Spleen Tyrosine Kinase Inhibitor Research Articles

Heterogeneity of Patient-Derived Acute Myeloid Leukemia Cells Subjected to SYK In Vitro Inhibition

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal prognosis. The cytoplasmic spleen tyrosine kinase (SYK) is highly expressed by hematopoietic cells and has emerged as a potential therapeutic target. In this study, we evaluated the in vitro antileukemic effects of five SYK inhibitors, fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021, in a consecutive AML patient cohort. All inhibitors demonstrated a concentration-dependent antiproliferative effect, although there was considerable heterogeneity among patients. For fostamatinib and TAK-659, the antiproliferative effects were significantly higher in FLT3 mutated patients compared to nonmutated patients. Fostamatinib, entospletinib, TAK-659, and RO9021 induced significant apoptosis in primary AML cells, although the proapoptotic effects of the SYK inhibitors were less pronounced than the antiproliferative effects. Finally, most of the SYK inhibitors caused a significant decrease in the release of cytokines and chemokines from primary AML cells, indicating a potent inhibitory effect on the release of these leukemic signaling molecules. We concluded that the SYK inhibitors had antileukemic effects in AML, although larger studies are strongly needed to identify which patient subsets will benefit most from such a treatment.

Population Pharmacokinetics of Mivavotinib (TAK-659), a Dual Spleen Tyrosine Kinase and FMS-Like Tyrosine Kinase 3 Inhibitor, in Patients With Advanced Solid Tumors or Hematologic Malignancies.

Mivavotinib (TAK-659), an orally administered, small-molecule, dual inhibitor of spleen tyrosine kinase and FMS-like tyrosine kinase 3 (SYK/FLT3), is under development for the treatment of patients with advanced malignancies. In this analysis, we evaluated the population pharmacokinetics (PK) of mivavotinib and its sources of variability (covariates) in adult patients with advanced solid tumors, or relapsed/refractory B-cell lymphomas or acute myeloid leukemia, using pooled data from 159 patients enrolled in 2 phase 1/2 clinical studies. A 2-compartment model with first-order linear elimination and a first-order absorption rate (and associated lag time) adequately described the PK of mivavotinib in this patient population. The population estimates of apparent clearance (CL/F)and apparent central compartment volume (Vc /F)were 31.6 L/h and 893 L, respectively, resulting in a half-life of ≈20 hours. In the final model, creatinine clearance was included as a covariate of CL/F, and sex as a covariate of Vc /F. Simulations showed that steady-state exposure to mivavotinib increased with decreasing renal function. Expanding eligibility by enrolling patients with moderate renal impairment in phase 1 increased the diversity of patients in early trials and allowed the model to inform dose adjustment in patients with moderate renal impairment in future trials. In addition, simulations showed median steady-state trough concentration of mivavotinib following 70 mg twice daily and 160 mg daily dosing to be commensurate with 100 ng/mL, the level leading to >90% FLT3 inhibition per ex vivo plasma immune assays and considered a potential exposure threshold required for FLT3-driven efficacy.

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

A Phase I Open Label Study of Fostamatinib, a SYK Inhibitor, in Patients with Lower-Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

