The discovery of potent and selective non-steroidal glucocorticoid receptor modulators, suitable for inhalation.

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

Quantitative assessment of sensitizing potency using a dose-response adaptation of GARDskin.

Hundreds of chemicals have been identified as skin sensitizers. These are chemicals that possess the ability to induce hypersensitivity reactions in humans, giving rise to a condition termed allergic contact dermatitis. The capacity to limit hazardous exposure to such chemicals depends upon the ability to accurately identify and characterize their skin sensitizing potency. This has traditionally been accomplished using animal models, but their widespread use offers challenges from both an ethical and a scientific perspective. Comprehensive efforts have been made by the scientific community to develop new approach methodologies (NAMs) capable of replacing in vivo assays, which have successfully yielded several methods that can identify skin sensitizers. However, there is still a lack of new approaches that can effectively measure skin sensitizing potency. We present a novel methodology for quantitative assessment of skin sensitizing potency, which is founded on the already established protocols of the GARDskin assay. This approach analyses dose-response relationships in the GARDskin assay to identify chemical-specific concentrations that are sufficient to induce a positive response in the assay. We here compare results for 22 skin sensitizers analyzed using this method with both human and LLNA potency reference data and show that the results correlate strongly and significantly with both metrics (rLLNA = 0.81, p = 9.1 × 10-5; rHuman = 0.74, p = 1.5 × 10-3). In conclusion, the results suggest that the proposed GARDskin dose-response methodology provides a novel non-animal approach for quantitative potency assessment, which could represent an important step towards reducing the need for in vivo experiments.

The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers-Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility.

Proactive identification and characterization of hazards attributable to chemicals are central aspects of risk assessments. Current legislations and trends in predictive toxicology advocate a transition from in vivo methods to nonanimal alternatives. For skin sensitization assessment, several OECD validated alternatives exist for hazard identification, but nonanimal methods capable of accurately characterizing the risks associated with sensitizing potency are still lacking. The GARD (Genomic Allergen Rapid Detection) platform utilizes exposure-induced gene expression profiles of a dendritic-like cell line in combination with machine learning to provide hazard classifications for different immunotoxicity endpoints. Recently, a novel genomic biomarker signature displaying promising potency-associated discrimination between weak and strong skin sensitizers was proposed. Here, we present the adaptation of the defined biomarker signature on a gene expression analysis platform suited for routine acquisition, confirm the validity of the proposed biomarkers, and define the GARDpotency assay for prediction of skin sensitizer potency. The performance of GARDpotency was validated in a blinded ring trial, in accordance with OECD guidance documents. The cumulative accuracy was estimated to 88.0% across 3 laboratories and 9 independent experiments. The within-laboratory reproducibility measures ranged between 62.5% and 88.9%, and the between-laboratory reproducibility was estimated to 61.1%. Currently, no direct or systematic cause for the observed inconsistencies between the laboratories has been identified. Further investigations into the sources of introduced variability will potentially allow for increased reproducibility. In conclusion, the in vitro GARDpotency assay constitutes a step forward for development of nonanimal alternatives for hazard characterization of skin sensitizers.

Fragment-Based Discovery of Pyrazolopyridones as JAK1 Inhibitors with Excellent Subtype Selectivity.

Herein, we report the discovery of a series of JAK1-selective kinase inhibitors with high potency and excellent JAK family subtype selectivity. A fragment screening hit 1 with a pyrazolopyridone core and a JAK1 bias was selected as the starting point for our fragment-based lead generation efforts. A two-stage strategy was chosen with the dual aims of improving potency and JAK1 selectivity: Optimization of the lipophilic ribose pocket-targeting substituent was followed by the introduction of a variety of P-loop-targeting functional groups. Combining the best moieties from both stages of the optimization afforded compound 40, which showed excellent potency and selectivity. Metabolism studies in vitro and in vivo together with an in vitro safety evaluation suggest that 40 may be a viable lead compound for the development of highly subtype-selective JAK1 inhibitors.

Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitizers: Ring Trial Results of Predictive Performance and Reproducibility.

Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in nonregulatory settings, considerable efforts have been made to develop nonanimal approaches to replace current animal testing. Genomic Allergen Rapid Detection (GARD™) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARD™skin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses, and provide a holistic readout of substance-induced DC activation. Here, results from an inter-laboratory ring trial of GARD™skin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1% and 88.9%, whereas the cumulative predictive accuracy across the 3 laboratories was 93.8%. It was concluded that GARD™skin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARD™skin.

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Fragment-Based Discovery of 6-Arylindazole JAK Inhibitors.

Janus kinase (JAK) inhibitors are emerging as novel and efficacious drugs for treating psoriasis and other inflammatory skin disorders, but their full potential is hampered by systemic side effects. To overcome this limitation, we set out to discover soft drug JAK inhibitors for topical use. A fragment screen yielded an indazole hit that was elaborated into a potent JAK inhibitor using structure-based design. Growing the fragment by installing a phenol moiety in the 6-position afforded a greatly improved potency. Fine-tuning the substituents on the phenol and sulfonamide moieties afforded a set of compounds with lead-like properties, but they were found to be phototoxic and unstable in the presence of light.

Agonist-specific patterns of beta 2-adrenoceptor responses in human airway cells during prolonged exposure.

BACKGROUND AND PURPOSE: Beta(2)-adrenoceptor agonists (beta(2)-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, beta(2)-adrenergic agonist stimulation induces desensitization of the beta(2)-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of beta(2)-agonists in cellular and in vitro tissue models. EXPERIMENTAL APPROACH: Beta(2)-adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro. KEY RESULTS: There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of beta(2)-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists. CONCLUSIONS AND IMPLICATIONS: There were clear differences in the ability of different beta(2) agonists to induce loss of receptor responsiveness at equi-effective concentrations.