Effects of CYP3A4 Inhibitors Ketoconazole and Verapamil and the CYP3A4 Inducer Rifampicin on the Pharmacokinetic Parameters of Fostamatinib: Results from In Vitro and Phase I Clinical Studies.

BACKGROUND: Fostamatinib (R788) is a spleen tyrosine kinase (SYK) inhibitor. The active metabolite of fostamatinib, R406, is primarily metabolized by CYP3A4. OBJECTIVES: The aim of this study was to characterize hepatic microsomal metabolism of R406 and confirm the role of CYP3A4 in R406 metabolism, determining whether co-administration of CYP3A4 inhibitors (ketoconazole, verapamil) or inducers (rifampicin) affects R406 pharmacokinetics. METHODS: R406 stability was determined using human hepatic microsomes. The CYP450 isoforms responsible for R406 metabolism in humans were identified using expressed CYP450 isoforms and specific chemical inhibitors. The ketoconazole interaction study (double-blind, randomized, placebo-controlled, two-period crossover) involved fostamatinib administration (single 80-mg dose), alone and with ketoconazole (200 mg twice daily). The verapamil and rifampicin interaction studies (open-label, two-period, fixed-sequence) involved fostamatinib administration (single 150-mg dose), alone and with immediate-release verapamil (80 mg three times daily) or rifampicin (600 mg once daily). Standard pharmacokinetic parameters were calculated in all studies. RESULTS/DISCUSSION: Hepatic microsomes showed time-dependent loss of R406 and formation of para-O-demethylated R406. Microsomal metabolism of R406 was markedly inhibited by CYP3A4 inhibitors and, in the expressed CYP450 studies, the rate of R406 disappearance was greatest with CYP3A4. In the clinical studies, co-administration of ketoconazole caused a 2-fold (CI 1.77-2.30) increase in R406 exposure. Verapamil increased R406 exposure (39% increase, CI 8-80), whereas rifampicin co-administration decreased exposure by 75% (CI 68-81). Fostamatinib was well tolerated. CONCLUSION: The oxidative metabolism of R406 is predominantly catalyzed by CYP3A4. In clinical studies, exposure to R406 is affected by concomitant administration of CYP3A4 inducers/inhibitors. These findings should be taken into account when considering co-prescription of fostamatinib with such agents.

Effects of fostamatinib (R788), an oral spleen tyrosine kinase inhibitor, on health-related quality of life in patients with active rheumatoid arthritis: analyses of patient-reported outcomes from a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To assess the influence of fostamatinib on patient-reported outcomes (PRO) in patients with active rheumatoid arthritis and an inadequate response to methotrexate (MTX). METHODS: Patients taking background MTX (N = 457) were enrolled in a phase II clinical trial (NCT00665925) and randomized equally to placebo, fostamatinib 100 mg twice daily (bid), or fostamatinib 150 mg once daily (qd) for 24 weeks. Self-administered PRO measures included pain, patient's global assessment (PtGA) of disease activity, physical function, health-related quality of life (HRQOL), and fatigue. Mean change from baseline and a responder analysis of the proportion of patients achieving a minimal clinically important difference were determined. RESULTS: At Week 24, there were statistically significant improvements in pain, PtGA, physical function, fatigue, and the physical component summary of the Medical Outcomes Study Short Form-36 (SF-36) for fostamatinib 100 mg bid compared with placebo. Mean (standard error) changes from baseline in the fostamatinib 100 mg bid group versus the placebo group were -31.3 (2.45) versus -17.8 (2.45), p < 0.001 for pain; -29.1 (2.26) versus -16.7 (2.42), p < 0.001 for PtGA; -0.647 (0.064) versus -0.343 (0.062), p < 0.001 for physical function; 7.40 (1.00) versus 4.50 (0.94), p < 0.05 for fatigue; 8.52 (0.77) versus 4.90 (0.78), p < 0.01 for SF-36 physical component score; and 3.99 (0.93) versus 3.71 (0.99), p = 0.83 for SF-36 mental component score. Patients receiving fostamatinib 150 mg qd showed improvements in some PRO, including physical function. CONCLUSION: Patients treated with fostamatinib 100 mg bid showed significant improvements in HRQOL outcomes.

Pharmacokinetics of fostamatinib, a spleen tyrosine kinase (SYK) inhibitor, in healthy human subjects following single and multiple oral dosing in three phase I studies.

