Tryptase Inhibitors Research Articles

Airway tryptase levels inform the lack of clinical efficacy of the tryptase inhibitor MTPS9579A in asthma.

Tryptase, a mast cell protease, has been identified as a potential therapeutic target in managing patients with refractory asthma. We assessed the efficacy, safety, pharmacokinetics, and pharmacodynamics of MTPS9579A, an anti-tryptase antibody, in a phase 2a randomized trial for patients with uncontrolled asthma and a phase 1c trial to understand activity within the lower respiratory tract. Phase 2a patients (n = 134) received 1800 mg MTPS9579A or placebo intravenously every 4 weeks for 48 weeks. The primary endpoint was time to the first composite exacerbation event. Phase 1c patients (n = 27) received one intravenous dose of 300 or 1800 mg MTPS9579A or placebo. Both trials measured MTPS9579A concentrations and effects on tryptase in serum and nasal lining fluid; phase 1c also analyzed bronchial lining fluid. MTPS9579A did not meet the primary endpoint (hazard ratio = 0.90; 95% CI: 0.55-1.47; p = 0.6835); exacerbation rates in the placebo group were low. Serum and nasal MTPS9579A pharmacokinetics and tryptase levels were consistent with data from healthy volunteers. However, in phase 1c patients, compared to nasal levels, MTPS9579A bronchial concentrations were 6.8-fold lower, and bronchial active and total tryptase levels were higher (119-fold and 30-fold, respectively). Pharmacokinetic/pharmacodynamic modeling predicted intravenous doses of 3800 mg every 4 weeks would be necessary to achieve 95% active tryptase inhibition from baseline. The MTPS9579A dose tested in the phase 2a study was insufficient to inhibit tryptase in bronchial lining fluid, likely contributing to the observed lack of efficacy.

Identification of Potential Tryptase Inhibitors from FDA-Approved Drugs Using Machine Learning, Molecular Docking, and Experimental Validation.

This study explores the innovative use of machine learning (ML) to identify novel tryptase inhibitors from a library of FDA-approved drugs, with subsequent confirmation via molecular docking and experimental validation. Tryptase, a significant mediator in inflammatory and allergic responses, presents a therapeutic target for various inflammatory diseases. However, the development of effective tryptase inhibitors has been challenging due to the enzyme's complex activation and regulation mechanisms. Utilizing a machine learning model, we screened an extensive FDA-approved drug library to identify potential tryptase inhibitors. The predicted compounds were then subjected to molecular docking to assess their binding affinity and conformation within the tryptase active site. Experimental validation was performed using RBL-2H3 cells, a rat basophilic leukemia cell line, where the efficacy of these compounds was evaluated based on their ability to inhibit tryptase activity and suppress β-hexosaminidase activity and histamine release. Our results demonstrated that several FDA-approved drugs, including landiolol, laninamivir, and cidofovir, significantly inhibited tryptase activity. Their efficacy was comparable to that of the FDA-approved mast cell stabilizer nedocromil and the investigational agent APC-366. These findings not only underscore the potential of ML in accelerating drug repurposing but also highlight the feasibility of this approach in identifying effective tryptase inhibitors. This research contributes to the field of drug discovery, offering a novel pathway to expedite the development of therapeutics for tryptase-related pathologies.

In-silico studies and docking of n-substituted isoindoline-1, 3-dioine analogues as anti-proliferative agents