INTRODUCTION: Ineffective hematopoiesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) have been associated to upregulated innate immune and inflammatory signals. Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is highly expressed in hematopoietic cells and mediates signaling through Toll-like receptors and pattern recognition receptors leading to activation of the ERK, AKT, NF-kB and p38 signaling cascades. Given prior data suggesting that inhibition of these signals might be associated with improved hematopoiesis in MDS we hypothesized SYK inhibition might have therapeutic efficacy in lower-risk MDS and CMML. METHODS: To study this we designed an open label phase I clinical trial of fostamatinib, a SYK inhibitor, for patients with lower-risk MDS or CMML (defined as those with a score <3.5 by the revised International Prognostic Scoring System [IPSS-R]) and no response to prior erythroid stimulating agents or hypomethylating agents (HMAs). Patients with MDS with del(5q) must have received prior therapy with lenalidomide and those with MDS with ring sideroblasts must have received luspatercept. Patients had to be transfusion dependent, defined as prior RBC or platelet transfusions within the past 28 days, or have hemoglobin <10g/dL or platelets <75x109/L. Patients with profound neutropenia (ANC <0.5x109/L) where excluded. The study includes an intra-patient dose escalation design by which patients start therapy at a dose of fostamatinib of 100mg po bid on days 1-28 of a 28-day cycle. If no response to therapy, by IWG 2006 response criteria, is observed after 8 weeks of therapy dose will be escalated to 150mg po bid on days 1-28 of a 28-day cycle and then, in the absence of response after an additional 8 weeks of therapy, to 200mg po bid on days 1-28 of a 28 day cycle. Patients achieving no response after the second dose escalation will be discontinued from study (Figure 1a). The primary endpoint of the study is to assess the safety and tolerability of fostamatinib. Secondary objectives included overall response rates, transfusion independence and survival outcomes. The study includes futility and safety stopping rules. This study is registered at clinicaltrials.gov (NCT05030675). RESULTS: To date, a total of 5 patients have been treated on study as of July 2022 including 3 patients with MDS and ring sideroblasts. Patient characteristics are detailed in Figure 1b. Median age is 71 years (range 59-82). Median number of prior therapies was 5 (range 4-6) including one patient with a prior allogeneic stem-cell transplant. Four (80%) patients had received prior therapy with HMA. Two patients were transfusion dependent of RBC at the time of enrollment with one patient who was eligible based on the presence of thrombocytopenia. At the current data cut off, three patients developed treatment emergent adverse events the main including grade 1-2 diarrhea or constipation. One patient developed COVID-19 infection during the course of therapy and had mild symptomatology. Fostamatinib was not discontinued in this patient. No episodes of expected on-target uncontrolled hypertension or transaminitis have been observed so far. To date, only 4 patients have completed at least 2 cycles of therapy (8 weeks) and are eligible for response, and only 2 patients have dose escalated beyond 100mg po bid: 1 currently at the 150mg po bid dose level, and 1 at the maximum dose of 200mg po bid. With a median follow up of only 2.7 months (95% CI 0-5.6), no responses have been observed. All 5 patients remain on study: 3 (60%) at the 100mg po bid dose, 1 (20%) at 150mg po bid and 1 (20%) at 200mg po bid. CONCLUSIONS: Preliminary data suggests that treatment with fostamatinib at doses of up to 200mg po bid can be safely administered in patients with lower-risk MDS who have failed available therapies. Enrollment is ongoing to determine the potential efficacy of doses of up to 200mg bid. Additional correlative studies to determine predictors of response are underway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fostamatinib for the Treatment of Japanese Patients with Primary Immune Thrombocytopenia: A Phase 3, Placebo-Controlled, Double-Blind, Parallel-Group Study

Background Fostamatinib, a spleen tyrosine kinase inhibitor, has been approved for the treatment of chronic immune thrombocytopenia (ITP) in the US, Canada, and Europe. We conducted a phase 3, multi-center study to evaluate the efficacy and safety of fostamatinib in Japanese patients with ITP. The study was composed of 3 treatment periods and 1 non-dosing period: a placebo-controlled, double-blind study of fostamatinib for 24 weeks, an open-label study of fostamatinib for 28 weeks, a non-dosing period of up to 4 weeks, and the voluntary open-label study of fostamatinib until official approval. Here we report the results of the 24-week, placebo-controlled, double-blind, parallel-group study. Methods The study enrolled Japanese patients, ≥20 years old, with ITP for ≥6 months, and who had failed or not tolerated ≥1 prior ITP treatment. The mean of 3 screening and baseline platelet counts had to be <30,000/μL, with a platelet count at each visit < 35,000/μL. Patients were randomised 2:1 to fostamatinib or placebo for 24 weeks, stratified by baseline platelet count (< or ≥ 15,000/μL). Dosing was at 100 mg bid for 4 weeks and then 150 mg bid if needed and tolerated. One concomitant treatment was allowed (corticosteroids, azathioprine or danazol). Results The study included 34 patients, fostamatinib (n=22) or placebo (n=12). See Table 1 for baseline characteristics. Stable responses (platelets ≥50,000/μL at ≥4 of 6 visit, weeks 14-24) were observed in 8 (36%) patients on fostamatinib and 0 patients on placebo (P=0.030) (Figure 1). Overall responses (platelets ≥50,000/μL at ≥1 visit, weeks 2-12) were seen in 10 (45%) patients on fostamatinib and 0 patients on placebo (P=0.006). The median platelet counts of stable responders were higher than those of non-responders and patients on placebo throughout weeks 2 to 24. The median platelet counts of responders on fostamatinib increased ≥50,000/μL from the early stage of fostamatinib administration and stayed at around 100,000/μL from weeks 14 to 24, whereas non-responders on fostamatinib and patients on placebo remained below 30,000/μL most of the time. No platelet overshoot (> 400,000/μL) was observed. Rescue medication was required less often with fostamatinib, and fewer bleeding symptoms occurred with fostamatinib than with placebo. Adverse events in ≥10% of patients on fostamatinib were diarrhoea, hypertension, and decreased neutrophil count. Most adverse events were mild or moderate and manageable. There was no difference in the incidence of infection between the fostamatinib and placebo group; no patients developed a severe infection. No thromboembolic events were reported. The safety profile was consistent with prior studies of fostamatinib. Conclusions This study demonstrated the efficacy and safety of fostamatinib in Japanese patients with ITP, including those who had failed or were intolerant to other approved ITP therapies. No new safety signals were identified in Japanese patients with ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparative Effectiveness of Fostamatinib Vs. Rituximab in Refractory Chronic Immune Thrombocytopenia: A Network Meta-Analysis