AIM: Fostamatinib (R788) is an orally dosed prodrug designed to deliver the active metabolite R940406 (R406), a spleen tyrosine kinase (SYK) inhibitor, for the treatment of rheumatoid arthritis. The objectives were to evaluate the human pharmacokinetic properties of fostamatinib and R406. METHOD: Three clinical studies were conducted in healthy subjects: (A) A single ascending dose study for R406 with doses ranging from 80-600 mg, (B) a single- and multiple-dose study of fostamatinib in aqueous suspension, with single doses ranging from 80-400 mg and multiple doses at 160 mg twice daily and (C) a study comparing suspension and tablet of fostamatinib, with the latter tested in both fed and fasted states. RESULTS: These studies demonstrated that when administered as a solution, R406 was rapidly absorbed. Increases in exposure were observed with doses up to 400 mg. A terminal half-life of 12-21 h was observed. Similar R406 exposure could be achieved with fostamatinib suspension and steady-state was achieved after 3-4 days following twice daily administration. Fostamatinib tablet and suspension exhibited similar R406 exposure. Upon co-administration with food, a delay in peak time and lower peak concentrations of R406 were observed but at the same time the overall exposure did not change. CONCLUSION: Fostamatinib demonstrates rapid and extensive conversion to R406, an inhibitor of SYK. Solid dosage forms of fostamatinib overcome the challenge of low aqueous solubility of R406. The PK profile of R406 could potentially allow once daily or twice daily oral administration of fostamatinib.

An oral Syk kinase inhibitor in the treatment of rheumatoid arthritis: a three-month randomized, placebo-controlled, phase II study in patients with active rheumatoid arthritis that did not respond to biologic agents.

OBJECTIVE: To assess the efficacy and safety of R788 (fostamatinib disodium), an inhibitor of spleen tyrosine kinase (Syk), in patients with active rheumatoid arthritis (RA) that did not respond to biologic therapies. METHODS: A total of 219 patients with active RA in whom treatment with biologic agents had failed were enrolled in a 3-month multicenter, randomized, double-blind, placebo-controlled trial of R788. The primary end point was the percentage of patients who met the American College of Rheumatology 20% improvement criteria (achieved an ACR20 response) at month 3. Secondary end points included changes in inflammation and damage, as assessed by magnetic resonance imaging (MRI), and changes in the Disease Activity Score. RESULTS: The ACR20 response in the R788 100 mg twice daily group was 38%, versus 37% in the placebo group, at month 3. No significant differences were achieved in the ACR20, ACR50, or ACR70 response levels at 3 months. There were differences between the groups from baseline to month 3 in the secondary end points C-reactive protein (CRP) level and synovitis score on MRI. There were baseline differences in steroid use, prior biologic use, and synovitis score on MRI between the R788 group and the placebo group that may have affected the outcomes. A high placebo response rate was seen in this trial, and exploratory analysis suggested that this may in part have been driven by patients who entered the trial with an elevated erythrocyte sedimentation rate but normal CRP level. CONCLUSION: Our findings indicate that there were no differences in the primary end point between the R788 and placebo groups. Differences were observed between the R788 and placebo groups in secondary end points, particularly in those patients who entered the study with an elevated CRP level.

Fostamatinib, a Syk-kinase inhibitor, does not affect methotrexate pharmacokinetics in patients with rheumatoid arthritis.

Fostamatinib (R788) is being investigated as an add-on therapy for the treatment of rheumatoid arthritis (RA) in patients with inadequate response to methotrexate (MTX). This study evaluated the potential pharmacokinetic interaction between R788 and MTX. Sixteen RA subjects on a stable weekly MTX regimen were enrolled and received MTX on days 1 and 8. Twelve subjects received 100 mg of R788 orally, and 4 subjects received a matching placebo twice daily from days 4 to 8 and once daily on days 3 and 9. Blood samples were collected on days 1 and 8 for MTX and 7-hydroxymethotrexate (7-OH-MTX), and days 3 and 9 for R788 and its active metabolite, R406. MTX and 7-OH-MTX pharmacokinetic parameters were similar on days 1 and 8. In the R788 group, the mean day 8 to day 1 ratios (90% confidence intervals) of maximum concentration and area under the plasma concentration-time curve estimates were 1.01 (0.85-1.20) and 1.12 (0.90-1.40) for MTX and 1.06 (0.82-1.35) and 1.06 (0.83-1.36) for 7-OH-MTX, respectively. Urinary excretion of MTX and 7-OH-MTX was also similar with or without R788, averaging 58% to 69% and 4% to 5% of the MTX dose, respectively. The data suggest that there is no clinically significant pharmacokinetic interaction of R788 and MTX in RA patients.

