p = 0.759). Additionally, common adverse reactions, including headache (RR = 0.648, p = 0.942), elevated AST/ALT (RR = 0.91, p = 0.161), and rhinitis (RR = 1.39, p = 0.337), did not show significant differences in incidence. This suggests that BTKi has a favorable safety profile with no significant adverse effects in the treatment of MS. IC50 analysis revealed that Evobrutinib had IC50 values of 42.49 μM (24 hours) and 17.68 μM (48 hours), Tolebrutinib had IC50 values of 25.13 μM (24 hours) and 9.77 μM (48 hours), Fenebrutinib had IC50 values of 20.87 μM (24 hours) and 20.63 μM (48 hours), and Orelabrutinib had IC50 values of 20.63 μM (24 hours) and 15.01 μM (48 hours). These results indicate that Tolebrutinib significantly inhibits cell proliferation at lower concentrations, whereas Evobrutinib requires higher concentrations for significant inhibitory effects. In vitro WB analysis showed that Evobrutinib, Tolebrutinib, Fenebrutinib, and Orelabrutinib significantly downregulated the expression of BTK and LMP1 proteins. In qPCR analysis, Evobrutinib and Tolebrutinib significantly downregulated the expression of EBNA2, LMP1, and CD40 genes, while Fenebrutinib had a significant effect on LMP1 expression. Discussion: This study addressed two key objectives: 1) to verify whether BTKi compounds affect molecules that are important in the EBV lifecycle in B cells, either directly because of viral origin or indirectly through their exploitation by the virus for survival; and 2) to assess whether there are differences between various BTKi compounds in their therapeutic effects on MS. Our results indicate that BTKis, including Evobrutinib, Tolebrutinib, Fenebrutinib, and Orelabrutinib, modulate the expression of EBV-related proteins such as LMP1 and EBNA2, which are involved in MS pathogenesis. These findings suggest that BTKis effectively suppress EBV-related immune activation, potentially slowing excessive immune responses and neurodegeneration in MS patients. Dose-response analysis aimed at assessing differences between the various BTKi compounds showed that Tolebrutinib significantly inhibited cell proliferation at lower concentrations, exhibiting strong immunomodulatory effects. In contrast, Evobrutinib required higher concentrations to achieve significant inhibitory effects. Despite the weaker inhibitory effect of Evobrutinib, its immunological and clinical effects may remain relevant. Systematic review and meta-analysis demonstrated that BTKis significantly reduced the number of new T1 Gd+ lesions and new/enlarged T2 lesions, indicating their effectiveness in suppressing central nervous system activity-related inflammation. BTKi treatment also significantly reduced relapse rates, further proving its potential in improving clinical function. The study further highlighted the critical role of treatment dosage in BTKi efficacy. Higher-dose groups (≥60mg QD) showed significantly better results than lower-dose groups (<60mg QD), suggesting that increasing the dosage could significantly improve treatment outcomes. Future clinical treatments should adjust drug doses based on the individual patient to achieve optimal efficacy. However, the low-dose group showed no statistical significance, indicating limited effectiveness for MS treatment, especially in reducing central inflammation and neurodegeneration. Regarding side effects and safety, no significant differences were found between the BTKi treatment and control groups. Common side effects such as headache, liver enzyme elevation, and upper respiratory symptoms showed no significant difference between the two groups, providing a solid safety foundation for BTKi clinical applications. However, the long-term side effects and different patient responses across MS subtypes require further investigation. Sensitivity analysis confirmed the overall consistency of treatment effects, and although heterogeneity was present, most studies indicated stable efficacy, particularly in the high-dose group. Future research should further evaluate the long-term effects of BTKi treatment, particularly in large-sample populations with different MS subtypes.