Evaluating the efficacy of upadacitinib in patients with moderate rheumatoid arthritis: a post-hoc analysis of the SELECT phase 3 trials.

Objectives: Moderately active RA is associated with poor patient outcomes. Despite this, some health systems have restricted access to advanced therapies to those with severe RA. There is also limited evidence of the efficacy of advanced therapies in the moderately active RA population. This post-hoc analysis from four phase 3 trials explored the efficacy of upadacitinib (UPA) for moderately active RA. Methods: Patients included in this analysis received UPA 15 mg once daily [monotherapy after switching from MTX or in combination with stable background conventional synthetic DMARDs (csDMARDs)] or placebo. Clinical, functional and radiographic outcomes were analysed separately for patients with moderate disease activity {28-joint count DAS using CRP [DAS28(CRP)] of >3.2 and ≤5.1} and severe disease activity [DAS28(CRP) >5.1]. Results: Patients with moderate disease activity who received UPA 15 mg (combination or monotherapy) after an inadequate response to biologic DMARDs and/or csDMARDs were significantly more likely to achieve a 20% improvement in the ACR response criteria, low disease activity status [DAS28(CRP) ≤ 3.2] or clinical remission [DAS28(CRP) < 2.6] by week 12/14 vs placebo. Statistically significant improvements in patient-reported functioning and pain from baseline were observed for UPA 15 mg vs placebo at week 12/14. Radiographic progression was also significantly reduced at week 26 compared with placebo. Similar improvements were observed for severe disease. Conclusion: This analysis provides support for the use of UPA for the treatment of patients with moderate RA. Trial registration: ClinicalTrials.gov: SELECT-NEXT: NCT02675426; SELECT-COMPARE: NCT02629159; SELECT-MONOTHERAPY: NCT02706951; SELECT-BEYOND: NCT02706847.

The inhibition of human lung fibroblast proliferation and differentiation by Gs-coupled receptors is not predicted by the magnitude of cAMP response.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrotic lung disease for which there is no cure. Current therapeutics are only able to slow disease progression, therefore there is a need to explore alternative, novel treatment options. There is increasing evidence that the 3', 5' cyclic adenosine monophosphate (cAMP) pathway is an important modulator in the development of fibrosis, with increasing levels of cAMP able to inhibit cellular processes associated with IPF. In this study we investigate the expression of Gs-coupled G protein-coupled receptors (GPCR) on human lung fibroblasts (HLF), and explore which can increase cAMP levels, and are most efficacious at inhibiting proliferation and differentiation. METHODS: Using TaqMan arrays we determined that fibroblasts express a range of Gs-coupled GPCR. The function of selected agonists at expressed receptors was then tested in a cAMP assay, and for their ability to inhibit fibroblast proliferation and differentiation. RESULTS: Expression analysis of GPCR showed that the prostacyclin, prostaglandin E2 (PGE2) receptor 2 and 4, melanocortin-1, ß2 adrenoceptor, adenosine 2B, dopamine-1, and adenosine 2A receptors were expressed in HLF. Measuring cAMP accumulation in the presence of selected Gs-coupled receptor ligands as well as an adenylyl cyclase activator and inhibitors of phosphodiesterase showed formoterol, PGE2, treprostinil and forskolin elicited maximal cAMP responses. The agonists that fully inhibited both fibroblast proliferation and differentiation, BAY60-6583 and MRE-269, were partial agonists in the cAMP accumulation assay. CONCLUSIONS: In this study we identified a number of ligands that act at a range of GPCR that increase cAMP and inhibit fibroblast proliferation and differentiation, suggesting that they may provide novel targets to develop new IPF treatments. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, the magnitude of the acute cAMP response is not a good predictor of the extent of the inhibitory effect. This highlights the importance of monitoring the kinetics and localisation of intracellular signals, as well as multiple pathways when profiling novel compounds, as population second messenger assays may not always predict phenotypic outcomes.

Comparing the cardiovascular therapeutic indices of glycopyrronium and tiotropium in an integrated rat pharmacokinetic, pharmacodynamic and safety model.

Long acting inhaled muscarinic receptor antagonists, such as tiotropium, are widely used as bronchodilator therapy for chronic obstructive pulmonary disease (COPD). Although this class of compounds is generally considered to be safe and well tolerated in COPD patients the cardiovascular safety of tiotropium has recently been questioned. We describe a rat in vivo model that allows the concurrent assessment of muscarinic antagonist potency, bronchodilator efficacy and a potential for side effects, and we use this model to compare tiotropium with NVA237 (glycopyrronium bromide), a recently approved inhaled muscarinic antagonist for COPD. Anaesthetized Brown Norway rats were dosed intratracheally at 1 or 6h prior to receiving increasing doses of intravenous methacholine. Changes in airway resistance and cardiovascular function were recorded and therapeutic indices were calculated against the ED50 values for the inhibition of methacholine-induced bronchoconstriction. At both time points studied, greater therapeutic indices for hypotension and bradycardia were observed with glycopyrronium (19.5 and 28.5 fold at 1h; >200 fold at 6h) than with tiotropium (1.5 and 4.2 fold at 1h; 4.6 and 5.5 fold at 6h). Pharmacokinetic, protein plasma binding and rat muscarinic receptor binding properties for both compounds were determined and used to generate an integrated model of systemic M2 muscarinic receptor occupancy, which predicted significantly higher M2 receptor blockade at ED50 doses with tiotropium than with glycopyrronium. In our preclinical model there was an improved safety profile for glycopyrronium when compared with tiotropium.