An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis.

BACKGROUND: Spleen tyrosine kinase (Syk) is an important modulator of immune signaling. The objective of this phase 2 study was to evaluate the efficacy and safety of R788, an oral inhibitor of Syk, in patients with active rheumatoid arthritis despite methotrexate therapy. METHODS: We enrolled 457 patients who had active rheumatoid arthritis despite long-term methotrexate therapy in a 6-month, double-blind, placebo-controlled trial. The primary outcome was the American College of Rheumatology (ACR) 20 response (which indicates at least a 20% reduction in the number of both tender and swollen joints and improvement in at least three of five other criteria) at month 6. RESULTS: R788, at a dose of 100 mg twice daily and at a dose of 150 mg once daily, was significantly superior to placebo at month 6 (ACR 20 response rates of 67% and 57%, respectively, vs. 35%; P<0.001 for the comparison of both doses with placebo). It was also significantly superior with respect to ACR 50, which indicates at least a 50% improvement (43% and 32% vs. 19%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.007 for the comparison of the 150-mg dose with placebo) and ACR 70 (28% and 14% vs. 10%; P<0.001 for the comparison of the 100-mg dose with placebo, P=0.34 for the comparison of the 150-mg dose with placebo). A clinically significant effect was noted by the end of the first week of treatment. Adverse effects included diarrhea (in 19% of subjects taking the 100-mg dose of R788 vs. 3% of those taking placebo), upper respiratory infections (14% vs. 7%), and neutropenia (6% vs. 1%). R788 was associated with an increase in systolic blood pressure of approximately 3 mm Hg between baseline and month 1, as compared with a decrease of 2 mm Hg with placebo; 23% of the patients taking R788 vs. 7% of the patients receiving placebo required the initiation of or a change in antihypertensive therapy. CONCLUSIONS: In this phase 2 study, a Syk inhibitor reduced disease activity in patients with rheumatoid arthritis; adverse events included diarrhea, hypertension, and neutropenia. Additional studies will be needed to further assess the safety and efficacy of Syk-inhibition therapy in patients with rheumatoid arthritis. (Funded by Rigel; ClinicalTrials.gov number, NCT00665925.)

Metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleen tyrosine kinase inhibitor R406 in humans: contribution of hepatic and gut bacterial processes to the overall biotransformation.

The metabolism of the spleen tyrosine kinase inhibitor N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine (R406) and its oral prodrug N4-(2,2-dimethyl-4-[(dihydrogenphosphonoxy)methyl]-3-oxo-5-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine disodium hexahydrate (R788, fostamatinib) was determined in vitro and in humans. R788 was rapidly converted to R406 by human intestinal microsomes, and only low levels of R788 were observed in plasma of human subjects after oral administration of (14)C-R788. R406 was the major drug-related compound in plasma from human subjects, and only low levels of metabolites were observed in plasma. The plasma metabolites of R406 were identified as a sulfate conjugate and glucuronide conjugate of the para-O-demethylated metabolite of R406 (R529) and a direct N-glucuronide conjugate of R406. Elimination of drug-related material into the urine accounted for 19% of the administered dose, and the major metabolite in urine from all the human subjects was the lactam N-glucuronide of R406. On average, 80% of the total drug was recovered in feces. Two drug-related peaks were observed; one peak was identified as R406, and the other peak was identified as a unique 3,5-benzene diol metabolite of R406. The 3,5-benzene diol metabolite appeared to result from the subsequent O-demethylations and dehydroxylation of R529 by anaerobic gut bacteria because only R529 was converted to this metabolite after the in vitro incubation with human fecal samples. These data indicate that the major fecal metabolite of R406 observed in humans is a product of a hepatic cytochrome P450-mediated O-demethylation and subsequent O-demethylations and dehydroxylation by gut bacteria.

Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen.

PURPOSE: Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. METHODS: Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. RESULTS: R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. CONCLUSIONS: R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

JAK3 inhibition significantly attenuates psoriasiform skin inflammation in CD18 mutant PL/J mice.

