Efficacy and Safety of the Bruton's Tyrosine Kinase Inhibitor Evobrutinib in Systemic Lupus Erythematosus: Results of a Phase II, Randomized, Double-Blind, Placebo-Controlled Dose-Ranging Trial.

OBJECTIVE: Evobrutinib is a highly selective, orally administered Bruton's tyrosine kinase (BTK) inhibitor. The objective of this phase II, multicenter, randomized, double-blind, placebo-controlled trial was to evaluate the efficacy and safety of evobrutinib in patients with active autoantibody-positive systemic lupus erythematosus (SLE). METHODS: Patients were diagnosed with SLE by either the Systemic Lupus International Collaborating Clinics criteria or at least four American College of Rheumatology criteria 6 months or more prior to screening, had an SLE Disease Activity Index-2000 score of 6 or more, were autoantibody-positive and on standard-of-care therapy. Randomization was 1:1:1:1 to oral evobrutinib 25 mg once daily (QD), 75 mg QD, 50 mg twice daily, or placebo. Primary efficacy endpoints were SLE responder index (SRI)-4 response at week 52 and SRI-6 response at week 52 in the high disease activity subpopulation. Safety endpoints included treatment-emergent adverse events (TEAEs). RESULTS: A total of 469 patients were randomized and received at least one dose of evobrutinib or placebo at the time of primary analysis. Mean (SD) age at baseline was 40.7 (±12.3) years; 94.9% of patients were female. Neither primary efficacy endpoint was met. All doses of evobrutinib were well tolerated, and there was no clear dose effect on the incidence of reported TEAEs, or serious TEAEs, including severe infections. CONCLUSION: This phase II, dose-ranging trial in SLE failed to show a treatment effect of evobrutinib versus placebo at any dose. Evobrutinib was generally well tolerated, with no dose effect observed for TEAEs. These results suggest that BTK inhibition does not appear to be an effective therapeutic intervention for patients with SLE.

PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

Behavioral Hyperventilation and Central Sleep Apnea in Two Children.

Behavioral hyperventilation is a rarely recognized cause of central sleep apnea (CSA) among children. We report two pediatric patients who presented with prolonged central sleep apnea secondary to behavioral hyperventilation. One patient also had a prolonged corrected QT (QT(C)) interval resulting from hyperventilation

TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease.

Linking acute infection to chronic lung disease. The role of IL-33-expressing epithelial progenitor cells.

Respiratory infection is a common feature of the major human airway diseases, such as asthma and chronic obstructive pulmonary disease, but the precise link between acute infection and chronic lung disease is still undefined. In a mouse model of this process, parainfluenza virus infection is followed by long-term induction of IL-33 expression and release and in turn innate immune cell generation of IL-13 and consequent airway disease signified by excess mucus formation. IL-33 induction was traceable to a subset of secretoglobin-positive airway epithelial cells linked to progenitor/stem cell function. In corresponding studies of humans with chronic obstructive pulmonary disease, an increase in IL-33 production was also detected in concert with up-regulation of IL-13 and airway mucus formation. In this case, increased IL-33 production was localized to a subset of airway basal cells that maintain an endogenous capacity for increased pluripotency and ATP-regulated release of IL-33 even ex vivo. The results provide evidence of a sustainable epithelial cell population that may be activated by environmental danger signals to release IL-33 and thereby lead to IL-13-dependent disease. The progenitor nature of this IL-33-expressing ATP-responsive cell population could explain an acquired susceptibility to chronic airway disease. The findings may therefore provide a new paradigm to explain the role of viral infection and the innate immune system in chronic lung disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production. Further studies are needed to address the basis for this type of postviral reprogramming and the means to correct it and thereby restore airway mucosal immune function to normal.

Myb permits multilineage airway epithelial cell differentiation.

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63(+) basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps are poorly defined. Here, we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb(+) cells were identified as p63(-) and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63(-) population with failed maturation of Foxj1(+) ciliated cells as well as Scbg1a1(+) and Muc5ac(+) secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb(+) cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63(-) Myb(+) population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease.

High-throughput screening normalized to biological response: application to antiviral drug discovery.

