Relationship between Pharmacokinetics and Pharmacodynamic Responses in Healthy Smokers Informs a Once-Daily Dosing Regimen for Nemiralisib.

Nemiralisib (GSK2269557), a potent inhaled inhibitor of phosphoinositide 3-kinase δ (PI3Kδ), is being developed for the treatment of respiratory disorders including chronic obstructive pulmonary disease. Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n = 56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1-6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-(4,5)-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% confidence interval [CI], 11%-34%) and 36% (90% CI, 11%-64%) after a single dose or after 14 days of treatment with nemiralisib, respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ∼3- to 4.5-fold (peak vs. trough) in plasma exposure after 14 days of nemiralisib treatment. The BAL analysis at trough confirmed higher levels of the drug in the lungs versus plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). A comparison of the drug levels in plasma and the reductions in sputum PIP3 showed a direct relationship between exposure and PIP3 reduction. These results demonstrated target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Safety, pharmacokinetics and dose-response characteristics of GSK2269557, an inhaled PI3Kδ inhibitor under development for the treatment of COPD.

BACKGROUND: While current therapies reduce symptoms in chronic obstructive pulmonary disease (COPD) patients, substantial unmet need remains and novel treatments are highly desired. Phosphoinositide 3-kinase δ (PI3Kδ) is a lipid kinase specifically expressed in leucocytes and involved in their recruitment and activation. This study evaluated the safety, pharmacokinetics (PK) and dose-response characteristics of inhaled GSK2269557, a PI3Kδ inhibitor, in moderate-to-severe COPD patients with stable disease. METHODS: In this randomised, double-blind, placebo controlled, parallel group study, patients received once daily inhaled treatment with GSK2269557 1000 µg or placebo for 14 days (Part A, primary aim safety, N = 28 patients). In part B of the study (primary aim pharmacodynamic dose-response, N = 36 patients), GSK2269557 100, 200, 500, 700, 1000, 2000 µg or placebo was given for 14 days. In both Part A and B, GSK2269557 was added to the usual maintenance therapy. Safety, PK assessments and induced sputum collection for cytokine analysis were conducted at baseline and after 7 and 14 days of treatment. Adverse events (AEs) were monitored throughout. RESULTS: In Part A, mean age was 61.7 years (SD 6.7), 29% were females, and mean FEV1% predicted was 59.7% (SD 11.4)2. In Part B, mean age was 63.3 years (SD 6.3), 44% were females, and mean FEV1% predicted was 56.5% (SD 11.5)2. GSK2269557 was well tolerated in both parts of the study; the most commonly reported AEs were cough and headache, with cough being reported with a greater incidence in the GSK2269557 groups vs. placebo (Part A: 19% vs. 14% and Part B: range of 0-80% for different doses vs. 0% on placebo). No drug-related serious AEs or clinically significant changes in any other safety parameters were reported. GSK2269557 was rapidly absorbed into plasma following all doses with a maximum peak at approximately 2 h. Following repeat administration, accumulation in plasma was approximately 2-3 fold from Day 1 to Day 7. At Day 14, relative to placebo, sputum interleukin (IL)-8 and IL-6 levels were reduced on average by 32% and 29% respectively after inhalation of GSK2269557 1000 µg in Part A. In Part B, although inhibition of both IL-8 and IL-6 levels was observed, the levels were variable and there was insufficient evidence to support a monotonic dose-response. CONCLUSIONS: In this study, inhaled GSK2269557 had an acceptable safety profile for progression into larger studies in COPD patients. Moreover, inhalation of GSK2269557 resulted in suppression of sputum IL-8 and IL-6 levels, consistent with the known anti-inflammatory activity of a PI3Kδ inhibitor. Inhibition of inflammatory cytokines in the airway compartment may contribute to the potential therapeutic benefit of a PI3Kδ inhibitor in chronically inflamed COPD patients.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

BACKGROUND: Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. METHODS: Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. RESULTS: The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. CONCLUSIONS: This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients.

