Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Efficacy and Pharmacodynamic Modeling of the BTK Inhibitor Evobrutinib in Autoimmune Disease Models.

Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.

Characterization of Novel PI3Kδ Inhibitors as Potential Therapeutics for SLE and Lupus Nephritis in Pre-Clinical Studies.

SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.

ASK1 promotes the contact hypersensitivity response through IL-17 production.

Contact hypersensitivity (CHS) is a form of delayed-type hypersensitivity triggered by the response to reactive haptens (sensitization) and subsequent challenge (elicitation). Here, we show that ASK1 promotes CHS and that suppression of ASK1 during the elicitation phase is sufficient to attenuate CHS. ASK1 knockout (KO) mice exhibited impaired 2,4-dinitrofluorobenzene (DNFB)-induced CHS. The suppression of ASK1 activity during the elicitation phase through a chemical genetic approach or a specific inhibitory compound significantly reduced the CHS response to a level similar to that observed in ASK1 KO mice. The reduced response was concomitant with the strong inhibition of production of IL-17, a cytokine that plays an important role in CHS and other inflammatory diseases, from sensitized lymph node cells. These results suggest that ASK1 is relevant to the overall CHS response during the elicitation phase and that ASK1 may be a promising therapeutic target for allergic contact dermatitis and other IL-17-related inflammatory diseases.

Modifications to the airway management trainer intubation head for training in difficult airway recognition through a comparative study with the Airsim® intubation head.

INTRODUCTION: The goal of this study was to explain some modifications to the Airway Management Trainer intubation head (case) that transform it into a difficult airway intubation head and to compare it with the Airsim intubation head (control). METHODS: Eight anesthesiologists (5 experienced and 3 residents) conducted 80 endotracheal intubations each, 10 intubations with each of the 2 airway training heads under 4 distinct clinical scenarios: normal airway, macroglossia (placing a squash ball under the tongue), cervical spine rigidity (with tape placed on the cervical spine to inhibit its extension), or both macroglossia and cervical spine rigidity. We used a Macintosh laryngoscope with a #3 blade and a 7-mm (internal diameter) endotracheal tube. The outcome variables included intubation time, ease of intubation (Likert scale), glottis visualization (Cormack-Lehane scale), and the need for maneuvers for intubation. The statistical tests used were the t test and the χ test. A P < 0.05 was considered statistically significant. RESULTS: The intubation was more difficult in the case, but this difference was only statistically significant in normal airway and combining macroglossia and spinal rigidity, and the time of intubation was longer in the case than in the control. In the evaluation of the glottic view grade when we combined macroglossia and cervical rigidity, the case presented significantly more cases of Cormack-Lehane grade 3. CONCLUSIONS: The present modifications proposed for the Airway Management Trainer are easy to complete to render it similar to the Airsim for training in difficult airway management.

Functional whole-genome analysis identifies Polo-like kinase 2 and poliovirus receptor as essential for neuronal differentiation upstream of the negative regulator alphaB-crystallin.

This study aimed at identifying transcriptional changes associated to neuronal differentiation induced by six distinct stimuli using whole-genome microarray hybridization analysis. Bioinformatics analyses revealed the clustering of these six stimuli into two categories, suggesting separate gene/pathway dependence. Treatment with specific inhibitors demonstrated the requirement of both Janus kinase and microtubule-associated protein kinase activation to trigger differentiation with nerve growth factor (NGF) and dibutyryl cAMP. Conversely, activation of protein kinase A, phosphatidylinositol-3-kinase alpha, and mammalian target of rapamycin, although required for dibutyryl cAMP-induced differentiation, exerted a negative feedback on NGF-induced differentiation. We identified Polo-like kinase 2 (Plk2) and poliovirus receptor (PVR) as indispensable for NGF-driven neuronal differentiation and alphaB-crystallin (Cryab) as an inhibitor of this process. Silencing of Plk2 or PVR blocked NGF-triggered differentiation and Cryab down-regulation, while silencing of Cryab enhanced NGF-induced differentiation. Our results position both Plk2 and PVR upstream of the negative regulator Cryab in the pathway(s) leading to neuronal differentiation triggered by NGF.

GLEPP1/protein-tyrosine phosphatase phi inhibitors block chemotaxis in vitro and in vivo and improve murine ulcerative colitis.

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase . Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.

BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity.

Bone morphogenetic proteins (BMPs) are potent regulators of several cellular events. We report that exposure of C2C12 cells to BMP2 leads to an increase in cell migration and a rapid rearrangement of the actin filaments into cortical protrusions. These effects required independent and parallel activation of the Cdc42 small GTPase and the alpha-isoform of the phosphoinositide 3-kinase (PI3Kalpha), because ectopic expression of a dominant-negative form of Cdc42 or distinct pharmacological PI3K inhibitors abrogated these responses. Furthermore, we demonstrate that BMP2 activates different group I and group II PAK isoforms as well as LIMK1 with similar kinetics to Cdc42 or PI3K activation. BMP2 activation of PAK and LIMK1, measured by either kinase activity or with antibodies raised against phosphorylated residues at their activation loops, were abolished by blocking PI3K-signaling pathways. Together, these findings suggest that Cdc42 and PI3K signals emanating from BMP receptors are involved in specific regulation of actin assembly and cell migration.