The process of conducting cell-based phenotypic screens can result in data sets from small libraries or portions of large libraries, making accurate hit picking from multiple data sets important for efficient drug discovery. Here, we describe a screen design and data analysis approach that allow for normalization not only between quadrants and plates but also between screens or batches in a robust, quantitative fashion, enabling hit selection from multiple data sets. We independently screened the MicroSource Spectrum and NCI Diversity Set II libraries using a cell-based phenotypic high-throughput screening (HTS) assay that uses an interferon-stimulated response element (ISRE)-driven luciferase-reporter assay to identify interferon (IFN) signal enhancers. Inclusion of a per-plate, per-quadrant IFN dose-response standard curve enabled conversion of ISRE activity to effective IFN concentrations. We identified 45 hits based on a combined z score ≥2.5 from the two libraries, and 25 of 35 available hits were validated in a compound concentration-response assay when tested using fresh compound. The results provide a basis for further analysis of chemical structure in relation to biological function. Together, the results establish an HTS method that can be extended to screening for any class of compounds that influence a quantifiable biological response for which a standard is available.

Clinical relevance of target identity and biology: implications for drug discovery and development.

Many of the most commonly used drugs precede techniques for target identification and drug specificity and were developed on the basis of efficacy and safety, an approach referred to as classical pharmacology and, more recently, phenotypic drug discovery. Although substantial gains have been made during the period of focus on target-based approaches, particularly in oncology, these approaches have suffered a high overall failure rate and lower productivity in terms of new drugs when compared with phenotypic approaches. This review considers the importance of target identity and biology in clinical practice from the prescriber's viewpoint. In evaluating influences on prescribing behavior, studies suggest that target identity and mechanism of action are not significant factors in drug choice. Rather, patients and providers consistently value efficacy, safety, and tolerability. Similarly, the Food and Drug Administration requires evidence of safety and efficacy for new drugs but does not require knowledge of drug target identity or target biology. Prescribers do favor drugs with novel mechanisms, but this preference is limited to diseases for which treatments are either not available or suboptimal. Thus, while understanding of drug target and target biology is important from a scientific perspective, it is not particularly important to prescribers, who prioritize efficacy and safety.

Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease.

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.

Primary ciliary dyskinesia-causing mutations in Amish and Mennonite communities.

OBJECTIVE: To determine whether individuals with primary ciliary dyskinesia (PCD) from unrelated Amish and Mennonite families harbor a single and unique founder mutation. STUDY DESIGN: Subjects from Amish and Mennonite communities in several states were enrolled in the study. All subjects were clinically characterized, and nasal nitric oxide levels were measured. Nasal epithelial scrapings were collected from several subjects for ciliary ultrastructural analyses. DNA was isolated from patients with PCD and their unaffected first- and second-degree relatives. Genome-wide homozygosity mapping, linkage analyses, targeted mutation analyses, and exome sequencing were performed. RESULTS: All subjects from Old-Order Amish communities from Pennsylvania were homozygous for a nonsense mutant DNAH5 allele, c.4348C>T (p.Q1450X). Two affected siblings from an unrelated Mennonite family in Arkansas were homozygous for the same nonsense DNAH5 mutation. Children with PCD from an Amish family from Wisconsin had biallelic DNAH5 mutations, c.4348C>T (p.Q1450X) and c.10815delT (p.P3606HfsX23), and mutations in other genes associated with PCD were also identified in this community. CONCLUSION: The Amish and Mennonite subjects from geographically dispersed and socially isolated communities had the same founder DNAH5 mutation, owing to the common heritage of these populations. However, disease-causing mutations in other PCD-associated genes were also found in affected individuals in these communities, illustrating the genetic heterogeneity in this consanguineous population.

IL-13-induced airway mucus production is attenuated by MAPK13 inhibition.

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.

Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia.

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

High throughput screening for small molecule enhancers of the interferon signaling pathway to drive next-generation antiviral drug discovery.

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z'>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

Host epithelial-viral interactions as cause and cure for asthma.

Research on the pathogenesis of asthma has concentrated on initial stimuli, genetic susceptibilities, adaptive immune responses, and end-organ alterations (particularly in airway mucous cells and smooth muscle) as critical steps leading to disease. Recent evidence indicates that the innate immune cell response to respiratory viruses also contributes to the development of inflammatory airway disease. We further develop this concept by raising the issue that the interaction between host airway epithelial cells and respiratory viruses is another aspect of innate immunity that is also a critical determinant of asthma. We also introduce a rationale for how antiviral performance at the epithelial cell level might be improved to prevent acute infectious illness and chronic inflammatory disease caused by respiratory viruses.

A New Electrospray Aerosol Generator with High Particle Transmission Efficiency.