Recently,isoindoline-1,3-dione compounds based folpet, phosmet, captonand thalidomide were developed because they have a comparable degree of anti-proliferative efficacy owing to their diverse mechanisms such as HDAC inhibitors, tryptase inhibitors, inhibits the mode of tnf-α, and angiogenesis inhibitors. It was investigated how the phthalimide pharmacophore interacted with molecules such tnf-α, HDAC, VEGF, EGF, and Tyrosine Kinase Angiogenesis. In order to assess the inhibitory activity against enzyme assay, a series of phthalimidepharmacophores with various substituent’s (Schiff's base) at the N-phenyl ring were submitted to protein-ligand docking investigations using the lib-dock method in the current work. All of the compounds' chemical structures were designed using Cambridge software, ChemBioOffice Ultra 12.0, and their molecular characteristics were determined using the online molecular modelling tool Molinspiration. Utilizing the Discovery Studio Client version 4.1, ADMETlab 2.0, and Lazar 1.4.2 softwares, ADME, Toxicity, and Molecular Docking investigations using the lib-dock method were carried out to evaluate the binding mode and interactions of synthetic hits at the binding site of receptors. Docking studies demonstrated that these sorts of ligands interacted mostly with TNF-α, HDAC, VEGF, EGF, Tyrosine kinase, and angiogenesis reports, among others, by forming hydrogen bonds and interacting hydrophobically with the domain. Our docking results indicate that compounds A1, A10, A11, A22, A26, and A28 demonstrated the greatest binding affinity with the corresponding proteins based on the predicted binding energy. These computer simulations have shown that phthalimide compounds with N-phenyl rings replaced can effectively suppress enzymatic assay.

Choroid plexus mast cells drive tumor-associated hydrocephalus

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH invivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

Mechanism of analgesic effect of fire needle on peripheral sensitization in rats with neuropathic pain induced by chemotherapy.

This study aims to explore the analgesic mechanism of fire needle on peripheral sensitization in rats with neuropathic pain(NP) induced by oxaliplatin, so as to investigate its mechanism in improving peri-pheral sensitization. Male SD rats aged 8 weeks were randomly divided into 4 groups：normal group(n=6), model group(n=6), fire needle group(n=6), and medication group(n=6). NP rat model was established by intraperitoneal injection of oxaliplatin(4 mg/kg) on days 1, 2, 8, 9, 15, 16, 22, and 23. For rats in the fire needle group, fire needle treatment was performed at the "Jiaji"(EX-B2) acupoints of the L4-L6 segments on days 24, 26, and 28, ie. 1 day, 3 and 5 days after modeling. The medication group received intraperitoneal injection of pregabalin(100 mg/kg). Mechanical pain thresholds of the rats were measured before modeling, after modeling and intervention. Serum contents of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and chemokine ligand 12(CXCL12) were detected by ELISA. Skin histopathology changes in the acupoint area were observed using HE staining. The number of mast cells in the skin of the acupoints was observed using toluidine blue staining. Immunohistochemical staining was performed to detect the postive expressions of transient receptor potential vanilloid 1(TRPV1), protease-activated receptor 2(PAR2) and tryptase(TPS) in the skin of the acupoint area. Western blot was used to detect the protein expressions of TRPV1 and PAR2 in the dorsal root ganglia(DRG). Compared with the normal group, the model group had decreased paw withdrawal threshold(PWT) after modeling(P<0.05), increased serum contents of IL-6, TNF-α, and CXCL12(P<0.05), increased number of mast cells in the acupoint area(P<0.05), and increased positive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05). Compared with the model group, the fire needle group and medication group had increased PWT after intervention(P<0.05), decreased serum contents of IL-6, TNF-α, and CXCL12, and postive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05)；while the medication group had decreased protein expressions of TRPV1 and PAR2 in DRG(P<0.05). HE staining showed thickened epidermis, disordered cellular arrangement, significant intercellular edema, and inflammatory cell infiltration in the model group. In the medication and fire needle groups, the epidermis was thinner, cellular arrangement was clearer, and the extent of tissue edema and inflammatory cell infiltration was reduced compared to the model group. Fire needle can improve mechanical pain threshold and reduce the contents of peripheral inflammatory factors in rats with oxaliplatin-induced NP. This effect may be related to the inhibition of mast cell activation and the inhibition of TPS, TRPV1 and PAR2 protein expressions, in the local areas of acupoints.

A mechanistic PK/PD model to enable dose selection of the potent anti-tryptase antibody (MTPS9579A) in patients with moderate-to-severe asthma.