Chemokine receptor CCR2 undergoes transportin1-dependent nuclear translocation.

Chemokines (CCs) are small chemoattractant cytokines involved in a wide variety of biological and pathological processes. Released by cells in the milieu, and extracellular matrix and activating signalling cascades upon binding to specific G protein-coupled receptors (GPCRs), they trigger many cellular events. In various pathologies, CCs are directly responsible for excessive recruitment of leukocytes to inflammatory sites and recent studies using chemokine receptor (CCR) antagonists permitted these molecules to reach the market for medical use. While interaction of CCs with their receptors has been extensively documented, downstream GPCR signalling cascades triggered by CC are less well understood. Given the pivotal role of chemokine receptor 2 (CCR2) in monocyte recruitment, activation and differentiation and its implication in several autoimmune-inflammatory pathologies, we searched for potential new CCR2-interacting proteins by engineering a modified CCR2 that we used as bait. Herein, we show the direct interaction of CCR2 with transportin1 (TRN1), which we demonstrate is followed by CCR2 receptor internalization. Further characterization of this novel interaction revealed that TRN1-binding to CCR2 increased upon time in agonist treated cells and promotes its nuclear translocation in a TRN1-dependent manner. Finally, we provide evidence that following translocation, the receptor localizes at the outer edge of the nuclear envelope where it is finally released from TRN1.

Isoform-specific functions of phosphoinositide 3-kinases: p110 delta but not p110 gamma promotes optimal allergic responses in vivo.

The leukocyte-enriched p110gamma and p110delta isoforms of PI3K have been shown to control in vitro degranulation of mast cells induced by cross-linking of the high affinity receptor of IgE (FcepsilonRI). However, the relative contribution of these PI3K isoforms in IgE-dependent allergic responses in vivo is controversial. A side-by-side comparative analysis of the role of p110gamma and p110delta in mast cell function, using genetic approaches and newly developed isoform-selective pharmacologic inhibitors, confirms that both PI3K isoforms play an important role in FcepsilonRI-activated mast cell degranulation in vitro. In vivo, however, only p110delta was found to be required for optimal IgE/Ag-dependent hypersensitivity responses in mice. These observations identify p110delta as a key therapeutic target among PI3K isoforms for allergy- and mast cell-related diseases.

Inactivation of PI3Kgamma and PI3Kdelta distorts T-cell development and causes multiple organ inflammation.

Mice lacking both the p110gamma and p110delta isoforms display severe impairment of thymocyte development. Here, we show that this phenotype is recapitulated in p110gamma-/-/p110delta(D910A/D910A) (p110gamma(KO)delta(D910A)) mice where the p110delta isoform has been inactivated by a point mutation. Moreover, we have examined the pathological consequences of the p110gammadelta deficiency, which include profound T-cell lymphopenia, T-cell and eosinophil infiltration of mucosal organs, elevated IgE levels, and a skewing toward Th2 immune responses. Using small-molecule selective inhibitors, we demonstrated that in mature T cells, p110delta, but not p110gamma, controls Th1 and Th2 cytokine secretion. Thus, the pathology in the p110gammadelta-deficient mice is likely to be secondary to a developmental block in the thymus that leads to lymphopenia-associated inflammatory responses.

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.

The p110delta catalytic isoform of PI3K is a key player in NK-cell development and cytokine secretion.

The signal transduction pathways that lead activated natural killer (NK) cells to produce cytokines, releases cytotoxic granules, or do both, are not clearly dissected. For example, phosphoinositide 3-kinases (PI3Ks) are key players in the execution of both functions, but the relative contribution of each isoform is unknown. We show here that the catalytic isoform p110delta, not p110gamma, was required for interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion, whereas neither was necessary for cytotoxicity. Yet, when both p110delta and p110gamma isoforms were inactivated by a combination of genetic and biochemical approaches, cytotoxicity was decreased. NK-cell numbers were also affected by the lack of p110delta but not p110gamma and more severely so in mice lacking both subunits. These results provide genetic evidence that p110delta is the dominant PI3K isoform for cytokine secretion by NK cells and suggest that PI3Ks cooperate during NK-cell development and cytotoxicity.

Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses.

Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases, many of which dephosphorylate threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). The regulated expression and activity of DUSP family members in different cells and tissues controls MAPK intensity and duration to determine the type of physiological response. For immune cells, DUSPs regulate responses in both positive and negative ways, and DUSP-deficient mice have been used to identify individual DUSPs as key regulators of immune responses. From a drug discovery perspective, DUSP family members are promising drug targets for manipulating MAPK-dependent immune responses in a cell-type and disease-context-dependent manner, to either boost or subdue immune responses in cancers, infectious diseases or inflammatory disorders.

PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.

PI(3)Kgamma has an important context-dependent role in neutrophil chemokinesis.

