3.1. The Role of Tyk in the Pathophysiology of ITP

The exact underlying mechanism of ITP has not been elucidated yet; however, pathological antibody-mediated platelet clearance [34], T cell-mediated platelet destruction [15], and impaired function of MK are the three major known mechanisms in ITP development [6]. The primary clearance site for antibody-coated platelets is the spleen, where they bind to tissue macrophages through Fc receptors (FcRs) [34] and this is based on a mechanism dependent on Fcγ- pathway [35].

Classical immunoreceptors, such as BCRs, T cell receptors (TCRs), and various FcRs, facilitate the adaptive detection of both self and foreign antigens, either through direct or indirect mechanisms [36]. All the aforementioned receptors interact with transmembrane proteins that feature cytoplasmic domains incorporating immunoreceptor tyrosine-based activation motifs (ITAMs). Dual phosphorylation of ITAMs leads to the activation of Syk, which ultimately drives downstream signaling pathways. More specifically, Syk modulates various biological processes depending on the cell type and the receptor interacting with it, including mast cell and basophil degranulation, proinflammatory cytokine production by macrophages, monocytes, and dendritic cells, as well as antibody production by B cells in response to specific antigens [29, 37]. Moreover, the activation of FcγRs on myeloid cells initiates a Syk-dependent signaling pathway, which facilitates the internalization of IgG-opsonized antigens, cells, or pathogens via phagocytosis [29]. IgG autoantibodies that bind to self-antigens on cells, such as platelets and red blood cells, can form immune complexes that subsequently activate FcγRs. This process contributes to the pathogenesis of autoimmune disorders, including ITP. Syk’s diverse functional activity depends on multiple phosphorylation sites. Several phosphorylated residues of Syk serve as essential structural elements for recruiting downstream signaling proteins, thereby coordinating cellular responses [37].

BTK is involved in the signaling pathway of FcγRs, BCR signaling, Toll-like receptor (TLR), B-cell activating factor (BAFF) signaling, and chemokine receptor signaling [38, 39]. The interaction of specific antigens with the BCR leads to the phosphorylation of ITAMs through Lck/Yes-related novel Tyk (LYN), a member of the SRC family of nonreceptor tyrosine kinases [40]. This sequence of events recruits Syk, a nonreceptor protein Tyk which phosphorylates BLNK, a B-cell linker protein that functions as a scaffold to facilitate the interaction of BTK and phospholipase C-γ2 (PLCγ2). Ultimately, phosphorylated PLCγ2 followed by activation of the nuclear factor of activated T cells (NFAT), nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) pathways, stimulate cell proliferation and differentiation, and production of antibody and cytokine [40]. Moreover, BAFF/BAFF-R interaction also prevents Syk inhibition, as well as promotes BTK activation, which plays a crucial role in B-cell survival, activation, and Ig production [39].

Both Syk and BTK have been identified as critical regulators for Fcγ-mediated platelet destruction and Fcγ -receptor-mediated signaling [34, 41]. It has been established that, in murine models of ITP, antiplatelet antibodies are necessary, but not sufficient for disease induction; functional phagocytic FcγRs are also critical [42]. In a murine model of the inflammatory response, severe thrombocytopenia occurred in ITP mice models injected with an antiplatelet antibody (mAB 6A6); however, FcRγ-deficient mice did not develop anemia or thrombocytopenia, indicating that FcR signaling is necessary for murine ITP development [29, 43].

Since BTK is present in numerous hematopoietic stem cells and has a crucial role in both the adaptive and innate immune responses, it is a key therapeutic target in autoimmune disorders [44, 45]. BTK inhibition was found to lead to positive outcomes in some autoimmune diseases, including ITP [40]. For instance, mice lacking BTK were found to be protected against SLE and autoimmune arthritis [46]. It has also been illustrated that orelabrutinib prescription in mice was associated with a significant elevation in platelet count [41]. Furthermore, another study exhibited that pretreatment with rilzabrutinib, a BTK inhibitor, in mouse models of ITP induced by IgG antibodies resulted in a dose-dependent and rapid diminution in platelet destruction, effectively preserving the mice from ITP [44].

Currently, Syk inhibitors are under investigation in clinical trials for immune-mediated diseases, including ITP, due to the important role of Syk-mediated opsonized platelet phagocytosis in ITP pathogenesis [29]. Syk inhibition has demonstrated effectiveness in mitigating antibody-driven pathology across various rodent disease models, such as vasculitis, arthritis, autoimmune hemolytic anemia (AIHA), glomerulonephritis, and acute lung injury [29]. Additionally, Syk inhibition has been shown to improve thrombocytopenia and increase platelet and red blood cell counts in a murine antibody-mediated model of ITP [47]. All these findings emphasize the role of BTK and Syk as crucial factors in ITP pathogenesis and highlight the potential of targeting these pathways as a therapeutic option.

3.2. Syk Inhibitors

Fostamatinib is the only Syk inhibitor that has received FDA approval for treating refractory ITP cases [48]. Fostamatinib inhibits the phosphorylation of Syk substrates, thereby preventing the activation of T cells and B cells [49, 50]. This leads to the suppression of antibody-mediated and immune complex-mediated pathways, which are critical mechanisms of platelet destruction in ITP. A meta-analysis conducted on randomized clinical trials investigating fostamatinib in refractory ITP patients demonstrated that this medication was associated with superior efficacy in comparison with conventional therapy alone in terms of obtaining stable platelet response by week 24 as well as platelet count ≥50,000/µL at weeks 12 and week 24 [51]. Moreover, adverse events reported in the meta-analysis, as mentioned earlier, included diarrhea, hypertension, and abnormal liver function tests. Overall, most adverse events with fostamatinib therapy were mild to moderate, highlighting the suitable safety profile of fostamatinib. Notably, ITP cases are generally at a greater risk of thromboembolic events; however, patients treated with fostamatinib demonstrated a lower incidence rate of thromboembolic events compared to older therapies [52]. Fostamatinib decreases the incidence of thromboembolic events by inhibiting all three major Syk-driven signaling contributing to pathogenic platelet aggregation, including FcyRIIA gamma globulin receptor, glycoprotein VI (GPVI) receptor, and C-type lectin-like II receptor CLEC-2. It is noteworthy that fostamatinib does not affect other platelet receptors involved in hemostasis, thereby preventing unwanted bleeding [53, 54].

Sovleplenib and cevidoplenib are two novel Syk inhibitors under investigation for ITP management. Sovleplenib is a small molecule, potent, and selective oral Syk inhibitor that has been studied in one randomized, double-blind, placebo-controlled, Phase 3 study and one randomized, multicenter, open-label, Phase 2 study. In a Phase 1b/2 study performed by Liu et al. [18], 45 cases with primary ITP with at least 6 months duration that failed or relapsed after previous first-line treatment were treated with either sovleplenib or placebo over two phases: dose-escalation and dose-expansion phases, each with an 8-week, double-blind, placebo-controlled period, followed by a 16-week, open-label period with sovleplenib. During the dose-escalation phase, 33 cases of patients were divided into five groups based on the dosage of sovleplenib: Group1 (G1) received a placebo (n = 9), while G2 (n = 6), G3 (n = 6), G4 (n = 6), and G5 (n = 6) received 100, 200, 300, and 400 mg, respectively. Twelve subjects assigned to the dose-expansion phase were recruited in the dose-expansion phase, of which 10 patients received sovleplenib and two underwent a placebo. A response was characterized by a platelet count increase to 30 × 10⁹/L or higher, with at least a twofold rise from baseline, observed consistently over two consecutive visits within the first 8 weeks of the trial period, without the administration of rescue therapy. The most significant rise in platelet count was noted in the sovleplenib 300 mg group, with increased platelet levels detectable from week 1 among responders. The number of patients who achieved the primary efficacy endpoint with sovleplenib was three in the 100 mg group (50%), three in the 200 mg group (50%), 10 in the 300 mg group (63%), and two in the 400 mg group (33%), whereas only one patient in the placebo group (9%) met this endpoint. Moreover, the sovleplenib 300 mg group exhibited the highest overall response rate during the 0–8 weeks period, followed by the 100 mg group. Additionally, the quickest response to treatment occurred in the sovleplenib 300 mg group with a median time of 1.1 weeks. Phase 2 of the study was conducted with oral sovleplenib at a dosage of 300 mg for a duration of 16 weeks. In the 300 mg sovleplenib group, the overall response rate was 80%, comprising 16 of 20 participants who either received continuous sovleplenib or transitioned from placebo. Among those on continuous 300 mg sovleplenib, the durable response rate was 31%, while it was 75% for the four participants who crossed over from placebo to sovleplenib between weeks 0 and 24. This higher response rate may have occurred because the crossover group lacked prior exposure to the drug, potentially enabling them for a more effective response when they began treatment. Moreover, the subjects who transitioned from placebo to sovleplenib may have had relatively higher platelet counts and less resistant disease at the time they received the treatment. The safety profile of sovleplenib in patients with primary ITP was generally consistent across doses ranging from 100 to 400 mg. This safety profile, along with the activity and pharmacokinetic findings during dose escalation, led the safety review committee to recommend 300 mg of sovleplenib once daily as the Phase 2 dose. Most adverse events were classified as mild to moderate in severity and included elevated aspartate aminotransferase (29%), alanine aminotransferase (27%), and blood lactate dehydrogenase (22%). There were no treatment-emergent adverse events (TEAEs) that led to treatment discontinuation or death. Furthermore, none of the patients treated with sovleplenib experienced thrombotic events associated with the medication.