The effect of phosphatidylinositol-3 kinase inhibition on matrix metalloproteinase-9 and reactive oxygen species release from chronic obstructive pulmonary disease neutrophils.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is characterised by increased neutrophilic inflammation. A potential novel anti-inflammatory target in COPD is phosphatidylinositol-3 kinase (PI3 kinase), which targets neutrophil function. This study evaluated the effects of selective PI3Kδ inhibition on COPD blood and sputum neutrophils both in the stable state and during exacerbations. METHODS: Blood and sputum neutrophils from stable and exacerbating COPD patients were cultured with the corticosteroid dexamethasone, a pan PI3 kinase inhibitor (ZSTK474), a δ selective PI3 kinase inhibitor (GSK045) and a p38 mitogen activated protein (MAP) kinase inhibitor (BIRB 796); matrix metalloproteinase (MMP)-9 and reactive oxygen species (ROS) release were analysed. RESULTS: PI3Kδ inhibition significantly reduced MMP-9, intracellular ROS and extracellular ROS release from blood neutrophils (45.6%, 30.1% and 47.4% respectively; p<0.05) and intracellular ROS release from sputum neutrophils (16.6%; p<0.05) in stable patients. PI3Kδ selective inhibition significantly reduced stimulated MMP-9 (36.4%; p<0.05) and unstimulated and stimulated ROS release (12.6 and 26.7%; p<0.05) from blood neutrophils from exacerbating patients. The effects of the p38 MAP kinase inhibitor and dexamethasone in these experiments were generally lower than PI3Kδ inhibition. CONCLUSION: PI3Kδ selective inhibition is a potential strategy for targeting glucocorticoid insensitive MMP-9 and ROS secretion from COPD neutrophils, both in the stable state and during exacerbations.

Allergen-specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression.

BACKGROUND: A sensitive measurement of low numbers of intracellular cytokine-expressing antigen-specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy. OBJECTIVE: To evaluate the applicability of this method for measuring changes in cytokine production by allergen-specific T cells in a clinical trial setting. METHODS: Ex vivo ragweed-specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1-immunostimulatory oligodeoxynucleotide conjugate (AIC)-treated (n=17) and placebo-treated (n=15) ragweed-allergic subjects both after pre- and post-treatment ragweed exposures. In vitro allergen-stimulated CD3(+)CD4(+)CD154(+) T cell intracellular IL-4, IL-5, IL-13, and IFN-gamma expression were evaluated by flow cytometry. RESULTS: Compared with the T helper type 2 (Th2) cytokine expression measured after pre-treatment ragweed exposures, placebo-treated subjects demonstrated a significantly elevated ragweed- and Amb a 1-specific T cell IL-4 and IL-13 co-expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed-specific IL-5 expression (P<0.001) following post-treatment ragweed exposures. In contrast, AIC-treated subjects demonstrated no increases in allergen-specific Th2 cytokine expression following post-treatment ragweed exposures. IFN-gamma expression remained low and un-changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1- and ragweed-stimulated intracellular Th2 cytokine responses. CONCLUSION: Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen-specific T cell subset frequencies in clinical studies. Antigen-specific cytokine expression moderately correlated with the reported levels of allergic symptoms.

Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation.

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.

Eosinophilic rhinitis accompanies the development of lower airway inflammation and hyper-reactivity in sensitized mice exposed to aerosolized allergen.

BACKGROUND: Allergic rhinitis is a risk factor for the development of asthma. About 80% of asthmatic patients also have rhinitis. However, the pattern of induction of allergic rhinitis and asthma remains unclear. OBJECTIVE: The purpose of this study was to investigate the development of upper airway inflammation in mice during the development of an asthma-like disease and after an acute allergen provocation. METHODS: BALB-c mice were sensitized intraperitoneally (i.p) to ovalbumin (OA, days 1-13) and were challenged with aerosols of either OA or saline on 8 consecutive days (days 33-40). In a second experiment, chronic exposure for 8 days was followed by 10 days of rest and then an acute nebulized allergen provocation was performed (day 50). Inflammatory parameters were investigated at different time-points. RESULTS: Upper and lower eosinophilic airway inflammation were simultaneously induced in the course of repeated inhalations of nebulized OA, as shown by analyses of nasal and broncho-alveolar lavage fluids and histological sections of the nose and bronchi. Mice that developed bronchial hyper-responsiveness also had increased thickness of the nasal mucosa on magnetic resonance image (MRI) scans. When chronic exposure was followed by acute allergen provocation, the latter caused a systemic increase in IL-5 levels, with a concomitant rise in blood and airway eosinophils, primarily in the nose. CONCLUSIONS: Simultaneous induction of eosinophilic inflammation in the nose and lungs was found in a mouse model of respiratory allergy. These findings support the viewpoint that upper and lower airway disease represent a continuum of inflammation involving one common airway and provide evidence for the concept of global airway inflammation after inhalation of allergen.

Airway epithelial Fas ligand expression: potential role in modulating bronchial inflammation.

Epithelium-derived Fas ligand is believed to modulate inflammation within various tissues. In this paper, we report findings that suggest a similar immunoregulatory role for Fas ligand in the lung. First, Fas ligand was localized to nonciliated, cuboidal airway epithelial cells (Clara cells) throughout the airways in the normal murine lung by employing nonisotopic in situ hybridization and immunohistochemistry. Second, gld mutant mice, which express a dysfunctional Fas ligand protein, were noted to develop prominent infiltration of inflammatory cells in submucosal and peribronchial regions of the upper and lower airways. Third, during allergic airway inflammation induced by ovalbumin in mice, cell-associated staining for Fas ligand mRNA and protein was markedly reduced in the airway epithelium. These data suggest that Clara cell-derived Fas ligand may control immune activity in the airway; thus alterations in this protective mechanism may be involved in the pathogenesis of certain inflammatory conditions of the airway, such as asthma.

Involvement of IL-16 in the induction of airway hyper-responsiveness and up-regulation of IgE in a murine model of allergic asthma.

Experiments were designed to investigate the role of IL-16 in a mouse model of allergic asthma. OVA-sensitized mice were repeatedly exposed to OVA or saline aerosols. Bronchoalveolar lavage fluid (BALF) was collected after the last aerosol, and the presence of IL-16 was evaluated using a migration assay with human lymphocytes. Migration of lymphocytes was significantly increased in the presence of cell-free BALF from OVA-challenged mice compared with BALF from saline-challenged controls. This response was significantly inhibited after addition of antibodies to IL-16, demonstrating the presence of IL-16 in BALF of OVA-challenged animals. Immunohistochemistry was performed and revealed IL-16 immunoreactivity particularly in airway epithelial cells but also in cellular infiltrates in OVA-challenged mice. IL-16 immunoreactivity was absent in nonsensitized animals; however, some reactivity was detected in epithelial cells of sensitized but saline-challenged mice, suggesting that sensitization induced IL-16 expression in airway epithelium. Treatment of mice with antibodies to IL-16 during the challenge period significantly suppressed up-regulation of OVA-specific IgE in OVA-challenged animals. Furthermore, antibodies to IL-16 significantly inhibited the development of airway hyper-responsiveness after repeated OVA inhalations, whereas the number of eosinophils in bronchoalveolar lavage or airway tissue was not affected. In conclusion, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Furthermore, IL-16 plays an important role in airway hyper-responsiveness and up-regulation of IgE but is not important for eosinophil accumulation in a mouse model of allergic asthma.

Development of airway hyperresponsiveness is dependent on interferon-gamma and independent of eosinophil infiltration.

In this study the role of interleukin (IL)4, IL5, interferon (IFN) gamma, and tumor necrosis factor (TNF) alpha in the development of airway hyperresponsiveness and inflammatory cell infiltration was investigated using a murine model for allergic asthma. Mice were sensitized with ovalbumin and subsequently challenged repeatedly with ovalbumin aerosols. During the challenge period, mice were treated with monoclonal antibodies directed against IL4, IL5, IFN gamma, or TNF alpha. Control antibody-treated mice showed airway hyperresponsiveness to methacholine and the presence of eosinophils in bronchoalveolar lavage (BAL). Treatment with antibodies to IFN gamma completely abolished development of airway hyperresponsiveness in ovalbumin-challenged animals. After treatment with antibodies to TNF alpha, airway hyperresponsiveness in the ovalbumin-challenged animals was partially but not significantly inhibited. Antibodies to IL4 or IL5 did not inhibit airway hyperresponsiveness. The presence of eosinophils in BAL of ovalbumin-challenged mice was completely inhibited after treatment with antibodies to IL5. Treatment with antibodies to IL4, IFN gamma, or TNF alpha had no effect on eosinophilia. Because IFN gamma and IL5 have either an effect on the induction of airway hyperresponsiveness or on the development of eosinophil infiltration, our results suggest that the two phenomena are differentially regulated.

Effect of dexamethasone and endogenous corticosterone on airway hyperresponsiveness and eosinophilia in the mouse.