Introduction Immune thrombocytopenia (ITP) is an autoimmune condition characterised by platelet destruction and insufficient platelet production. The American Society of Hematology ITP guidelines recommend rituximab, splenectomy, and thrombopoietin receptor agonists (TPO-RAs) as primary treatment options in patients with chronic ITP (Neunert C, et al. 2019). Fostamatinib, a spleen tyrosine kinase inhibitor, is approved for the treatment of chronic ITP in patients who have had an insufficient response to a previous treatment (FDA 2018, EMA 2020). Recent real-world evidence suggests that fostamatinib is used after a previous TPO-RA line (Hughes DM, et al. 2021). The objective of this network meta-analysis (NMA) was to estimate the relative efficacy of fostamatinib compared with current standard treatments after TPO-RAs in patients with refractory chronic ITP for overall platelet response. Methods A systematic literature review was conducted in 2019 and updated in July 2021. Six identified studies informed the NMA, including 2 placebo-controlled trials of fostamatinib (Bussel J, et al. 2018), 2 placebo-controlled trials of rituximab (Arnold DM, et al. 2012, Ghanima W, et al. 2015), 1 randomised trial of 3 rituximab regimens (Zwaginga JJ, et al. 2015) and 1 non-randomised study of 2 rituximab regimens (Zaja F, et al. 2012) (Figure 1). No studies assessing additional comparators of interest were found that could inform the NMA. The outcome of interest was overall platelet response, however definitions varied across studies in terms of platelet count thresholds and assessment time points, and several analyses were therefore performed to investigate sensitivity in variation in outcome definition. Analysis 1 used the published definitions for each study. Analysis 2 used an alternative standardised definition (platelet count >30,000/µL by week 4) for the fostamatinib trials, and the published definitions for each of the remaining studies. Analysis 3 used the alternative standardised definition for all studies and was restricted to those with results available: Bussel 2018 and Ghanima 2015. Possible treatment effect modifiers were evaluated by 8 British haematologists via a Delphi panel. None were identified and it was assumed that the differing baseline characteristics between studies did not have a significant impact on the outcome of interest. Random-effect models were employed in the analyses. The Odds Ratio (OR) for outcome occurrence of each treatment compared to fostamatinib together with the 95% credible intervals (CrI), 95% prediction intervals (PrI), median rank and probability of being the best treatment were estimated and are displayed in forest plots for each analysis. ORs<1 indicate reduced response relative to fostamatinib. All analyses were performed following the guidance of the National Institute for Health and Care Excellence Decision Support Unit and were implemented with WinBUGS and R. Results In analysis 1, pairwise comparisons indicated that fostamatinib was statistically significantly more effective than placebo and all rituximab regimens with ORs for comparators vs. fostamatinib ranging from 0.11 to 0.20 (Figure 2A). In analysis 2, fostamatinib was associated with improved response vs. placebo and all rituximab regimens (OR range 0.14 - 0.25) but statistical significance was only reached vs. placebo and rituximab 4 x 100 mg/week (Figure 2B). In analysis 3, pairwise comparisons suggested increased efficacy of fostamatinib relative to placebo and rituximab 4 x 375 mg/m2/week with ORs of 0.25 and 0.34, respectively. However, OR was only statistically significant vs. placebo (Figure 2C). The probability of fostamatinib being the most effective treatment ranged 0.91 - 0.97 across analyses; median rank for fostamatinib was 1 in all analyses (Figure 2). The between study standard deviations indicated moderate heterogeneity in treatment effects between studies. Conclusions The results from all analyses indicate that fostamatinib was associated with improved overall platelet response relative to rituximab at all regimens evaluated (4 x 375 mg/m2/week, 2/4 x 375 mg/m2/week, 2 x 750 mg/m2/week, 4 x 100 mg/week), and placebo. The use of rituximab offered no additional benefit with regards to achieving a platelet response than being on placebo. Fostamatinib may be a better alternative than rituximab to increase platelet counts in patients with refractory chronic ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterisation of Platelet Sensitivity in Severe COVID-19

Background: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterised by a hyperinflammatory and prothrombotic state. In the most severe cases, patients can develop lung complications such as pneumonitis and acute respiratory distress syndrome, frequently complicated by venous and arterial thrombosis. Recent studies suggest that platelets play a role as immune mediators, and may therefore contribute to the development of hyperinflammation and thrombosis in severe COVID-19 patients. The expression and signalling of Glycoprotein VI on platelets are dependent on its association and cross-linking with the dimeric Fc receptor γ-chain, which, upon platelet activation, leads to a signalling cascade involving the Spleen Tyrosine Kinase (SYK). Fostamatinib, a potent SYK inhibitor, has been approved for treatment of Immune Thrombocytopenia (ITP), and has been implicated in abrogating thrombosis by mitigating SYK signalling in platelets. We have recently shown that SYK inhibition can abrogate the thrombosis caused by cross-linking of COVID-19 antibodies. In this study, we assessed platelet reactivity using the Platelet Phenomic Analysis (PPAnalysis), which identifies platelet sensitivity and functional response as independent measures of platelet reactivity by flow cytometry. We hypothesised that platelets in patients with severe COVID-19 are hypersensitive to platelet agonists, in particular to the collagen-related peptide agonist, which involves the SYK pathway of platelet activation. Methods: Patients with COVID-19 pneumonia, recruited to the Multi-Arm Trial of Inflammatory Signal inhibitors for COVID-19 (MATIS) clinical trial - an interventional study assessing the impact of Fostamatinib (FOS) or Ruxolitinib (RUX) against Standard of Care (SOC) to prevent escalation of COVID-19 from moderate (WHO grades 3 and 4) to severe (WHO grade 5) - were included in this study, REC reference 20/HRA/2618. Samples were collected in citrated vacutainers, taken at day 0, before starting study medication or SOC, and at days 7, 14 and 28, where possible. Samples were processed immediately to obtain platelet-rich plasma (PRP). PRP was labelled with fluorescein isothiocyanate (FITC)-conjugated anti-fibrinogen and PE-Cy5-conjugated P-Selectin antibodies, and stimulated with increasing concentrations of the platelet agonists, adenosine diphosphate (ADP; 0.03-30 µM), collagen-related peptide (CRP; 0.003-3 µg/mL), and thrombin receptor activator peptide 6 (TRAP-6; 0.05-15 µM). Changes in platelet sensitivity, measured as LogEC50, were analysed using R. Results: 40 of 182 patients recruited into MATIS were included. A total of 50 COVID-19 samples (24 at day 0, 14 at day 7, 2 at day 14, and 10 at day 28) and 59 healthy control samples were processed using PPAnalysis. The 24 samples taken at day 0 showed extensive variability in platelet sensitivities between the responses to 3 agonists, with outliers both above and below the 95% centile of controls: 0-4% (P-Selectin response) and 13-42% (fibrinogen response) of patients displayed higher platelet sensitivity (below the 95% centile for controls), and 4-42% (P-Selectin response) and 8-16% (fibrinogen response) had lower sensitivity (above the 95% centile for controls), (Figure 1). Of the 9 patients with samples also taken at day 7, no patients showed an increase in platelet sensitivity from the P-Selectin response to all agonists. 5 patients showed reduced sensitivity. 2 patients in this cohort required non-invasive ventilation (NIV). Conversely, fibrinogen response to all agonists showed increased sensitivity at day 7 in 6 patients, with 1 patient requiring NIV, and reduced sensitivity was observed in 2 patients, both requiring NIV (Figure 2). Conclusions: Patients with severe COVID-19 display a large variability in platelet function, with either higher or lower platelet sensitivity to the agonists at day 0 when compared to controls. This implicates a role for aberrant platelet reactivity in COVID-19 and its associated prothrombotic state, and could contribute to the morbidity and mortality of patients. Interpretation of day 7 results at present is limited due to small numbers. However, it does show utility of the PPAnalysis, as a useful tool in the stratification of different patient cohorts, and in studying the associations between platelet function and disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Safety of Fostamatinib in Elderly Patients with Immune Thrombocytopenia: A Single-Center, Real-World Case Series