Hu et al. [17] conducted a study to assess the efficacy and safety of sovleplenib in patients with chronic primary ITP who had a median of four prior treatment lines, including agents such as anti-CD20 antibodies and TPO or TPO-RAs. The study was conducted in two phases: in the double-blind phase of the study, oral sovleplenib with a dosage of 300 mg was compared with a matched placebo in 188 adults for a duration of 24 weeks. As a result, the study achieved its primary endpoint, showing that the durable response (a platelet count of ≥50 × 10⁹/L on at least four of six scheduled visits between weeks 14 and 24) rate was significantly greater in the sovleplenib group compared to the placebo group. Moreover, at all postbaseline visits, the median platelet counts were higher in the sovleplenib group than in the placebo group. Sovleplenib also demonstrated a significantly higher overall response rate compared to placebo during both the 0–12 week and 0–24 weeks periods. A post hoc analysis also showed that the proportion of patients with a platelet count of 30 × 10⁹/L or more was significantly greater in the sovleplenib group than in the placebo group across both time intervals. During the double-blind phase, patients receiving sovleplenib required substantially less rescue medication than those on placebo. Additionally, more patients in the sovleplenib group either reduced or discontinued their baseline concomitant anti-ITP treatment (n = 11%, 27%) compared to the placebo group (n = 2%, 10%). The ability of the treatment to sustain stable platelet levels likely contributed to this outcome, as patients receiving sovleplenib were less prone to experiencing rapid platelet count declines that would require rescue therapy. This indicates that sovleplenib not only provided direct benefits in increasing platelet counts but also exerted a broader positive effect by reducing the need for additional medications, potentially improving the burden of long-term therapy. Additionally, sovleplenib induced a faster response, with a median time to response of 8 days, compared to 30 days for a placebo. Notably, sovleplenib demonstrated a more rapid onset of action, with a median time to response of 8 days, whereas fostamatinib [55] (with biweekly visits) and TPO [56] or TPO-RAs [57] (with weekly visits) typically required more than 14 days to elicit a response. The incidence of bleeding, as measured by the WHO bleeding score, showed a decreasing trend from baseline to week 24 in the sovleplenib group. Regarding safety, most adverse events were mild to moderate, and sovleplenib was tolerable. Notably, 125 out of 126 cases in both groups experienced TEAEs of any grade. Moreover, four patients (3%) in the sovleplenib group experienced TEAEs that led to discontinuation, including increased blood creatinine (Grade 1), weight gain (Grade 1), and hemorrhage (Grade 3), each affecting one patient, while another patient experienced elevated alanine aminotransferase (Grade 3) and aspartate aminotransferase (Grade 2). In the open-label phase of the investigations, cases of both groups who did not respond in the first 12 weeks of the study could terminate the double-blinded phase and enroll in the open-label study receiving sovleplenib 300 mg once daily, which is still ongoing (NCT05029635).

Cevidoplenib is another novel Syk inhibitor that is under investigation in patients suffering from persistent or chronic ITP who failed or relapsed after at least one prior therapy (NCT04056195) [58]. Cevidoplenib is a selective Syk inhibitor engineered to target downstream signaling of B-cell and FcRs without affecting T-cell functions or other pathways involved in cytokine-mediated inflammation. The preliminary results of this Phase 2 trial have illustrated that the platelet response, defined as a count of at least 30,000/µL and doubled their baseline total at any point during the treatment period without the use of rescue medication, occurred in 64% of cases underwent 400 mg of cevidoplenib compared to 33% of subjects who received placebo.

3.3. BTK Inhibitors

As of yet, two BTK inhibitors, rilzabrutinib and orelabrutinib, have been examined in ITP management.

