RESUMO
Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.
RESUMO
Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.
Assuntos
Asma/tratamento farmacológico , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Asma/induzido quimicamente , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Estrutura Molecular , Ovalbumina , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Ratos Endogâmicos BN , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Tiazóis/síntese química , Tiazóis/metabolismo , Tiazóis/farmacocinéticaRESUMO
PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 > 9), and by careful manipulation of the pKa and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Relação Estrutura-Atividade , Administração por Inalação , Animais , Disponibilidade Biológica , Técnicas de Química Sintética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/administração & dosagem , Meia-Vida , Isoenzimas/antagonistas & inibidores , Camundongos Transgênicos , Permeabilidade , Ratos , Solubilidade , Tiazóis/químicaRESUMO
BACKGROUND: In a dog with joint pain, it is important to determine whether it has suppurative joint disease, characterized by exudation of neutrophils in the synovial fluid, or not, as this affects choice of diagnostic tests and treatments. The aim of this study was to evaluate whether measurement of serum C-reactive protein (CRP) concentration could be used to discriminate between dogs with suppurative arthritis and osteoarthritis (OA). Furthermore, the concentrations of serum and synovial fluid interleukin (IL) 6 concentrations were measured in dogs with joint disease and in healthy dogs, and were correlated to serum CRP concentrations. METHODS: Dogs with joint pain were enrolled prospectively and were classified to have suppurative arthritis or OA based on synovial fluid analysis and radiographic/arthroscopic findings. Healthy Beagles were enrolled as a comparative group. CRP and IL-6 concentrations were measured with canine-specific immunoassays. The performance of CRP concentration in discriminating between dogs with suppurative arthritis and OA was evaluated using a previously established clinical decision limit for CRP (20 mg/l), and by receiver operator characteristic (ROC) curve and logistic regression analysis. Comparisons of CRP and IL-6 concentrations between groups were performed using t-tests, and correlations by Spearman rank correlation coefficients. RESULTS: Samples were obtained from 31 dogs with suppurative arthritis, 34 dogs with OA, and 17 healthy dogs. Sixty-two out of 65 dogs with joint disease were correctly classified using the clinical decision limit for CRP. Evaluation of ROC curve and regression analysis indicated that serum CRP concentrations could discriminate between suppurative arthritis and OA. Dogs with suppurative arthritis had higher serum CRP and serum and synovial fluid IL-6 concentrations compared to dogs with OA (p < 0.001). Dogs with OA had higher synovial fluid IL-6 concentrations (p < 0.001), but not higher serum CRP (p = 0.29) or serum IL-6 (p = 0.07) concentrations, compared to healthy dogs. There was a positive correlation between synovial fluid IL-6 and serum CRP concentrations (rs = 0.733, p < 0.001), and between serum IL-6 and serum CRP concentrations (rs = 0.729, p < 0.001). CONCLUSION: CRP concentration was found to discriminate well between dogs with suppurative arthritis and OA.
Assuntos
Artrite Infecciosa/veterinária , Proteína C-Reativa/metabolismo , Doenças do Cão/diagnóstico , Osteoartrite/veterinária , Animais , Artrite Infecciosa/sangue , Diagnóstico Diferencial , Doenças do Cão/sangue , Cães , Feminino , Masculino , Osteoartrite/sangue , Estudos Prospectivos , Líquido Sinovial/metabolismoRESUMO
Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.