1. Mice were sensitized by 7 intraperitoneal injections of ovalbumin without adjuvant (10 micrograms in 0.5 ml of sterile saline) on alternate days and after 3 weeks exposed to either ovalbumin (2 mg ml-1 in sterile saline) or saline aerosol for 5 min on 8 consecutive days. One day before the first challenge, animals were injected intraperitoneally on a daily basis with vehicle (0.25 ml sterile saline), dexamethasone (0.5 mg kg-1) or metyrapone (30 mg kg-1). 2. In vehicle-treated ovalbumin-sensitized animals ovalbumin challenge induced a significant increase of airway responsiveness to metacholine both in vitro (27%, P < 0.05) and in vivo (40%, P < 0.05) compared to saline-challenged mice. Virtually no eosinophils could be detected after saline challenge, whereas the numbers of eosinophils were significantly increased (P < 0.01) at both 3 and 24 h after the last ovalbumin challenge (5.48 +/- 3.8 x 10(3) and 9.13 +/- 1.7 x 10(3) cells, respectively). Furthermore, a significant increase in ovalbumin-specific immunoglobulin E level (583 +/- 103 units ml-1, P < 0.05) was observed after ovalbumin challenge compared to saline challenge (201 +/- 38 units ml-1). 3. Plasma corticosterone level was significantly reduced (-92%, P < 0.001) after treatment with metyrapone. Treatment with metyrapone significantly increased eosinophil infiltration (17.4 +/- 9.93 x 10(3) and 18.7 +/- 2.57 x 10(3) cells, P < 0.05 at 3 h and 24 h, respectively) and potentiated airway hyperresponsiveness to methacholine compared to vehicle-treated ovalbumin-challenged animals. Dexamethasone inhibited both in vitro and in vivo hyperresponsiveness as well as antigen-induced infiltration of eosinophils (0, P < 0.05 and 0.7 +/- 0.33 x 10(3) cells, P < 0.05 at 3 h and 24 h, respectively). Metyrapone as well as dexamethasone did not affect the increase in ovalbumin-specific immunoglobulin E levels after ovalbumin challenge (565 +/- 70 units/ml-1; P < 0.05; 552 +/- 48 units ml-1, P < 0.05 respectively). 4. From these data it can be concluded that exogenously applied corticosteroids can inhibit eosinophil infiltration as well as airway hyperresponsiveness. Vise versa, endogenously produced corticosteroids play a down-regulating role on the induction of both eosinophil infiltration and airway hyperresponsiveness.

Lung inflammation and epithelial changes in a murine model of atopic asthma.

A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer.

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice.

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.

Repeated measurement of respiratory function and bronchoconstriction in unanesthetized mice.

A noninvasive forced oscillation technique was used to determine respiratory function in unanesthetized and spontaneously breathing mice. Pseudorandom noise pressure variations in a frequency range of 16-208 Hz were applied to the body surface, and the flow response was measured at the nose. From the pressure-flow relationship, respiratory transfer impedance was calculated. Study of intra-animal variability on a short- and a long-term basis revealed that the real part of respiratory transfer impedance was reproducible within 9%. The imaginary part appeared less reproducible (within 22%). Furthermore, bronchoconstrictive responses were investigated and analyzed by evaluation of respiratory resistance as measured at 16 Hz (Rrs16). During the first 15 min after ovalbumin challenge in ovalbumin-sensitized mice, Rrs16 was significantly increased [49 +/- 7% (SE)]. Inhalation of methacholine in untreated mice induced an increase in Rrs16 of 75 +/- 16% (SE). In saline-challenged animals, no significant changes were observed. This method enables evaluation of long-term respiratory function in mice and appeared to be a sensitive measure for bronchoconstriction.

Minimal immunological changes in structurally malformed rats after prenatal exposure to cyclophosphamide.

In order to compare the sensitivities of morphological and immunological parameters in a teratology study, effects in day 20 rat fetuses were studied after a single exposure to the immunosuppressive cytostatic agent cyclophosphamide (CP) on either day 11 or day 15 of gestation. Teratological methods included evaluation of external and skeletal morphology. Furthermore histology, immunohistochemistry and flow cytometry were performed on fetal thymus, liver and spleen. Immune function was assayed using the Trichinella spiralis infection model. Treatment resulted in dose-dependent gross morphological malformations, and in addition in overt skeletal anomalies such as brachygnathia, wavy ribs, and lordosis. In contrast, the immunological parameters tested revealed only minimal differences between treated and control groups. These results suggest either a remarkable recovery of the immune system after treatment, or a relatively high resistance of the immune system to the present treatment.