Functional desensitization of the ß 2 adrenoceptor is not dependent on agonist efficacy.

Chronic treatment with ß 2 adrenoceptor agonists is recommended as a first-line maintenance therapy for chronic obstructive pulmonary disease (COPD). However, a potential consequence of long-term treatment may be the loss of functional response (tachyphylaxis) over time. In this study, we have investigated the tendency of such agonists, with a range of efficacies, to develop functional desensitization to cAMP responses in primary human bronchial smooth muscle cells following prolonged agonist exposure. The data show that upon repeat exposure, all agonists produced functional desensitization to the same degree and rate. In addition, ß 2 adrenoceptor internalization and ß-arrestin-2 recruitment were monitored using ß 2·eGFP visualization and the PathHunter™ ß-arrestin-2 assay, respectively. All agonists were capable of causing robust receptor internalization and ß-arrestin-2 recruitment, the rate of which was influenced by agonist efficacy, as measured in those assays. In summary, although a relationship exists between agonist efficacy and the rate of both receptor internalization and ß-arrestin-2 recruitment, there is no correlation between agonist efficacy and the rate or extent of functional desensitization.

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

The antiallergic mast cell stabilizers lodoxamide and bufrolin as the first high and equipotent agonists of human and rat GPR35.

Lack of high potency agonists has restricted analysis of the G protein-coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

The use of cold plasma technology to reduce carryover in screening assays.

The accurate transfer of biological reagents represents a fundamental step in the drug screening process, and the elimination of carryover is critical for the generation of accurate measurements of biological activity. The introduction of automated liquid robotics into screening laboratories has transformed the drug screening process, enabling accurate and reproducible transfer of liquids to become a high-throughput activity, but has also introduced a new challenge for drug discoverers: to establish screening workflows that limit analyte carryover for the generation of high-quality screening data. The widespread use of pipetting tips on automated liquid handlers often necessitates the use of optimized wash protocols for removing contaminants and frequently requires the use and disposal of large quantities of organic solvents. Furthermore, many chemical and biological reagents are recalcitrant to removal from pipetting tips by treatment with organic solvents. The use of cold atmospheric plasma technology provides an alternative approach for removal of contaminants and offers many advantages over traditional decontamination protocols commonly used during biological screening. This report describes the evaluation of a cold plasma tip-cleaning system for reducing carryover in a range of biological screening assays requiring the transfer of low molecular weight compound, nucleic acid, and bacterial liquid transfers. The validation of this technology for biological screening assays is presented, and the impact of this technology for screening workflows is discussed.

Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates.

BACKGROUND: Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function. METHODS: The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates. RESULTS: The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates. CONCLUSIONS: The data described in this report provides insight into the appropriate choice of cell models for investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.

The Influence of receptor kinetics on the onset and duration of action and the therapeutic index of NVA237 and tiotropium.

Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [3H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3-4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M3) versus muscarinic type 2 (M2) receptors, with a faster off rate from M2 versus M3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors.

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

The effect of ICER on screening methods involving CRE-mediated reporter gene expression.

We describe a mechanism whereby increasing levels of cAMP in Chinese hamster ovary (CHO) and other cell lines lead to a significant repression in cAMP response element (CRE)-mediated luciferase reporter gene expression. This effect was shown to be mediated by a modulatory factor located downstream of cyclic AMP (cAMP), which displayed the temporal regulation pattern of an immediate early gene. The expression of this inducible cAMP early repressor (ICER) was shown to be coincident with the time and concentration dependency of the repression of CRE-mediated luciferase gene expression on the treatment of CHO cells with forskolin. Furthermore, this phenomenon was also observed in JEG and GH3 cell lines (both previously reported to express ICER), but not in COS-7 cells, which do not express ICER. These studies suggest that, in certain cell lines, expression of ICER can be induced at pharmacologically elevated cAMP levels, leading to a potent inhibition of CRE-mediated gene expression. We therefore conclude that screening methodologies employing such CRE-linked reporter genes (particularly in high-throughput screening assays) may produce false functional responses in certain cell lines. Moreover, such effects are likely to be exacerbated in screening assays in which receptors either are overexpressed or high concentrations of potent cAMP-elevating compounds are used.