Rilzabrutinib is an orally available, selective, and reversible covalent inhibitor targeting BTK, particularly developed for immune-mediated disease management [30]. Its therapeutic action operates via two primary mechanisms: (1) suppression of B-cell activation and (2) inhibition of FcγR-driven phagocytosis of antibody-coated cells in the spleen and liver. Rilzabrutinib has depicted favorable outcomes in two clinical trials published to date. In the open-label, dose-finding Phase 1/2 clinical trial carried out by Kuter et al. [31], the efficacy and safety of rilzabrutinib were investigated in 60 patients with chronic or refractory ITP who did not respond to prior medications. Patients received rilzabrutinib at one of the following dosing regimens: 200 mg once daily, 400 mg once daily, 300 mg twice daily, or 400 mg twice daily for a duration of 24 weeks. Consequently, 40% of participants achieved the primary endpoint, which was described as a platelet count of ≥50 × 10⁹/L with an increase of at least 20 × 10⁹/L from the pretreatment baseline. Distinctively, higher doses of rilzabrutinib were associated with superior outcomes, with 18 out of 45 patients who underwent rilzabrutinib at the highest dose achieving the primary endpoint. Key therapeutic objectives for individuals with ITP include achieving a rapid elevation in platelet count to levels sufficient to prevent bleeding and maintaining this response over time. In this regard, rilzabrutinib therapy resulted in rapid platelet responses, with clinically significant platelet increases observed in 40% of patients, with the median time to obtain a platelet count of at least 50 × 10⁹/L was 11.5 days. Considering safety, all TEAEs were mild to moderate, and the investigation reported no treatment-related bleeding, severe adverse events, infections, deaths, or typical signs and symptoms commonly associated with other irreversible BTK inhibitors.

In the long-term extension of the prior open-label Phase 1/2 clinical trial, 16 patients who responded during the initial 24-week investigation continued rilzabrutinib treatment at a dosage of 400 mg twice daily [32]. After a median treatment duration of 1.3 years, 11 out of 16 subjects remained on rilzabrutinib therapy, with 93% of these patients achieving a platelet count of ≥50 × 10⁹/L in over half of their monthly assessments. Moreover, five out of 11 subjects who were prescribed concomitant therapy for their ITP could cease their concomitant medications. With respect to safety, three cases experienced mild to moderate TEAEs, including Grade 2 upper respiratory tract infection, rhinorrhea, vulvovaginal dryness, and Grade 1 cough and diarrhea. Moreover, serious AEs were reported in three patients; all events were determined to be unrelated to the study treatment. Importantly, rilzabrutinib was not associated with bleeding, arrhythmia, or other AEs typically linked to nonselective BTK inhibitors. Furthermore, despite a very low rate of bleeding at the start of the study, bleeding scores improved even further with continued rilzabrutinib treatment in the long-term extension. Compared to other treatments, in an open-label extension study, romiplostim treatment resulted in achieving a platelet response of ≥50 × 10⁹/L at least once in 95% of patients, with the response being sustained for a median of 92% of visits [59]. However, rescue medication was required in 33% of patients, and TEAEs occurred in 35% of patients, with 8% classified as serious. Notably, bleeding events were observed in 57% of patients, while thrombotic events occurred in 6.5%. In another clinical trial conducted on eltrombopag [60], a remarkable proportion, 32% of patients, experienced Grade ≥3 AEs (6% had thromboembolic events) despite clinical effectiveness. Currently, rilzabrutinib at a dosage of 400 mg twice daily is under investigation in adolescents and adults with ITP in a multicenter, double-blind, and placebo-controlled Phase III study (LUNA 3, NCT04562766) [30]. The upcoming results of this study are still awaited.

Orelabrutinib, a next-generation BTK inhibitor, offers enhanced selectivity and sustained inhibition of BTK while minimizing off-target impacts [61]. In a Phase 2 clinical trial carried out by Yan et al. [33], 33 patients with chronic or persistent ITP were assessed for the effectiveness and tolerability of Orelabrutinib therapy. Patients were divided into two groups based on the Orelabrutinib dosage (G1:30 mg and G2:50 mg). Out of the 18 patients in the 30 mg group, 13 had their dosage increased to 50 mg by week 4. The primary endpoint of platelet response was reached by 12 patients (36%): six out of 15 patients in G2 and six out of 18 patients in G1, including two of the 13 patients who transitioned from G1 to G2. The sustained response rate was 27% in G2 and 33% in G1, including two of the 13 patients who transitioned from G1 to G2. Of the 12 patients who reached the primary endpoint, 83% maintained a sustained response. G2 achieved a notably quicker median time to an initial platelet count increase than G1. G2 had the lowest rate of patients using concomitant rescue therapy, with six out of 15 patients (40%) reported to have done so. Notably, in cases that had previously shown a response to glucocorticoids or IVIG, the primary endpoint response rate in the 50 mg group was reported in 75% of patients. This finding highlighted that orelabrutinib may demonstrate elevated efficacy in individuals who have responded to prior first-line treatments. The efficacy results of orelabrutinib were similar to those of rilzabrutinib in a Phase 1/2 study, which showed primary and sustained response rates of 40% and 28%, respectively [32]. A total of 28 patients (85%) reported side effects, the majority of which were mild (Grades 1 or 2), and only 24% were deemed treatment-related. One patient in G1 had a TRAE that required treatment cessation due to Grade 2 pneumonia. No serious or Grade 3 TRAEs or fatalities were reported.