Tryptase, a protease implicated in asthma pathology, is secreted from mast cells upon activation during an inflammatory allergic response. MTPS9579A is a novel monoclonal antibody that inhibits tryptase activity by irreversibly dissociating the active tetramer into inactive monomers. This study assessed the relationship between MTPS9579A concentrations in healthy subjects and tryptase levels in serum and nasal mucosal lining fluid from healthy subjects and patients with moderate-to-severe asthma. These data were used to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model that quantitatively inter-relates MTPS9579A exposure and inhibition of active tryptase in the airway of patients with asthma. From initial estimates of airway tryptase levels and drug partitioning, the PK/PD model predicted almost complete neutralization of active tryptase in the airway of patients with asthma with MTPS9579A doses of 900 mg and greater, administered intravenously (i.v.) once every 4 weeks (q4w). Suppression of active tryptase during an asthma exacerbation event was also evaluated using the model by simulating the administration of MTPS9579A during a 100-fold increase in tryptase secretion in the local tissue. The PK/PD model predicted that 1800 mg MTPS9579A i.v. q4w results in 95.7% suppression of active tryptase at the steady-state trough concentration. Understanding how the exposure-response relationship of MTPS9579A in healthy subjects translates to patients with asthma is critical for future clinical studies assessing tryptase inhibition in the airway of patients with moderate-to-severe asthma.

Mast cell tryptase-PAR2 pathway in proliferation of prostatic stromal cells reacted with Trichomonas vaginalis.

We investigated whether tryptase released from mast cells activated by prostate stromal cells (PSC) reacted with Trichomonas vaginalis (Tv) promoted the proliferation of PSC through protease- activated receptor 2 (PAR2). Conditioned medium of PSC was prepared by stimulating them with Tv (Trichomonad-conditioned medium (TCM)), and mast cell-conditioned medium were prepared by incubating them with TCM (mast cell-TCM (M-TCM)). Mast cells incubated with TCM migrated more efficiently and produced more β-hexosaminidase and tryptase. M-TCM containing tryptase increased the proliferation of PSC, while inhibition of tryptase decreased proliferation. Expression of signalling molecules such as PAR2, p-ERK, COX-2, 15d-PGJ2 and PPARγ, known to be involved in the tryptase-PAR2 pathway, increased in response to M-TCM, and blocking any of these signals decreased proliferation, indicating that tryptase-PAR2 signalling is involved in the proliferation of PSC. Inhibition of tryptase and PAR2 led to reduced expression of PAR2, p-ERK, COX-2, 15d-PGJ2 and PPARγ, while inhibition of ERK or COX-2 reduced the expression of COX-2, 15d-PGJ2 and PPARγ indicating that the tryptase-PAR2 pathway proceeds in the order p-ERK, COX-2, 15d-PGJ2 , and PPARγ. These results show that interaction between PSC stimulated with Tv and mast cells causes proliferation of PSC through the tryptase-PAR2 pathway.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Mast Cells Positive for c-Kit Receptor and Tryptase Correlate with Angiogenesis in Cancerous and Adjacent Normal Pancreatic Tissue.

Background: Mast cells (MCs) contain proangiogenic factors, in particular tryptase, associated with increased angiogenesis in several tumours. With special reference to pancreatic cancer, few data have been published on the role of MCs in angiogenesis in both pancreatic ductal adenocarcinoma tissue (PDAT) and adjacent normal tissue (ANT). In this study, density of mast cells positive for c-Kit receptor (MCDP-c-KitR), density of mast cells positive for tryptase (MCDPT), area of mast cells positive for tryptase (MCAPT), and angiogenesis in terms of microvascular density (MVD) and endothelial area (EA) were evaluated in a total of 45 PDAT patients with stage T2–3N0–1M0. Results: For each analysed tissue parameter, the mean ± standard deviation was evaluated in both PDAT and ANT and differences were evaluated by Student’s t-test (p ranged from 0.001 to 0.005). Each analysed tissue parameter was then correlated to each other one by Pearson t-test analysis (p ranged from 0.01 to 0.03). No other correlation among MCDP-c-KitR, MCDPT, MCAPT, MVD, EA and the main clinical–pathological characteristics was found. Conclusions: Our results suggest that tissue parameters increased from ANT to PDAT and that mast cells are strongly associated with angiogenesis in PDAT. On this basis, the inhibition of MCs through tyrosine kinase inhibitors, such as masitinib, or inhibition of tryptase by gabexate mesylate may become potential novel antiangiogenetic approaches in pancreatic cancer therapy.

Stabilizing mast cells improves acute lung injury after orthotopic liver transplantation via promotion of apoptosis in polymorphonuclear neutrophils.

Postoperative pulmonary complications including acute lung injury (ALI) and acute respiratory distress syndrome have contributed to mortality and morbidity of orthotopic liver transplantation (OLT) with unclear mechanisms. Mast cells (MCs) and polymorphonuclear neutrophils (PMNs) are the main inflammatory cells and participants in the process of ALI. The present study was designed to investigate the role of MCs and PMNs and their potential relation to ALI following OLT. Rat orthotopic autologous liver transplantation (OALT) model was designed to determine lung injury at different time points after liver reperfusion. We also evaluated the function of MCs and the effect of tumor necrosis factor-α (TNF-α) and tryptase on ALI and PMN apoptosis in rats subjected to OALT. Histological scores and inflammatory factor levels as well as PMN apoptosis were measured. Rats suffered from ALI after OALT, which was demonstrated by a collapse of the pulmonary architecture, pulmonary edema, and infiltration of inflammatory cells in alveolar and interstitial spaces, as well as increased levels of proinflammatory cytokines. ALI maximized at 8 h after OALT. However, PMN apoptosis lagged behind the pulmonary injury and maximized at 16 h after OALT, when the acute inflammation resolution initiated. MC stabilization, and tryptase and TNF-α inhibitors could significantly decrease the lung pathophysiologic scores accompanied by an increase in PMN apoptosis. ALI after OALT was associated with MC activation and PMN apoptosis. ALI progression might be affected by delayed PMN apoptosis, which was related to MC activation. Induction of PMN apoptosis might alleviate ALI after OALT.

A natural product biflavonoid scaffold with anti-tryptase activity.

Tryptase is a serine protease that is released from mast cells during allergic responses. Tryptase inhibitors are being explored as treatments for allergic inflammation in the skin and respiratory system, most notably asthma. Here we report direct tryptase inhibition by natural product compounds. Candidate inhibitors were identified by computational screening of a large (98,000 compounds) virtual library of natural product compounds for tryptase enzymatic site binding. Biochemical assays were used to validate the predicted anti-tryptase activity in vitro, revealing a high (four out of six) success rate for predicting binding using the computational docking model. We further assess tryptase inhibition by a biflavonoid scaffold, whose structure-activity relationship is partially defined by assessing the potency of structurally similar analogs.

A role for mast cells in geographic atrophy.

Mast cells (MCs) are the initial responders of innate immunity and their degranulation contribute to various etiologies. While the abundance of MCs in the choroid implies their fundamental importance in the eye, little is known about the significance of MCs and their degranulation in choroid. The cause of geographic atrophy (GA), a progressive dry form of age-related macular degeneration is elusive and there is currently no therapy for this blinding disorder. Here we demonstrate in both human GA and a rat model for GA, that MC degranulation and MC-derived tryptase are central to disease progression. Retinal pigment epithelium degeneration followed by retinal and choroidal thinning, characteristic phenotypes of GA, were driven by continuous choroidal MC stimulation and activation in a slow release fashion in the rat. Genetic manipulation of MCs, pharmacological intervention targeting MC degranulation with ketotifen fumarate or inhibition of MC-derived tryptase with APC 366 prevented all of GA-like phenotypes following MC degranulation in the rat model. Our results demonstrate the fundamental role of choroidal MC involvement in GA disease etiology, and will provide new opportunities for understanding GA pathology and identifying novel therapies targeting MCs.

Mast Cell Tryptase Promotes Inflammatory Bowel Disease-Induced Intestinal Fibrosis.