JAK3, a member of the Janus kinase family, is predominantly expressed in hemopoietic cells and binds specifically to the common gamma chain of a subfamily of cytokine receptors that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies suggest that this tyrosine kinase plays key roles in mediating T cell functions, and inhibition of JAK3 has been shown to prevent graft rejection and decrease the severity of arthritis in rodent models. However, the functions of JAK3 in the development of skin immune responses and diseases such as psoriasis have not been determined. CD18 mutant PL/J mice develop spontaneous T cell-dependent psoriasiform skin disease with several similarities to human psoriasis. In this study, we treated mice with established skin disease with R348, a small molecule inhibitor of JAK3, and observed a marked attenuation of skin lesions following 6 wk of treatment. Histological analyses revealed major reductions of both epidermal and dermal lesion severity scores in R348-treated CD18-deficient PL/J mice compared with vehicle controls, which was associated with decreased CD4(+) T cell infiltration. In addition, systemic levels of IL-17, IL-22, IL-23, and TNF-alpha were significantly lower in mice receiving the compound, and T cells isolated from R348-treated mice also showed reduced phosphorylation of Stat5 after stimulation with IL-2. These findings suggest that small-molecule inhibitors of JAK3 may be useful in the treatment of inflammatory skin diseases such as psoriasis and strongly implicate JAK signaling events as important in the pathogenesis of this disease.

Developmental toxicity associated with receptor tyrosine kinase Ret inhibition in reproductive toxicity testing.

BACKGROUND: Urogenital abnormalities are among the most common of all human birth defects. In developmental toxicity studies with the Syk kinase inhibitor R788, a spectrum of findings, including renal agenesis, were observed. R788 has also been found to inhibit the receptor tyrosine kinase Ret. Ret kinase is known to be an essential component in the signaling pathway required for renal organogenesis and ureteric duct formation. Previously known is that mutant mice without the c-ret gene, develop urogenital malformations including renal agenesis. METHODS: In GLP developmental toxicity studies, gravid rabbits were treated orally with R788 at doses of 0, 10, 22, and 50 mg/kg/day (gestation days 7-19) and gravid rats received 0, 5, 12.5, and 25 mg/kg/day (gestation days 6-17) by the same route. The activity of R406 against Ret kinase was assessed in biochemical and cell-based assays. RESULTS: A dose-dependent increase in malformations, including renal and ureteric agenesis and a specific major vessel anomaly, retroesophageal right subclavian artery, was observed in both the rat and rabbit. R788 proved to be a potent inhibitor of Ret kinase. CONCLUSIONS: R788 promoted a spectrum of developmental toxicity, including renal and ureteric agenesis and a specific major vessel abnormality, retroesophageal right subclavian artery, in two different species. These effects are likely the result of inhibition of Ret kinase given its importance in the normal ontogeny of the urogenital and cardiovascular systems across species.

Treatment of rheumatoid arthritis with a Syk kinase inhibitor: a twelve-week, randomized, placebo-controlled trial.

OBJECTIVE: Spleen tyrosine kinase (Syk) has been identified as an important modulator of immune signaling in B cells and cells bearing Fcgamma-activating receptors. R788, a prodrug of active metabolite R406, has been shown to be an inhibitor of Syk kinase, active in a variety of in vitro and in vivo models, suggesting potential activity in the treatment of rheumatoid arthritis (RA). METHODS: We enrolled 189 patients with active RA despite methotrexate therapy in a 3-month, multicenter, ascending-dose, double-blind, placebo-controlled trial. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. RESULTS: Twice-daily oral doses of 100 mg and 150 mg of R788 were significantly superior to placebo or twice-daily oral doses of 50 mg at week 12 (ACR20 achieved in 65% and 72% versus 38% and 32% of patients, respectively [P < 0.01]). ACR50 (achieved in 49% and 57% versus 19% and 17% of patients, respectively) and ACR70 (achieved in 33% and 40% versus 4% and 2% of patients, respectively) scores showed a similar pattern. Clinical effect was noted as early as 1 week after initiation of therapy. Reductions in serum interleukin-6 and matrix metalloproteinase 3 levels also occurred as early as week 1 in the groups receiving 100 mg and 150 mg R788. The major adverse effects were gastrointestinal side effects (predominantly diarrhea) and neutropenia (<1,500/mm3), both of which were dose related. CONCLUSION: These results indicate that an inhibitor of Syk kinase produces significant clinical benefits at 12 weeks in a population of patients with active RA receiving methotrexate therapy. Syk kinase may be an important new therapeutic target in RA and related autoimmune conditions.

An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus.

OBJECTIVE: To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)-dependent signaling, could modulate disease in lupus-prone (NZB x NZW)F1 (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. METHODS: R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24-34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. RESULTS: When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. CONCLUSION: We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.

Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor.