Background: Fostamatinib is an oral small molecule spleen tyrosine kinase (Syk) inhibitor that was approved for treatment of adult patients with immune thrombocytopenia (ITP) in second-line therapy [1]. Syk inhibition prevents cytoskeletal rearrangements during phagocytosis allowing platelet survival in ITP. The efficacy of fostamatinib in adults with chronic ITP has been established in phase 3 clinical trials [2], and further studies have established fostamatinib as an effective second-line treatment [3]. However, fostamatinib treatment in elderly patients with ITP has not been well established. These individuals pose a particular challenge with a higher risk of bleeding [4], having multiple comorbidities, and may be at increased risk of toxicity from treatments such as fostamatinib. Methods: We performed a retrospective review of all elderly patients (age greater than or equal to 65 years) who had started on fostamatinib between June 1, 2021 and May 31, 2022 for the treatment of ITP at a single tertiary care centre to evaluate its efficacy and safety. Results: Seven patients, median age 80 years (range 78 - 94), four women and three men, all Caucasian background, with various comorbidities started fostamatinib 100mg oral twice daily as second or subsequent line therapy. Patients had a diagnosis of ITP for a median 6 years (range approximately 6 months - 30 years), had six comorbidities (range 2 - 14), and experienced 2 unique prior lines of ITP therapy (range 1 to 6). Over 1290 days of fostamatinib exposure, two patients required dose escalation to 150mg oral twice daily while five patients remained on initial starting dose of 100mg twice daily in partial or complete response. The median platelet count at time of initiating fostamatinib was 25 x 109/L (range less than 10 - 193). Median time to response (defined any first platelet count greater than or equal to 30 x 109/L) was 19 days (range 0 - 181 days) with two patients responding rapidly (5 days and 19 days). Two patients required dose escalation, rescue therapy, and these same two patients discontinued fostamatinib after 175 days and 216 days of treatment. Treatment was tolerated in all patients with no thromboembolic events observed. One death was noted and unrelated to treatment. In keeping with the current recommendations, dose reduction was not required in any of the patients despite their advanced age. Conclusions: Fostamatinib was effective and safe for the majority of our very elderly patients with ITP in second and subsequent line therapies over a cumulative 1290 days of exposure.

A novel selective spleen tyrosine kinase inhibitor SKI-O-703 (cevidoplenib) ameliorates lupus nephritis and serum-induced arthritis in murine models.

Spleen tyrosine kinase (Syk) plays a pivotal role in the activation of B cells and innate inflammatory cells by transducing immune receptor-triggered signals. Dysregulated activity of Syk is implicated in the development of antibody-mediated autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the effect of Syk inhibition on such diseases remains to be fully evaluated. We have developed a novel selective Syk inhibitor, SKI-O-592, and its orally bioavailable salt form, SKI-O-703 (cevidoplenib). To examine the efficacy of SKI-O-703 on the progression of SLE, New Zealand black/white mice at the autoimmunity-established phase were administrated orally with SKI-O-703 for 16 weeks. Levels of IgG autoantibody, proteinuria, and glomerulonephritis fell significantly, and this was associated with hypoactivation of follicular B cells via the germinal center. In a model of serum-transferred arthritis, SKI-O-703 significantly ameliorated synovitis, with fewer neutrophils and macrophages infiltrated into the synovial tissue. This effect was recapitulated when mice otherwise refractory to anti-TNF therapy were treated by TNF blockade combined with a suboptimal dose of SKI-O-703. These results demonstrate that the novel selective Syk inhibitor SKI-O-703 attenuates the progression of autoantibody-mediated autoimmune diseases by inhibiting both autoantibody-producing and autoantibody-sensing cells.

The Efficacy and Safety of Fostamatinib in Elderly Patients with Immune Thrombocytopenia: A Single-Center, Real-World Case Series.

