Cpd 1 specifically impairs human Th17 cell polarization and Th17-signature cytokine production.

<p>(A) Total CD4⁺ T-cells were incubated with the RORγt inhibitor and were activated under Th17 cell favoring conditions for 72 hrs followed by quantification of IL-17A production. Data are representative of five experiments with triplicate readings. (B) Th17 cells were treated with PMA/ionomycin and Brefeldin A for 4 hrs and frequencies of IL-17A producing cells in gated CD4⁺ T-cells were determined by intracellular staining. Data represent duplicate measurements of two independent experiments. (C and D) Supernatants from polarized Th17 cells were taken to determine IL-17F and IL-22 cytokine concentrations. Naïve (E) and memory (F) human CD4⁺ T-cells were purified from PBMCs and were cultured under Th17 skewing conditions for 7 days in the presence of cpd 1. IL-17A production was quantified by ELISA. Representative examples of concentration-response curves from two experiments with duplicate readings are shown. (G) Human CD4⁺ T-cells were stimulated with anti-CD3 plus anti-CD28 antibodies only (Th0) or incubated with IL-12 and anti-IL-4 antibody (Th1) or with IL-4 and anti-IFN-γ antibody (Th2). After 48 hrs, Th subset signature cytokines including IL-2, IFN-γ and IL-13 were analyzed by ELISAs. Representative examples from two experiments with triplicate readings are shown.</p

Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis <i>in vivo</i>

<div><p>Retinoic acid receptor-related-orphan-receptor-C (RORγt) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of RORγt in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the <i>in vitro</i> and <i>in vivo</i> pharmacology of a potent and selective small-molecular-weight RORγt inverse agonist. The compound binds to the ligand binding domain (LBD) of RORγt leading to displacement of a co-activator peptide. We show for the first time that a RORγt inverse agonist down-regulates permissive histone H3 acetylation and methylation at the <i>IL17A</i> and <i>IL23R</i> promoter regions, thereby providing insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and γδT-cells and attenuated transcription of RORγt target genes. The inhibitor showed good <i>in vivo</i> efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in <i>ex vivo</i> recall assays. In summary, we demonstrate that inhibiting RORγt by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.</p></div

Downregulated RORγt-regulated target gene expression by cpd 1.

<p>(A-F) Purified human CD4⁺ T-cells were polarized towards Th17 cells and were treated at the beginning of the cell culture with various concentrations of cpd 1 or with DMSO control (Co). After 72 hrs, mRNA was extracted and transcript levels were quantified by RT-PCR. Gene expression was normalized to β-glucoronidase levels and expressed as arbitrary units. All graphs are representative of three independent experiments containing three technical replicates.</p

Cpd 1 blocks IL-17A production by differentiated Th17 cells and by Tc17 and γδT-cells.

<p>(A) Human CD4⁺ T-cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of Th17 cell promoting conditions for 7 days without compound, followed by extensive washing of cells to remove IL-17A and re-stimulation with anti-CD3 plus anti-CD28 antibodies in the presence of cpd 1. After 3 days, supernatants were collected and IL-17A concentrations were determined. (B) Purified CD8⁺ T-cells were stimulated with anti-CD3 and anti-CD28 antibodies under Th17-polarizing cytokines for 72 hrs, and IL-17A production was quantified. (C) Human γδ T-cells were incubated with cpd 1 and stimulated with PMA/ionomycin for 24 hrs, followed by quantification of IL-17A concentration by ELISA. Representative examples from three independent experiments with triplicate readings are shown.</p

Cpd 1 ameliorates antigen-induced arthritis (AiA) responses in Lewis rats.

<p>(A) Rats were immunized twice with methylated BSA (mBSA). Three weeks later, the right knees of the animals were challenged with mBSA in 5% glucose, while the left knees were injected with vehicle (5% glucose). Starting just prior to antigen challenge, cpd 1 was administered twice daily via oral gavage at the indicated doses for 7 days and knee swelling was monitored. The mean ± SEM of the swelling ratios between antigen-challenged and vehicle injected knees are shown (<i>n</i> = 5). Results are representative of two experiments with 5 rats per treatment group. (B) Draining lymph node cells were prepared 7 days after mBSA challenge and stimulated <i>ex vivo</i> with mBSA (200 μg/ml). Frequencies of IL-17A secreting antigen-specific cells were quantified by ELISPOT after 6 h. (C) Diluted sublingual whole blood cells originating from cpd 1- or vehicle-treated animals were re-stimulated with mBSA (50 μg/ml) and after 96 hrs, IL17A concentrations were determined by ELISA. Each point represents mean ± SEM from an individual rat (n = 4–5 readings/animal). *, P < 0.05; **, P < 0.01; ***, P < 0.001 Dunnett´s test.</p

Cpd 1 inhibits IL-17A production by whole blood cells and blocks rat Th17 differentiation.

<p>(A and B) Diluted human whole blood cells were stimulated with ConA and IL-23 in the presence of cpd 1. After 72 hrs, IL-17A (A) or IL-2 (B) production was measured by ELSA. Data are representative from five (A) or two (B) independent experiments with triplicate measurements. (C) Purified CD4⁺ Tcells originating from splenocytes from Lewis rats were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of Th17-skewing conditions. After 72 hrs, IL-17A concentrations in supernatants were determined by ELISA. Representative example of concentration-response curve from two experiments with triplicate readings is shown.</p

Pathophysiological Consequences of a Break in S1P1-Dependent Homeostasis of Vascular Permeability Revealed by S1P1 Competitive Antagonism

<div><p>Rational</p><p>Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist.</p><p>Results</p><p>NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3–4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences.</p><p>Conclusions</p><p>Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates.</p></div

NIBR-0213-induced vascular leakage in the lungs evaluated by MRI.

<p><b>(A)</b> Axial MRI sections through the chest of one animal at approximately the same anatomical location acquired before (baseline) and at different time points with respect to beginning of treatment with NIBR-0213 (30 mg/kg BID). The white and red arrows display fluid signals in the lungs and the pleura, respectively, elicited by the compound. <b>(B)</b> Differential MRI signal volumes (mean ± s.e.m; n = 4–8) in the lungs and pleura. For the lungs, differential volumes (<i>i</i>.<i>e</i>. baseline-subtracted) are presented. <b>(C)</b> MRI signals (means ± s.e.m, n = 3) in the lungs and pleura at 24 h and 96 h after beginning of treatment with NIBR-0213 (3, 10 or 30 mg/kg BID) or FTY720 (1 mg/kg QD). For the lungs, differential signal volumes (<i>i</i>.<i>e</i>. baseline-subtracted) are presented. * p<0.05.</p

Chronic microscopic changes provoked by NIBR-0213 in rat lungs.

<p>An overview of chronic changes with alveolar degeneration /regeneration and accumulation of macrophages (<b>A:</b> 10X, H&E.) and of fibrotic foci (<b>B:</b> 12X, H&E.) observed in the lungs of a rat treated orally over 2 weeks with NIBR-0213 at 300 and 100 mg/kg QD, respectively.</p

Acute microscopic changes provoked by NIBR-0213 in rat hearts.

<p>Perivascular edema (<b>A:</b> 20X, H&E.) and focal degenerative myocytes with interstitial inflammatory cells and fibrosis (<b>B:</b> 20X, H&E.) observed in the hearts of a rat treated orally over 2 weeks with NIBR-0213 at 100 mg/kg QD.</p