Setting the Bar High: Barcode Medication Administration in the Ambulatory Surgery Setting

https://scholarlycommons.baptisthealth.net/dh-bestpractice-2023/1001/thumbnail.jp

Pharmacological inhibition of ROR gamma t suppresses the Th17 pathway and alleviates antigen-induced arthritis in-vivo

Retinoic acid receptor-related-orphan-receptor-C (ROR gammat) 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 RORgammat in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim to inhibit the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in-vitro and in-vivo pharmacology of a potent and selective small-molecular-weight RORgammat inverse agonist. The compound binds to the ligand binding domain (LBD) of RORgammat leading to displacement of a co-activator peptide. We show for the first time that a RORgammat inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of RORgammat-dependent genes. Consitent with this, the compound effectively reduced IL-17A production by polarized human gamma delta T-cells and by T-cells and attenuated transcription of RORgammat target genes. The inhibitor showed good in-vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex-vivo recall assays. In summary, we demonstrate that inhibiting RORgammat 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

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

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

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

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