A new single-capillary electrospray (ES) aerosol generator has been developed for monodisperse particle production with maximal transmission efficiency. The new generator consists of both a spray chamber in a point-to-orifice-plate configuration and a charge reduction chamber that can hold up to 4 Nuclespot ionizers (Model P-2042, NRD Inc.). The 2 chambers are partitioned by an orifice plate. To optimize the particle transmission efficiency of the prototype, a systematic study was performed on the generator by varying the system setup and operation. Two key dimensions of the generator setup, the orifice diameter and the distance from the capillary tip to the orifice plate, were varied. Fluorescence analysis was applied to characterize the loss of ES-generated particles at different locations of the prototype. It was found that particle loss in the generator could be reduced by either increasing the orifice diameter or decreasing the distance between the capillary tip and the orifice plate. Increasing either the total radioactivity of the ionizers or the flowrate of the particle carrier gas also further decreased the particle loss in the system. The maximum particle transmission efficiency of 88.0% was obtained with the spray chamber fully opened to the charge reduction chamber, the capillary tip at the same level as the orifice plate, and 4 bipolar ionizers installed.

Immune pathways for translating viral infection into chronic airway disease.

To better understand the immune basis for chronic inflammatory lung disease, we analyzed a mouse model of lung disease that develops after respiratory viral infection. The disease that develops in this model is similar to asthma and chronic obstructive pulmonary disease (COPD) in humans and is manifested after the inciting virus has been cleared to trace levels. The model thereby mimics the relationship of paramyxoviral infection to the development of childhood asthma in humans. When the acute lung disease appears in this model (at 3 weeks after viral inoculation), it depends on an immune axis that is initiated by expression and activation of the high-affinity IgE receptor (FcvarepsilonRI) on conventional lung dendritic cells (cDCs) to recruit interleukin (IL)-13-producing CD4(+) T cells to the lower airways. However, when the chronic lung disease develops fully (at 7 weeks after inoculation), it is driven instead by an innate immune axis that relies on invariant natural killer T (iNKT) cells that are programmed to activate macrophages to produce IL-13. The interaction between iNKT cells and macrophages depends on contact between the semi-invariant Valpha14Jalpha18-TCR on lung iNKT cells and the oligomorphic MHC-like protein CD1d on macrophages as well as NKT cell production of IL-13 that binds to the IL-13 receptor (IL-13R) on the macrophage. This innate immune axis is also activated in the lungs of humans with severe asthma or COPD based on detection of increased numbers of iNKT cells and alternatively activated IL-13-producing macrophages in the lung. Together, the findings identify an adaptive immune response that mediates acute disease and an innate immune response that drives chronic inflammatory lung disease in experimental and clinical settings.

The role of CLCA proteins in inflammatory airway disease.

Inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) exhibit stereotyped traits that are variably expressed in each person. In experimental mouse models of chronic lung disease, these individual disease traits can be genetically segregated and thereby linked to distinct determinants. Functional genomic analysis indicates that at least one of these traits, mucous cell metaplasia, depends on members of the calcium-activated chloride channel (CLCA) gene family. Here we review advances in the biochemistry of the CLCA family and the evidence of a role for CLCA family members in the development of mucous cell metaplasia and possibly airway hyperreactivity in experimental models and in humans. On the basis of this information, we develop the model that CLCA proteins are not integral membrane proteins with ion channel function but instead are secreted signaling molecules that specifically regulate airway target cells in healthy and disease conditions.

Effects of time, albuterol, and budesonide on the shape of the flow-volume loop in children with asthma.

BACKGROUND: Assessment of asthma through spirometric analysis in children is challenging because of often normal FEV(1) values. OBJECTIVE: We used Mead's slope ratio (SR; (dV /dV)/(V /V)) to analyze the shape of the flow-volume loop. METHODS: We analyzed the effects of time, albuterol, and budesonide on FEV(1), FEV(1)/forced vital capacity (FVC) ratio, forced expiratory flow from 25% to 75% of expired volume, and Mead's SR both early (between 75% and 50% of FVC, SR61) and late (between 75% and 50% of FVC, SR35) in exhalation in the Childhood Asthma Management Program cohort at baseline, 4 months, and the end of the study in participants who received either inhaled placebo or budesonide twice daily. RESULTS: In the placebo group both SR61 and SR35 improved over time. Bronchodilator consistently improved both SR61 and SR35, without change in degree of improvement over time. Similarly, in the budesonide group time and bronchodilator each independently improved both SR61 and SR35. At 4 months and the end of the study, patients receiving budesonide had significant improvements in SR61 relative to patients receiving placebo, which was independent of bronchodilator effect. Budesonide and placebo were not different with respect to prebronchodilator or postbronchodilator SR35. CONCLUSION: Budesonide-treated patients have less concave flow-volume loops when compared with placebo-treated patients. Time and bronchodilator also make the flow-volume loop less concave. Furthermore, it appears that there are discrete bronchodilator- and corticosteroid-responsive components of airflow obstruction in pediatric asthma.