Methotrexate and a spleen tyrosine kinase inhibitor cooperate to inhibit responses to peripheral blood B cells in rheumatoid arthritis

Abstract

Background: Selective disruption of the spleen tyrosine kinase (Syk) represents a novel strategy to control B-cell functional responses by inhibition of B-cell antigen receptor (BCR) signaling. PRT062607 (P505-15) is a highly selective small molecule Syk inhibitor that potently suppresses B-cell function in human and rodent blood, and reduces inflammation in rodent models of rheumatoid arthritis (RA).

Aims: In this study, we sought to determine the potency of Syk inhibition by PRT062607 in whole blood from RA patients, and elucidate covariates that affect the potency of immune-regulation by this compound.

Materials and methods: Whole blood was collected from 30 patients diagnosed with RA as part of a single-center outpatient study. Disease severity, serum protein markers of inflammation, and co-medications were related to each other, and to PRT062607 activity in ex vivo Syk-mediated immune function assays.

Results: We report here that PRT062607 exhibited greater potency in suppressing BCR mediated B-cell functional responses in whole blood from RA patients who received stable methotrexate (MTX) therapy. We demonstrate that the B-cell functional response to BCR ligation is influenced by cytokines and JAK/STAT signaling.

Discussion: MTX is a known cytokine modulating agent, and this mechanism may act in concert with PRT062607 to control B-cell function.

Conclusion: These data have important implications for the co-administration of Syk inhibitors and MTX for the treatment of RA.

Keywords: B cells; methotrexate; rheumatoid arthritis; spleen tyrosine kinase.

Figure 1

Syk-independent mechanism(s) influence BCR-mediated B-cell activation in whole blood from RA patients. The PRT062607 concentration-effect relationship in the basophil degranulation assay (A) and B-cell activation assay (B) is shown for healthy normal volunteers (n = 13 and 17, respectively) and in RA patients (n = 28 and 31, respectively). PRT062607 concentration is depicted on the x-axis in μmol/L, and the corresponding percent inhibition of immune cell activation on the y-axis. Data represent means ± SEM. The IC₅₀ derived from each concentration-effect relationship is shown.

Figure 2

The dependency of BCR-mediated B-cell activation on Syk is affected by disease activity and treatment with MTX. DAS28-CRP (A), DAS28-ESR (B) scores were used to group patient data in three categories of disease activity; Remission/Mild (by DAS28-CRP n = 11, by DAS28-ESR n = 7), Moderate (by DAS28-CRP n = 13, by DAS28-ESR n = 15), and Severe (by DAS28-CRP n = 8, by DAS28-ESR n = 10). PRT062607 concentration (x-axis) by percent inhibition of B-cell activation (y-axis; mean ± SEM) is shown, along with the IC₅₀ and 95% confidence interval. (C) The concentration-effect relationship was compared in RA patients that received (MTX; n = 18) or did not receive (No MTX; n = 14) stable MTX therapy. The IC₅₀ and 95% confidence interval for each group are shown. Data are represented as mean ± SEM. (D) RA patients with severe activity as defined by DAS28-ESR scores were separated into two groups based on treatment with MTX. Raw data are shown (n = 5 per group) with a curvefit.

Figure 3

Serum cytokines and markers of inflammation change in accordance with disease severity in RA patients. Data depict serum protein concentration (pg/mL) as it relates to disease activity defined by DAS28-ESR as remission/mild (Mild), Moderate, and Severe. The shaded box represents the first and third quartile of the population, and the whiskers extend to the 1.5 interquartile range. The median is shown as the horizontal black bar and the mean by the closed circle. The specific serum protein measured is listed at the top of each figure.

Figure 4

Treatment with MTX is associated with significant decreases in serum IL2 and IL17A. Serum cytokines and protein markers of inflammation were compared between RA patients on stable MTX therapy (MTX) or not receiving MTX (No MTX). Statistically significant differences between the two groups were determined by the Wilcoxon test (P ≤ 0.05). Raw data (black dots) are overlaid with the box and whisker plots that represent the first and third quartile of the population (shaded box), and the whiskers extend to the 1.5 interquartile range. The black bar represents the median and large shaded circle the mean. Serum concentration of each protein is plotted on the y-axis as pg/mL.

Figure 5

Cytokines and JAK/STAT signaling influence BCR-mediated B-cell activation. (A) Change from baseline in B-cell CD69 upregulation following BCR stimulation is compared between RA patients on stable MTX therapy (MTX) or not receiving MTX (No MTX). Raw data (block dots) are overlaid with box and whisker plots that represent the CD69 MFI on the y-axis. The shaded box represents the first and third quartile of the population, and the whiskers extend to the 1.5 interquartile range. The black bar represents the median and large shaded circle the mean. (B) The effect of costimulation of the BCR with IL2 or IL4 on B-cell activation is shown. B-cell CD69 MFI is plotted on the y-axis, and represented in the box and whisker plots. The stimulation conditions are shown on the x-axis. (C) The effect of Syk (Syki), JAK (JAKi), and combined Syk/JAK inhibition (Syki/JAKi) on B-cell activation is shown. CD69 MFI normalized to% of vehicle control is plotted on the y-axis (mean ± SEM), and the concentration of each inhibitor (0.1–3 μmol/L) is shown on the x-axis. The asterisks represent significant differences comparing combined Syk/JAK inhibition to Syk inhibition alone at matching concentrations. (D) The PRT062607 concentration-effect relationship in response to BCR stimulation alone (Anti-BCR) or costimulation of the BCR with IL2 (Anti-BCR + IL2; left panel), IL4 (Anti-BCR + IL4; center panel), or IL2 and IL4 (Anti-BCR + IL2/4; right panel) is shown. Percent inhibition of CD69 MFI relative to vehicle control is plotted on the y-axis, and concentration of PRT062607 in μmol/L on the x-axis. The dashed line across each panel represents the point of 100% inhibition, and asterisks represent statistical differences by Wilcoxon test (P < 0.05). The inset box and whisker plots depict the 1 and 3 μmol/L PRT062607 concentrations only.