Spleen tyrosine kinase (Syk), a key mediator of immunoreceptor signaling in inflammatory cells, is essential for immune complex-mediated signal transduction initiated by activated receptors for immunoglobulin G. In collagen-induced arthritis, R788/R406, a novel and potent small molecule Syk inhibitor suppressed clinical arthritis, bone erosions, pannus formation, and synovitis. Serum anti-collagen type II antibody levels were unaltered, while the half-life of exogenous antibody was extended when co-administered with R406. Expression of the targeted kinase (Syk) in synovial tissue correlated with the joint level of inflammatory cell infiltrates and was virtually undetectable in treated rats. Syk inhibition suppressed synovial cytokines and cartilage oligomeric matrix protein (COMP) in serum, suggesting a sensitive and reliable biomarker for R406 activity. These results highlight the role of activating Fcgamma receptors in inflammatory synovitis and suggest that interruption of the signaling cascade with a novel Syk inhibitor may be a useful addition to immunosuppressive disease-modifying anti-rheumatic drugs currently used in the treatment of human autoimmune diseases such as rheumatoid arthritis.

Immunotoxicity assessment for the novel Spleen tyrosine kinase inhibitor R406.

Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects.

R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation.

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

An intranasal Syk-kinase inhibitor (R112) improves the symptoms of seasonal allergic rhinitis in a park environment.

BACKGROUND: R112 inhibits Syk kinase, a transducer of signaling through the Fcepsilon receptor of mast cells, blocking mast cell responses to allergic stimuli. OBJECTIVE: Examine the efficacy and safety of intranasal R112 in volunteers with symptomatic seasonal allergic rhinitis compared with a placebo in a park setting. METHODS: In this double-blind, placebo-controlled study of 319 volunteers with seasonal allergic rhinitis, 160 were randomized to intranasal R112 and 159 to a vehicle control during 2 days at 2 separate locations in spring 2004. Subjects were evaluated for symptoms of allergic rhinitis (i.e., sneezes, runny nose/sniffles, itchy nose, stuffy nose) on the basis of a possible maximum score of 32 for the Global Symptom Complex (GSC) scale. The primary outcome evaluated was the difference in the reduction in GSC (area under the curve over a period of 8 hours) from baseline between R112 and vehicle placebo. RESULTS: At baseline, the combined GSC was approximately 18/32 and equal between treatment groups. After 8 hours (dosing 3 mg/nostril every 4 hours x 2), R112 significantly reduced the GSC compared with placebo (7 vs 5.4 units, respectively; P = .0005). Each individual symptom combined to form the GSC was also significantly improved in the R112 group compared with control ( P < .05). As early as 45 minutes after dosing, R112 showed a significant improvement in symptoms over placebo, and the duration of action exceeded 4 hours. Adverse effects were indistinguishable between the groups and clinically insignificant. CONCLUSION: Intranasal R112 was effective in this park study and is a promising new treatment for seasonal allergic rhinitis.

Targeting Syk as a treatment for allergic and autoimmune disorders.

Recent advances in our understanding of allergic and autoimmune disorders have begun to translate into novel, effective and safe medicines for these common maladies. Examples include an anti-IgE monoclonal antibody recently approved for severe asthmatics and the TNF-alpha antagonists that have demonstrated their ability to suppress rheumatoid arthritis, Crohn's disease and other chronic inflammatory processes. However, protein therapies are difficult and expensive to develop, manufacture and administer. Clearly, there is also a need for small-molecule inhibitors of novel targets that have safe and effective characteristics. Syk is an intracellular protein tyrosine kinase that was discovered 15 years ago as a key mediator of immunoreceptor signalling in a host of inflammatory cells including B cells, mast cells, macrophages and neutrophils. These immunoreceptors, including Fc receptors and the B-cell receptor, are important for both allergic diseases and antibody-mediated autoimmune diseases and thus pharmacologically interfering with Syk could conceivably treat these disorders. In addition, as Syk is positioned upstream in the cell signalling pathway, therapies targeting Syk may be more advantageous relative to drugs that inhibit a single downstream event. Syk inhibition during an allergic or asthmatic response will block three mast cell functions: the release of preformed mediators such as histamine, the production of lipid mediators such as leukotrienes and prostaglandins and the secretion of cytokines. In contrast, commonly used antihistamines or leukotriene receptor antagonists target only a single mediator of this complex cascade. Despite its expression in platelets and other non-haematopoietic cells, the role of Syk in regulating vascular homeostasis and other housekeeping functions is minimal or masked by redundant Syk-independent pathways. This suggests that targeting Syk would be an optimal approach to effectively treat a multitude of chronic inflammatory diseases without undue toxicity.