Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin

<div><p>Background</p><p>Psoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy.</p><p>Methods and Findings</p><p>The preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in <i>ex vivo</i> lesional psoriatic skin.</p><p>Conclusions</p><p>Our work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.</p></div

Effects of C-type natriuretic peptide on rat cardiac contractility

1. Natriuretic peptide receptors have been found in different heart preparations. However, the role of natriuretic peptides in the regulation of cardiac contractility remains largely elusive and was, therefore, studied here. 2. The rate of relaxation of electrically stimulated, isolated rat papillary muscles was enhanced (114.4±1.4%, P<0.01) after addition of C-type natriuretic peptide (CNP; 1 μM). Time to peak tension decreased in parallel (88±3 and 75±2 msec before and 5 min after addition of CNP, respectively, P<0.01). On the other hand, the rate of contraction slowly decreased when CNP was added to the papillary muscles. These results show that CNP displays a positive lusitropic effect associated with a negative inotropic effect. The effects of CNP were mimicked by 8-bromo-guanosine 3′,5′ cyclic monophosphate. 3. Addition of CNP to isolated adult rat cardiomyocytes, induced a 25 fold increase in guanosine 3′,5′ cyclic monophosphate (cGMP) levels and stimulated the phosphorylation of phospholamban and troponin I, two proteins involved in the regulation of cardiac contractility. The levels of adenosine 3′,5′ cyclic monophosphate (cAMP) were not affected by the addition of CNP to the myocytes. The CNP-dependent phospholamban phosphorylation was accompanied by the activation of the sarcoplasmic reticulum Ca(2+)-ATPase. 4. In summary, CNP exerts a positive lusitropic effect, in rat papillary muscles. The putative mechanism involved in the lusitropism induced by this peptide, a cGMP-dependent enhancement of the rate of relaxation with a slowly developing negative inotropic effect, seems different to that described for catecholamines

Co-activation of AMPK and mTORC1 Induce Cytotoxicity in Acute Myeloid Leukemia

International audienceGraphical Abstract Highlights d AMPK activation blocks AML propagation without toxicity to normal hematopoiesis d Cytotoxicity induced by an AMPK activator (GSK621) involves autophagy in AML d Co-activation of AMPK and mTORC1 is synthetically lethal in AML d AMPK and mTORC1 crosstalk requires eIF2a/ATF4 signalin

Suppression of Th17-type cytokine production following topical application.

<p><i>Ex vivo</i> human skin was cultured in Franz Cell chambers for a total of 48 hours. GSK2981278 was applied to the dry surface of the skin at time zero followed 24 hrs later by activation of skin resident immune cells under Th17 polarizing conditions. The experimental schema is shown in panel A. Skin sections were harvested after 24 hrs of stimulation (48 hrs of compound treatment) and analyzed for relative gene expression of <i>il17a</i> (B) or <i>il17f</i> (C). Data are shown as the percent maximum expression of each cytokine as compared to Th17-stimulated samples treated topically with vehicle only.</p

GSK2981278 attenuates inflammation in a mouse model of psoriasis.

<p>(A) Mice were treated topically with placebo or 1% GSK2981278 (1278) in ointment, and with imiquimod (IMQ) or petrolatum (vehicle). At study’s end (day +9 of IMQ treatment), back skin was imaged and stained (H&E). (B) Mean epidermal thickness is shown across 6–9 mice per treatment group. (C-E) Changes to local cytokine expression was determined following topical application of 1% or 0.1% compound in a simple ethanolic solution (60:40 ethanol:water). (C) Description of study groups for panels D-E. Skin cytokine levels on day +3 (D) or day +9 (E). Data reflect the mean ± SEM gene expression level across 6–9 mice per treatment group. Significant inhibition was determined by Student’s <i>t</i> test. (*p≤0.05;**p≤0.01).</p

Skin Resident Immune Cell Activation (sRICA) leads to pro-inflammatory cytokine responses that are reduced by GSK2981278.

<p>(A) Skin explants were cultured ≥4 days under the indicated conditions. Explants were analyzed for tissue integrity by H&E. (B) Samples were pre-treated with 10 μM compound (closed bar) or vehicle (DMSO; open bars–set to 100%) for 1 day prior to 24–48 hrs of Th17 stimulation. Relative transcript levels were determined by qRT-PCR. (C) Samples were treated as in B, then analyzed daily for tissue integrity by H&E. Images are representative at least 3 independent experiments. (D) Samples were treated as in B. Graphs show the mean percent maximum stimulation of 3 independent experiments. Significant inhibition was determined by Student’s <i>t</i> test. (*p≤0.05; **p≤0.01; ***p≤0.001).</p

Treatment of psoriatic tissue with the RORγ inverse agonist GSK2981278 reduces proinflammatory cytokine levels.

<p>Three psoriatic skin biopsies were obtained via 3-4mm punch biopsy and placed in Unisol buffer for overnight shipment. Upon arrival, biopsy sections were placed in Cornification media without stimulation for 12–14 hours with either 0.2% DMSO or 10 μM compound. The percent inhibition of each biomarker compared to culture with DMSO alone is shown. Each point represents an individual patient sample. The percent maximum expression ± SEM for each analyte assayed of each tissue was calculated relative to time zero. Significant inhibition was determined by Paired <i>t</i>-test. (**p≤0.01).</p

Discovery of Epigenetic Regulator I‑BET762: Lead Optimization to Afford a Clinical Candidate Inhibitor of the BET Bromodomains

The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure–activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers

The Discovery of I‑BET726 (GSK1324726A), a Potent Tetrahydroquinoline ApoA1 Up-Regulator and Selective BET Bromodomain Inhibitor

Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure–activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors