RESUMEN
BACKGROUND: Application of basophil activation test (BAT) in clinical trials requires assay validity. Whether assay variability differs between healthy and asthmatic subjects is mostly unknown. This study compares basophil stimulation using blood from healthy and asthmatic subjects with or without inhibition of spleen tyrosine kinase (SYK). METHODS: Whole blood of healthy and mild asthmatic subjects was stimulated with anti-dinitrophenyl (DNP) IgE/DNP bovine serum albumin and anti-IgE. Basophil activation was detected by CD63 and CD203c expression. CD63 expression levels were compared with serum IgE levels. Three operators repeated experiments with three subjects each from both groups at 3 days to observe assay precision. The effect of the SYK inhibitor BI 1002494 was assessed in BAT for both healthy and asthmatic subjects. RESULTS: BAT was reproducible in both groups. Acceptance criteria of <25% CV were mostly fulfilled. Stimulation with anti-DNP (p < 0.001, r = -0.80) but not anti-IgE (p = 0.74, r = 0.05) was related to serum IgE with levels > 200 IU/ml limiting anti-DNP stimulation. BI 1002494 IC50 values were 497 nM and 1080 nM in healthy and 287 nM and 683 nM in asthmatics for anti-DNP and anti-IgE stimulation, respectively. CONCLUSION: BAT, performed with blood from healthy or asthmatic subjects, is a robust test for the measurement of a physiological response in clinical trials. Blood from asthmatic donors with serum IgE > 200 IU/ml is less feasible when using anti-DNP stimulation. SYK inhibition was not affected by disease status.
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Prueba de Desgranulación de los Basófilos , Inmunoglobulina E , Basófilos , Citometría de Flujo , Humanos , Naftiridinas , Pirrolidinonas , Quinasa Syk , Tetraspanina 30RESUMEN
AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of BI 655064 in healthy Chinese and Japanese subjects after administration of single doses of 80-240 mg and multiple dosing of 240 mg once weekly over 4 weeks. METHODS: Two phase 1, double-blind, placebo-controlled studies were conducted (single-rising doses of BI 655064 in Chinese/Japanese male subjects [n = 12 per BI 655064 dose group] or repeated 240 mg BI 655064 in Chinese male subjects [n = 9]). Plasma samples were collected to investigate BI 655064 pharmacokinetics, pharmacodynamics (CD40 receptor occupancy [RO]) and immunogenicity, along with the safety and tolerability of BI 655064. RESULTS: BI 655064 showed good overall tolerability following single-dose administration of 80-240 mg and repeated administration of 240 mg BI 655064 over 4 weeks. More Chinese subjects reported adverse events compared with Japanese subjects following single-dose administration (59.4% vs 3.1%). BI 655064 exhibited nonlinear, saturable kinetics, with higher doses resulting in slower apparent clearance (0.514-0.713 mL min-1 ), and disproportionately higher total exposure (AUC0-inf ; 5610-7780 µg·h mL-1 ) and maximum plasma concentration (15 700-21 300 ng mL-1 ) with 240 mg BI 655064. Ninety percent inhibition of CD40 RO was achieved with doses ≥120 mg, and a direct relationship between BI 655064 plasma concentration and inhibition of CD40 RO was observed. Most subjects had a positive treatment-emergent antidrug antibody response. CONCLUSIONS: BI 655064 pharmacokinetic and safety profiles in East Asian male subjects were consistent with those observed in a Western population. No adjustments in the BI 655064 dosing recommendations are warranted for future clinical trials.
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Área Bajo la Curva , Anticuerpos Monoclonales Humanizados , China , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Voluntarios Sanos , Humanos , Japón , MasculinoRESUMEN
AIMS: To evaluate the safety, pharmacokinetics and pharmacodynamics of single- and multiple-rising doses (MRDs) of BI 705564 and establish proof of mechanism. METHODS: BI 705564 was studied in 2 placebo-controlled, Phase I clinical trials testing single-rising doses (1-160 mg) and MRDs (1-80 mg) of BI 705564 over 14 days in healthy male volunteers. Blood samples were analysed for BI 705564 plasma concentration, Bruton's tyrosine kinase (BTK) target occupancy (TO) and CD69 expression in B cells stimulated ex vivo. A substudy was conducted in allergic, otherwise healthy, MRD participants. Safety was assessed in both studies. RESULTS: All doses of BI 705564 were well tolerated. Geometric mean BI 705564 plasma terminal half-life ranged from 10.1 to 16.9 hours across tested doses, with no relevant accumulation after multiple dosing. Doses ≥20 mg resulted in ≥85% average TO that was maintained for ≥48 hours after single-dose administration. Functional effects of BTK signalling were demonstrated by dose-dependent inhibition of CD69 expression. In allergic participants, BI 705564 treatment showed a trend in wheal size reduction in a skin prick test and complete inhibition of basophil activation. Mild bleeding-related adverse events were observed with BI 705564; bleeding time increased in 1/12 participants (8.3%) who received placebo vs 26/48 (54.2%) treated with BI 705564. CONCLUSION: BI 705564 showed efficient target engagement through durable TO and inhibition of ex vivo B-cell activation, and proof of mechanism through effects on allergic skin responses. Mild bleeding-related adverse events were probably related to inhibition of platelet aggregation by BTK inhibition.
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Linfocitos B , Agregación Plaquetaria , Agammaglobulinemia Tirosina Quinasa , Voluntarios Sanos , Humanos , Masculino , Inhibidores de Proteínas Quinasas/efectos adversos , Transducción de SeñalRESUMEN
BI 1021958, a novel antagonist of the chemoattractant-receptor-homologous molecule (CRTH2), targets airway inflammation in asthma by inhibiting prostaglandin binding to CRTH2 receptors. Two phase 1 studies assessed BI 1021958 safety/tolerability and pharmacokinetics (PK)/pharmacodynamics (PD) following single doses in healthy men and multiple doses in men/women with well-controlled asthma. Studies 1 had 2 parts: a placebo-controlled, fixed-sequence, single-blind, single-rising-dose part (n = 56) and a randomized, 2-way crossover, open-label, repeated-dose part studying the food effect on PK/PD (n = 12). Study 2 was a placebo-controlled, single-center, double-blind multiple-rising-dose study (n = 84). Primary end points were safety/tolerability and PK/PD (both studies); secondary end points were eosinophil shape change (ESC; study 1) and dose proportionality/linearity following first dose and at steady state (study 2). BI 1021958 was adequately tolerated in both studies; adverse events were infrequent, generally mild to moderate, and occurred similarly in treatment groups. Maximum measured concentration (Cmax ) was achieved in ≤2.5 hours in study 1 and ≤2.0 hours in study 2. BI 1021958 exposure increased proportionally with dose. In study 1, following a single 60-mg dose, AUC parameters and Cmax were 20% and 15% lower, respectively, after a high-fat meal compared with the fasted state. After ≥60-mg single doses (study 1) and >40-mg multiple doses (study 2), >95% ESC inhibition was observed for ≥24 hours. PK/PD was similar in healthy subjects and subjects with well-controlled asthma. Data support further investigation of CRTH2 antagonists for the treatment of asthma.
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Antiasmáticos/farmacocinética , Asma/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Administración Oral , Adulto , Antiasmáticos/efectos adversos , Área Bajo la Curva , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Eosinófilos/efectos de los fármacos , Eosinófilos/fisiología , Femenino , Interacciones Alimento-Droga/fisiología , Semivida , Humanos , Masculino , Método Simple CiegoRESUMEN
Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1ß, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3(+)CD4(+) Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans.
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Inflamación/etiología , Interleucina-10/genética , Receptores Toll-Like/agonistas , Resistencia de las Vías Respiratorias/inmunología , Animales , Asma/tratamiento farmacológico , Asma/inmunología , Relación Dosis-Respuesta a Droga , Eosinofilia/inmunología , Femenino , Inflamación/inmunología , Glicoproteínas de Membrana/agonistas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Ovalbúmina/inmunología , Factores de Tiempo , Receptor Toll-Like 7/agonistasRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the pathogenesis of acute and chronic lung disease as a major regulator governing the functions of granulocyte and macrophage lineage populations. Chronic obstructive pulmonary disease (COPD) is a disease characterized by lung inflammation with accumulation of neutrophils and increased levels of pro-inflammatory cytokines including GM-CSF in the patient's lungs. We used intranasal administration of lipopolysaccharide (LPS) to mice to induce a disease that resembles COPD with pulmonary inflammation, neutrophil recruitment and release of pro-inflammatory mediators in the bronchoalveolar lavage fluid of the diseased mice. 2 h prior to LPS administration, mice were systemically treated with the murine GM-CSF neutralizing antibody mAb 22E9 per intraperitoneal injection. Intranasal challenge with LPS-induced an increase of total cell number and of neutrophils in the bronchoalveolar lavage fluid. Elevated levels of tumor necrosis factor alpha (TNF-alpha), keratinocyte cytokine and macrophage inflammatory protein-2 (MIP-2) were also observed at this time point. GM-CSF was no longer detectable in bronchoalveolar lavage fluid at 24 h due to its early expression with a peak reached 6 h after LPS challenge. Pretreatment of mice with GM-CSF neutralizing antibody dose-dependently inhibited the accumulation of neutrophils and reduced TNF-alpha and MIP-2 protein levels in bronchoalveolar lavage fluid. These data suggest that neutralization of GM-CSF may represent a novel treatment modality for lung inflammation and in particular for COPD.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Lipopolisacáridos/farmacología , Neumonía/inmunología , Neumonía/metabolismo , Animales , Anticuerpos/farmacología , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Neumonía/inducido químicamente , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Patient response to asthma therapy is consistently observed to be heterogeneous. Pharmacogenomics is the study of inherited differences in interindividual drug disposition and effects, with the goal of selecting the optimal drug therapy and dosage for each patient. This review will cover selected examples of gene polymorphisms that influence the outcome of asthma therapy, and whole-genome expression studies using microarray technology that have shown tremendous potential for benefiting asthma pharmacogenomics. The utility of the mouse as an experimental system for pharmacogenomic discovery will also be discussed in the context of asthma therapy.