Fostamatinib is a small molecule spleen tyrosine kinase (Syk) inhibitor that was approved for the treatment of adult patients with immune thrombocytopenia (ITP) in second-line therapy. Syk inhibition prevents cytoskeletal rearrangements during phagocytosis, allowing platelet survival in ITP. However, fostamatinib treatment in elderly patients with ITP has not been well established. We performed a retrospective review of all elderly patients (age greater than or equal to 65 years) who had started on fostamatinib for the treatment of ITP at a single tertiary care centre to evaluate its efficacy and safety. Seven patients, median age 80 years (range 78-94), four women and three men, all of Caucasian background, with various comorbidities, started fostamatinib 100 mg orally twice daily as second or subsequent line therapy. Patients had a diagnosis of ITP for a median of 6 years (range approximately 6 months-30 years), had six comorbidities (range 2-14), and experienced 2 unique prior lines of ITP therapy (range 1 to 6). Over 1290 days of fostamatinib exposure, two patients required dose escalation to 150 mg orally twice daily, while five patients remained on the initial starting dose of 100 mg twice daily. The median platelet count at the time of initiating fostamatinib was 25 × 109/L (range less than 10-193). The median time to response (defined as any first platelet count greater than or equal to 30 × 109/L) was 19 days (range 0-181 days), with two patients responding rapidly (5 days and 19 days). Two patients required dose escalation and rescue therapy, and these same two patients discontinued fostamatinib after 175 days and 216 days of treatment. Treatment was tolerated in all patients with no thromboembolic events observed. One death was noted and unrelated to treatment. Overall, fostamatinib was effective and safe for the majority of these very elderly patients with ITP.

The role of organ-deposited IgG in the pathogenesis of multi-organ and tissue damage in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), often known simply as lupus, is a severe chronic autoimmune disease that is characterized by multi-organ and tissue damage and high levels of autoantibodies in serum. We have recently investigated, using animal models, the role of organ-deposited IgG autoantibodies in the pathogenesis of organ and tissue damage in SLE. We found that intra-organ injection of serum from mice with lupus (i.e., lupus mice) into healthy mice triggered inflammation in tissue and organs but that serum from other healthy mice did not, and that the severity of inflammation was related to the dose of serum injected. Immunohistochemistry showed that a large number of IgG molecules are deposited at the site of organ and tissue damage in lupus mice, and that IgG is a major contributor to the development of tissue inflammation triggered by serum from lupus mice or patients. The development of tissue inflammation induced by IgG in serum from lupus mice requires the presence of monocytes/macrophages, but not of lymphocytes or neutrophils; tumor necrosis factor (TNF)/tumor necrosis factor receptor 1 (TNFR1) and interleukin 1 (IL-1) also play essential roles in the development of tissue inflammation triggered by IgG. In addition, it has been found that TNFR1 inhibitors can suppress skin injury in lupus mice and that spleen tyrosine kinase (Syk) inhibitors, which can block the signaling transduction of IgG/Fc gamma receptors (FcγRs), can prevent and treat skin injury and kidney damage in lupus mice. We have also observed that lupus IgG might protect against bone erosion. Based on these results, we conclude that IgG plays a crucial role in the development of organ and tissue damage in SLE and in protecting bone erosion and arthritis, and we suggest that the IgG/FcγR signaling pathway is an important therapeutic target in SLE.

Evaluation of the Synergistic Potential of Simultaneous Pan- or Isoform-Specific BET and SYK Inhibition in B-Cell Lymphoma: An In Vitro Approach.

Simple SummaryB-cell lymphomas represent the majority of non-Hodgkin lymphomas and are the most common lymphoid malignancies in the Western world. Genetic alterations or epigenetic modulations can lead to tumor initiation and tumor progression. Aside from standard care, targeted, individualized therapies can be highly effective. Here, we evaluated the impact of simultaneous specific inhibition of two key regulators involved in B lymphoid tumor progression. Spleen tyrosine kinase (SYK) is a B-cell receptor-associated kinase acting as a proto-oncogene in B-cell malignancies, while bromodomain and extra-terminal domain (BET) proteins are epigenetic reader proteins involved in histone recognition and transcription regulation. The simultaneous inhibition of SYK and BET showed enhanced anti-proliferative effects, as well as inducing a distinct combination-specific gene expression profile, suggesting SYK and BET inhibition as a promising combination in the treatment of B-cell lymphoma.Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature.