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

[Perioperative care and treatment of chronic pain in Catalonia, Spain]. / Medicina perioperatoria y tratamiento del dolor crónico en Cataluña.

BACKGROUND AND OBJECTIVE: Perioperative care and chronic pain therapy are anesthesia practices that aim to improve patient safety and well-being. The purpose of this arm of the ANESCAT survey of anesthesia practices in Catalonia, Spain, in 2003 was to describe and quantify these 2 services. PATIENTS AND METHOD: Taking relevant responses to the ANESCAT 2003 questionnaires, we analyzed type of preanesthetic assessment, use of specialized techniques for postoperative analgesia, and provision of postoperative critical care. We also estimated type and volume of procedures performed by anesthesiologists to treat chronic pain. RESULTS: A preanesthetic assessment visit took place in an outpatient clinic setting in 47.4% of cases, on a hospital ward in 18.3%, and in the operating room waiting area in 34.3%. Specialized analgesic techniques were provided for 7.6% of postoperative patients, a rate that would put the estimated number of such procedures at 30,321 per year. Postoperative critical care was afforded to 7.7% of patients in the sample, a rate that extrapolates to anesthesia in 44,686 patients in Catalonia annually. Patients benefiting from specialized analgesic techniques and postoperative critical care were older, had worse physical status classifications, and were anesthetized for more aggressive procedures. Approximately 120,000 medical acts related to treatment for chronic pain were undertaken by anesthetists in Catalonia in 2003. CONCLUSIONS: The findings of ANESCAT 2003 reveal the considerable scope of Catalan anesthetists' practice in perioperative care management and chronic pain therapy, but more human resources and organizational attention are still needed for ideal workload management.

Medicina perioperatoria y tratamiento del dolor crónico en Cataluña / Perioperative care and treatment of chronic pain in Catalonia, Spain

Fundamento y objetivo: La medicina perioperatoria y el tratamiento del dolor crónico son actividades practicadas por anestesiólogos dirigidas a mejorar la seguridad y el bienestar de los pacientes. El objetivo de este capítulo fue cuantificar y describir estas actividades en Cataluña (España) en el año 2003. Pacientes y método: De los datos recogidos en la encuesta de actividad anestésica ANESCAT 2003 se cuantificaron el tipo de evaluación preanestésica, la administración de técnicas especiales de analgesia postoperatoria (TEAP) y los cuidados críticos postoperatorios (CCP). Asimismo, se estimaron el tipo y el volumen de actividades relacionadas con el tratamiento del dolor crónico realizado por anestesiólogos. Resultados: La visita preanestésica se realizó en una consulta ambulatoria en el 47,4% de los casos, en hospitalización en el 18,3% y en el antequirófano en el 34,3%. Un 7,6% de los pacientes intervenidos quirúrgicamente recibió TEAP, lo que permite estimar en 30.321 el número anual de pacientes. De todos los pacientes a los que se administró anestesia en Cataluña en 2003, un 7,7% recibió CCP, lo que significa una estimación anual de 44.686 pacientes. El perfil de los pacientes que se beneficiaron de las TEAP y los CCP fue: aquellos de más edad, con peor estado físico y anestesiados para procedimientos más agresivos. Unos 120.000 actos médicos relacionados con el tratamiento del dolor crónico fueron realizados por anestesiólogos en 2003 en Cataluña. Conclusiones: ANESCAT 2003 revela la importante dedicación de los anestesiólogos catalanes en la medicina perioperatoria y en el tratamiento del dolor crónico, pero todavía es necesario dedicar más recursos humanos y organizativos para que se alcance un nivel óptimo

Background and objective: Perioperative care and chronic pain therapy are anesthesia practices that aim to improve patient safety and well-being. The purpose of this arm of the ANESCAT survey of anesthesia practices in Catalonia, Spain, in 2003 was to describe and quantify these 2 services. Patients and method: Taking relevant responses to the ANESCAT 2003 questionnaires, we analyzed type of preanesthetic assessment, use of specialized techniques for postoperative analgesia, and provision of postoperative critical care. We also estimated type and volume of procedures performed by anesthesiologists to treat chronic pain. Results: A preanesthetic assessment visit took place in an outpatient clinic setting in 47.4% of cases, on a hospital ward in 18.3%, and in the operating room waiting area in 34.3%. Specialized analgesic techniques were provided for 7.6% of postoperative patients, a rate that would put the estimated number of such procedures at 30,321 per year. Postoperative critical care was afforded to 7.7% of patients in the sample, a rate that extrapolates to anesthesia in 44,686 patients in Catalonia annually. Patients benefiting from specialized analgesic techniques and postoperative critical care were older, had worse physical status classifications, and were anesthetized for more aggressive procedures. Approximately 120,000 medical acts related to treatment for chronic pain were undertaken by anesthetists in Catalonia in 2003. Conclusions: The findings of ANESCAT 2003 reveal the considerable scope of Catalan anesthetists' practice in perioperative care management and chronic pain therapy, but more human resources and organizational attention are still needed for ideal workload management