Selective IRAK4 Inhibition Attenuates Disease in Murine Lupus Models and Demonstrates Steroid Sparing Activity

Abstract

The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.

FIGURE 1.

Inhibition of TLR-induced IKKα/β and ERK phosphorylation by BMS-986126. PBMCs were cultured with different concentrations of BMS-986126 and stimulated with either 100 ng/ml LPS (A) or 10 μg/ml LTA (B) for 20 min. Some groups were stimulated with 10 μg/ml gardiquimod for 30 min (C). Lysates were analyzed using the Sally Sue ProteinSimple system. The pIKKα/β and pERK bands were normalized to actin bands for each group. Percentage inhibition relative to the vehicle control is shown under each band. Cropped bands for pIKKα/β, pERK, and actin are shown. One representative experiment of two is shown.

FIGURE 2.

Inhibition of TLR-induced cytokines by BMS-986126 in mice. Mice were dosed orally with either vehicle or the indicated doses of BMS-986126 at 30 min prior to challenge either with 25 mg/kg LTA i.p. (A), 2.5 μg CpG-ODN i.v. (B), or 5 mg/kg gardiquimod i.p (C and D). Ninety minutes after LTA challenge, mice were bled and plasma concentrations of IL-6 were determined by ELISA. Mice were bled 2 and 8 h after CpG challenge, and plasma levels of IFN-α were measured by ELISA. Plasma concentrations of IFN-α (C) and IL-6 (D) were measured at 2 and 8 h after challenge with gardiquimod. Mean and SEM are plotted. Data are from one experiment with eight animals per group. **p < 0.001, ***p < 0.0001, one-way ANOVA with a Dunnett test.

FIGURE 3.

Inhibition of TLR7-induced skin inflammation by BMS-986126. C57BL/6 mice were dosed orally with vehicle or BMS-986126 at doses of 0.3, 1, 3, and 10 mg/kg/d. Imiquimod cream was applied daily on the shaved backs of the mice. Changes in skin thickness (A), scaling (B), and erythema (C) were monitored daily. At termination of the study, splenomegaly was assessed by spleen weight normalized by body weight (D). Skin histology (E) was also assessed at end of study. For (A)–(C), difference from vehicle at day 6 was assessed by two-way ANOVA with the Bonferroni test. For (D) and (E), difference from vehicle on day 6 was assessed by one-way ANOVA with a Dunnett test. Means and SEM for each group are plotted. Data are from one experiment with eight animals per group. *p < 0.01, **p < 0.01, ***p < 0.0001.

FIGURE 4.

Inhibition of disease activity in MRL/lpr mice by BMS-986126. Mice were dosed orally either with vehicle or 0.3, 1, 3, or 10 mg/kg/d BMS-986126, or with 10 mg/kg/d prednisolone. Following 8 wk of dosing, multiple biomarkers were assessed, including total urine protein levels (A), urine NGAL concentrations (B), blood urea nitrogen concentrations (C), anti-dsDNA Ab titers (D), plasma IL-10 concentrations (E), and plasma IL-12p40 concentrations (F). In the spleen, the numbers of IFN-α⁺ pDCs (G) and IL-6⁺ myeloid cells (H) were determined by flow cytometry. Intensity of Ig deposition in the kidney (I) and kidney histology as visualized by H&E staining with ×20 magnification (J and K) were also assessed at the end of study. Asterisks indicate glomerular changes characterized by mesangial thickening, cellular infiltration, and sclerosis/fibrosis. Preonset values are from MRL/lpr mice at 8 wk of age. Means and SEM for each group are plotted. Data are from one experiment with 10 animals per group. *p < 0.01, **p < 0.001, ***p < 0.0001 versus vehicle by one-way ANOVA with a Dunnett test.

FIGURE 5.

Inhibition of disease activity in NZB/NZW mice by BMS-986126. Mice were dosed orally with vehicle or 0.3, 1, 3, or 10 mg/kg/d BMS-986126, or with 1 mg/kg/d dexamethasone (DEX). Following 25 wk of dosing, several biomarkers of disease activity were assessed, including protein (A) and NGAL (B) concentrations in urine, plasma titers of dsDNA-specific autoantibodies (C), plasma levels of IL-12p40 (D), numbers of splenic IFN-α⁺ pDCs (E), expression of IFIT1 mRNA in blood (F), intensity of Ig deposition in the kidney (G), and kidney histology as visualized by H&E staining with ×20 magnification (H and I). Asterisks indicate glomerular changes characterized by mesangial thickening, cellular infiltration, and sclerosis/fibrosis. Preonset values are from NZB/NZW mice at 10 wk of age. Means and SEM for each group are plotted. Data are from one experiment with 14 animals per group. *p < 0.01, **p < 0.001, ***p < 0.0001 versus vehicle by one-way ANOVA with a Dunnett test.

FIGURE 6.

Inhibition of TLR7- and TLR9-induced gene expression by BMS-986126. PBMCs were stimulated in duplicate with the TLR7 agonists gardiquimod (0.5 μg/ml) and heat-inactivated influenza virus (0.02 μl) or the TLR9 agonist ODN2216 (300 nM) in the presence and absence of different concentrations of BMS-986126. Expression of the target genes IFIT1 (A) and MX1 (B) were measured by qPCR, normalized to the housekeeping gene GAPDH, and compared with unstimulated levels to determine fold induction. Data are expressed as percentage inhibition of fold induction relative to a DMSO control for one representative donor of three. (C) PBMCs from either healthy controls (NHV) or lupus patients (SLE) were stimulated with either a TLR7 agonist (gardiquimod, 0.5 μg/ml) or a TLR9 agonist (ODN2216, 300 nM) in the presence and absence of BMS-986126. The IC₅₀s for compound-dependent inhibition of IFIT1 from four to five independent donors are plotted. (D) Heat map of PBMCs from two donors stimulated in triplicate with gardiquimod (5 μg/ml) in the presence and absence of 5 μM BMS-986126. The top 196 genes significantly inhibited by BMS-986126 in both donors are shown. (E) PBMCs from three healthy controls were stimulated with a complex of anti-RNP Abs purified from lupus patients mixed with necrotic cell lysates in the presence and absence of BMS-986126. Fold induction of normalized IFIT1 expression is plotted. One representative experiment of three is shown.

FIGURE 7.

Synergistic inhibition of TLR7- and TLR9-induced IFN-regulated genes by the combination of BMS-986126 and prednisolone. PBMCs from a healthy control donor were stimulated in vitro either with the TLR9 agonist ODN2216 (300 nM) (A) or the TLR7 agonist heat-inactivated influenza virus (0.02 μl) (B) in the presence and absence of suboptimal concentrations of prednisolone, BMS-986126, and the combination of both. Expression of IFIT1 was quantitated by real-time PCR and normalized to the housekeeping gene GAPDH. Data represent fold induction relative to unstimulated controls. One representative experiment of three is shown. Comparisons were performed as shown using a one-way Student t test. *p < 0.01, **p < 0.001, ***p < 0.0001. (C and D) MRL/lpr mice were dosed orally with suboptimal doses of BMS-986126 (0.3 or 1 mg/kg/d), two doses of prednisolone (pred, 1 or 10 mg/kg/d), or the combination of BMS-986126 with prednisolone (0.3 + 1; 1 + 1). Following 8 wk of dosing, serum titers of dsDNA-specific autoantibodies (C) and urine protein levels (D) were measured. Mean and SEM for each group are plotted. Data are from one experiment with 10 animals per group. *p < 0.01, **p < 0.001, ***p < 0.0001 versus vehicle by one way ANOVA with Dunnett’s test.