Role of different types of spleen tyrosine kinase (SYK) inhibitors on reducing inflammation in human umbilical vein endothelial cell (HUVECs) previously induced by repetitive oxidative stress

Role of different types of spleen tyrosine kinase (SYK) inhibitors on reducing inflammation in human umbilical vein endothelial cell (HUVECs) previously induced by repetitive oxidative stress

Role of different types of spleen tyrosine kinase (SYK) inhibitors on reducing inflammation in human umbilical vein endothelial cell (HUVECs) previously induced by repetitive oxidative stress

Role of different types of spleen tyrosine kinase (SYK) inhibitors on reducing inflammation in human umbilical vein endothelial cell (HUVECs) previously induced by repetitive oxidative stress

Clinical Pharmacokinetics and Pharmacodynamics of Fostamatinib and Its Active Moiety R406.

Fostamatinib is the first approved spleen tyrosine kinase inhibitor for chronic immune thrombocytopenia. This review summarizes the clinical development, pharmacokinetics, pharmacodynamics, drug–drug interactions, adverse events, and comprehensive analyses of fostamatinib. While integrating these findings, we discuss the fostering and improvement of fostamatinib for further clinical applications. Fostamatinib is designed as a prodrug and cleavage of its active moiety R406 in the intestine. As R406 is the major product in the blood, this review mainly discusses the pharmacokinetics and pharmacodynamics of R406. It is metabolized by cytochrome 3A4 and UGT1A9 in the liver and is dominantly excreted in feces after anaerobic modification by the gut microbiota. As fostamatinib and R406 strongly inhibit the breast cancer resistance protein, the interaction with those substrates, particularly statins, should be carefully monitored. In patients with immune thrombocytopenia, fostamatinib administration started at 100 mg twice daily, and most patients increased to 150 mg twice daily in the clinical trial. Although responders showed a higher R406 concentration than non-responders, the correlation between R406 exposure and achievement of the platelet count as a pharmacodynamic marker was uncertain in the pharmacokinetic/pharmacodynamic analysis. Additionally, R406 concentration was almost halved in patients with a heavy body weight; hence, the exposure-efficacy study for suitable dosing should be continued with post-marketing data. In contrast, the pharmacokinetic/pharmacodynamic analysis for exposure safety revealed that R406 exposure significantly correlated with the incidence of hypertension. Even though the influence of elevated exposure on other toxicities, including diarrhea and neutropenia, is still unclear, careful management is required with dose escalation to avoid toxicity-related discontinuation.

3D-QSAR-Based Pharmacophore Modeling, Virtual Screening, and Molecular Dynamics Simulations for the Identification of Spleen Tyrosine Kinase Inhibitors

Spleen tyrosine kinase (SYK) is an essential mediator of immune cell signaling and has been anticipated as a therapeutic target for autoimmune diseases, notably rheumatoid arthritis, allergic rhinitis, asthma, and cancers. Significant attempts have been undertaken in recent years to develop SYK inhibitors; however, limited success has been achieved due to poor pharmacokinetics and adverse effects of inhibitors. The primary goal of this research was to identify potential inhibitors having high affinity, selectivity based on key molecular interactions, and good drug-like properties than the available inhibitor, fostamatinib. In this study, a 3D-QSAR model was built for SYK based on known inhibitor IC50 values. The best pharmacophore model was then used as a 3D query to screen a drug-like database to retrieve hits with novel chemical scaffolds. The obtained compounds were subjected to binding affinity prediction using the molecular docking approach, and the results were subsequently validated using molecular dynamics (MD) simulations. The simulated compounds were ranked according to binding free energy (ΔG), and the binding affinity was compared with fostamatinib. The binding mode analysis of selected compounds revealed that the hit compounds form hydrogen bond interactions with hinge region residue Ala451, glycine-rich loop residue Lys375, Ser379, and DFG motif Asp512. Identified hits were also observed to form a desirable interaction with Pro455 and Asn457, the rare feature observed in SYK inhibitors. Therefore, we argue that identified hit compounds ZINC98363745, ZINC98365358, ZINC98364133, and ZINC08789982 may help in drug design against SYK.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

Antithrombotic Effects of Fostamatinib in Combination with Conventional Antiplatelet Drugs.

