Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis

Abstract

Background: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition.

Methods: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa.

Results: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays.

Conclusions: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease.

FIGURE 1

TOP1210 is a potent inhibitor of the innate immune responses in LPS-stimulated human PBMCs and macrophages. TOP1210 inhibits IL-8 release by LPS-stimulated PBMCs (A) and macrophages (B), and also LPS-stimulated TNF-α release by macrophages (C), with greater potency than any of the selective kinases tested. Generally, compared to TOP1210, BIRB-796, dasatinib, and BAY61-3606 have weak potency and efficacy in both PBMCs and macrophages. Graphs represent means of at least 3 independent experiments ± SEM.

FIGURE 2

TOP1210 is a potent inhibitor of an adaptive immune response in anti-CD3/anti-CD28–stimulated T-cell release of IFN-γ (A) and IL-2 (B). Both TOP1210 and dasatinib inhibit IFN-γ release with similar potency, whereas TOP1210 is approximately 15-fold weaker than dasatinib as an inhibitor of IL-2 release. BAY61-3606 demonstrates weaker inhibition, whereas BIRB-796 has little-to-no effect. Graphs represent means of at least 3 independent experiments ± SEM.

FIGURE 3

TOP1210 is a potent inhibitor of IL-1β–stimulated IL-8 release from HT29 cells. Selective kinase inhibitors achieve less than 50% inhibition and are weak inhibitors of IL-8 release from HT29 cells. Graph represents means of at least 3 independent experiments ± SEM.

FIGURE 4

Combination of selective kinase inhibitors leads to more than additive inhibitory effects in reducing IL-8 secretion from IL-1β–stimulated HT29 cells. BIRB-796, BAY-61-3606, and dasatinib individually induce concentration-dependent inhibition of IL-8 secretion but with limited efficacy (A). BIRB-796 in combination with BAY-61-3606 (100 ng/mL) and/or dasatinib (100 ng/mL) produces concentration-dependent inhibition of IL-8 secretion with improved efficacy and potency (B). Compared to the lack of effect of the same concentrations when used individually, BIRB-796 (1 ng/mL)/BAY-61-3606 (100 ng/mL)/dasatinib (100 ng/mL) combination treatment is effective in reducing IL-8 concentration in a more than additive manner (C). Graphs represent means of at least 3 independent experiments ± SEM.

FIGURE 5

TOP1210 is more potent than selective kinase inhibitors in downregulating proinflammatory cytokine release from TNF-α–stimulated UC mucosal myofibroblasts. Effect of different concentrations of TOP1210, BIRB-796 (BIRB), dasatinib (DAS), or BAY-61-3606 (BAY) on the release of IL 6 and IL-8, expressed as mean percent inhibition compared with stimulated control (DMSO; 20-ng/mL recombinant human TNF-α + DMSO, 0.5% v/v). Bars represent mean ± SEM of at least 3 independent experiments. *P < 0.01 versus stimulated control; **P < 0.001 versus stimulated control.

FIGURE 6

TOP1210 is superior to selective kinase inhibitors alone in downregulating proinflammatory cytokine release from UC biopsies. TOP1210 activity is comparable to a combination of all three selective inhibitors and reduces proinflammatory cytokine release from UC biopsies in a concentration-dependent manner. Effect of 1 μg/mL TOP1210, 1 μg/mL BIRB-796 (BIRB), 1 μg/mL dasatinib (DAS), and 1 μg/mL BAY-61-3606 (BAY), or the combination of the 3 selective kinase inhibitors (A) or TOP1210 (0.001–1 μg/mL) (B) on the release of IL-1β, IL-6, and IL-8, expressed as mean percent inhibition compared with control (DMSO; DMSO, 0.5% v/v), in organ culture supernatants of inflamed colonic biopsies from patients with UC. Bars represent mean ± SEM of at least 3 independent experiments. *P < 0.05 versus DMSO; **P < 0.005 versus DMSO; ***P < 0.0005 versus DMSO.