New antithrombotic medications with less effect on haemostasis are needed for the long-term treatment of acute coronary syndromes (ACS). The platelet receptor glycoprotein VI (GPVI) is critical in atherothrombosis, mediating platelet activation at atherosclerotic plaque. The inhibition of spleen tyrosine kinase (Syk) has been shown to block GPVI-mediated platelet function. The aim of our study was to investigate if the Syk inhibitor fostamatinib could be repurposed as an antiplatelet drug, either alone or in combination with conventional antiplatelet therapy. The effect of the active metabolite of fostamatinib (R406) was assessed on platelet activation and function induced by atherosclerotic plaque and a range of agonists in the presence and absence of the commonly used antiplatelet agents aspirin and ticagrelor. The effects were determined ex vivo using blood from healthy volunteers and aspirin- and ticagrelor-treated patients with ACS. Fostamatinib was also assessed in murine models of thrombosis. R406 mildly inhibited platelet responses induced by atherosclerotic plaque homogenate, likely due to GPVI inhibition. The anti-GPVI effects of R406 were amplified by the commonly-used antiplatelet medications aspirin and ticagrelor; however, the effects of R406 were concentration-dependent and diminished in the presence of plasma proteins, which may explain why fostamatinib did not significantly inhibit thrombosis in murine models. For the first time, we demonstrate that the Syk inhibitor R406 provides mild inhibition of platelet responses induced by atherosclerotic plaque and that this is mildly amplified by aspirin and ticagrelor.

Pharmacological Inhibition of Spleen Tyrosine Kinase Suppressed Neuroinflammation and Cognitive Dysfunction in LPS-Induced Neurodegeneration Model.

Tyrosine-protein kinase (Syk) plays a potential role in neuroinflammation and adaptive immune responses in several neurodegenerative conditions. Seeing the significant role of Syk in the pathophysiology of neurodegeneration, several pharmacological inhibitors have been developed. One of the known inhibitors of Syk is BAY61-3606, which has shown efficacies in Alzheimer’s disease (AD) through regulating amyloid production. However, little is known about its efficacies in neuroinflammation and neurodegeneration. Our finding showed that Syk expression was up-regulated by lipopolysaccharide (LPS)-dependent manner, and BAY61-3606 significantly suppressed the activated microglia (ionized calcium-binding adaptor molecule 1 [Iba-1]) and the inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin 1-beta [IL-1β], IL-6) and other inflammatory mediators (nuclear factor kappa B [NF-κB], cyclooxygenase-2 [Cox-2], and inducible nitric axide synthase [iNOS]) in the lipopolysaccharide (LPS)-treated in vivo and in vitro models. Moreover, BAY61-3606 significantly reduced microglia-mediated neuronal cell death by regulating the expression of Cytochrome C and Bim (B-cell lymphoma 2 [BCL-2] interacting mediator of cell death) in the LPS-treated mice brain and HT22 cells. Furthermore, the expression of synaptic markers, synaptosomal-associated protein, 25 kDa (SNAP25), synaptophysin (Syp), and postsynaptic density protein-95 (PSD95) in LPS-challenged mice showed that BAY61-3606 significantly recovered the synaptic markers. Finally, we have analyzed the effects of BAY61-3606 against memory and cognitive dysfunctions in the LPS injected mice. The Y-maze test and Passive avoidance test suggested that BAY61-3606 significantly protected against LPS-induced cognitive and memory dysfunctions. The current findings not only highlight the mechanisms of Syk in the pathophysiology of neuro-inflammation, but also support the therapeutic efficacy of BAY61-3606 in the management of neurodegeneration.

Evaluating fostamatinib disodium as a treatment option for immune thrombocytopenia in adult patients

ABSTRACT Introduction Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased platelet destruction and decreased platelet production, leading to thrombocytopenia with or without bleeding manifestations. The majority of patients experiencing treatment need will eventually need secondary treatment following first-line therapy with steroids. In 2018, the oral spleen tyrosine kinase inhibitor fostamatinib received US Food and Drug Administration approval for ITP patients with an insufficient response to a previous treatment. Areas covered This review outlines pharmacological characteristics of fostamatinib and provides an overview of its efficacy and safety results in phase II and III trials, followed by the expert opinion of the authors. Expert opinion Increasing knowledge on the role of different players and mechanisms in the pathophysiology of autoimmune disorders, in general, and of ITP, in particular, has led to the development of several new treatment options, as illustrated by the introduction of fostamatinib in the treatment of ITP. However, lacking direct comparison with other recent treatment options (in particular, thrombopoietin receptor agonists), its use should be evaluated critically taking into account the unique toxicity and potential drug-drug interaction profile.