Intestinal fibrosis is the final pathological outcome of chronic intestinal inflammation without specific therapeutic drugs, which leads to ileus and surgical intervention. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM). The role of mast cells (MCs), which are members of the sentinel immune cell population, is unknown in intestinal fibrosis. In this study, we analyzed changes in MCs, tryptase proteins, and ECM components in human fibrotic and control patient intestines. We constructed dextran sodium sulfate-induced intestinal fibrosis models using wild-type mice, MC-reconstituted mice, and MC-deficient mice to explore the role of MCs and tryptase in intestinal fibrosis. The roles and mechanisms of MCs and tryptase on fibroblasts were evaluated using human MCs (HMC-1 and LAD-2), commercial tryptase proteins, human colon fibroblasts (CCD-18Co fibroblasts), the tryptase inhibitor APC366, and the protease-activated receptor-2 (PAR-2) antagonist ENMD-1068. Regardless of whether the colon was a human colon or a mouse colon, the fibrotic intestinal tissue had increased MC infiltration and a higher expression of ECM proteins or genes than that of the control group. The dextran sodium sulfate-induced intestinal fibrosis in MC-deficient mice was alleviated compared with that in wild-type mice. After MC reconstruction in MC-deficient mice, the alleviating effect disappeared. Tryptase, as a content stored in MC granules, was released into fibrotic intestinal tissues in the form of degranulation, resulting in an increased expression of tryptase. Compared with the control group, the tryptase inhibition group (the APC366 group) had reduced intestinal fibrosis. The CCD-18Co fibroblasts, when cocultured with MCs or treated with tryptase proteins, were activated to differentiate into myofibroblasts and secrete more ECM proteins (such as collagen and fibronectin). The underlying mechanism of fibroblast activation by tryptase was the activation of the PAR-2/Akt/mTOR pathway. We found that MC tryptase promotes inflammatory bowel disease-induced intestinal fibrosis. The underlying mechanism is that tryptase promotes the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/ mTOR pathway of fibroblasts.

Mast cells positive to c-kit receptor and to tryptase in normal to cancer pancreatic tissue and the correlation with angiogenesis.

e16502 Background: In the last years published studies demonstrated that mast cells (MCs) contain pro-angiogenic factors associated with tumoral angiogenesis. Tryptase is the most abundant and potent pro-angiogenic substance contained in MCs secretory granules and it can be released in tumour microenvironment. Up to now few data have been published on the role of MCs in angiogenesis in both pancreatic ductal adenocarcinoma cancer tissue (PDACT) and adjacent normal tissue (ANT). Methods: In this study MCs density positive to C-Kit receptor (MCDP-C-KitR), MCs density positive to tryptase (MCDPT), MCs area positive to tryptase (MCAPT), angiogenesis in terms of microvascular density (MVD) and endothelial area (EA) were evaluated by immunohistochemistry and image analysis in both PDACT and ANT. All tissue samples were obtained from 45 patients with stage T2-3N0-1M0 (by AJCC for Pancreas Cancer Staging 7th Edition) who had undergone surgery. For each analyzed tissue parameter mean ± standard deviation was evaluated in both PDACT and ANT and differences were evaluated by Student t-test. Each analysed tissue parameter was then correlated each other by Pearson t-test analysis. Results: The mean value ± standard deviation (SD) regarding MCDP-C-KitR, MCDPT, MCAPT, MVD and EA in TT was 14.69±4.57, 13.31± 4,23, 171.41±62,39 µ2, 29.11±7.93, 201.82±70.05 µ2 respectively and the mean value ± SD in ANT was 5.61 ± 2.39, 5.13 ± 2.03, 54.43 ± 16.73 µ2, 11.45 ± 4.96, 67.60 ± 21.96 µ2, respectively. Differences in terms of mean value ± SD between PDACT and ANT were significant for each analyzed tissue biomarker (p ranged from 0,001 to 0,004 by t-test analysis; Table). Data demonstrated that MCDP-C-KitR MCDPT, MCAPT, MVD and EA significantly increased from ANT to PCT. In PCT it was showed a correlation between MCDP-C-KitR and MCDPT (r = 0.87, p = 0.01), MCDP-C-KitR and MVD (r = 0.74, p = 0.02), MCDP-C-KitR and MCAPT (r = 0.81, p = 0.01), MCDPT and MVD (r = 0.72, p = 0.02), MCD-C-KitR and EA (r = 0.73, p = 0.02), MCDPT and MCAPT (r = 0.85, p = 0.01), MCAPT and MVD (r = 0.76, p = 0.02), MCAPT and EA (r = 0.66, p = 0.03), MCDPT and EA (r = 0.69, p = 0.03), MVD and EA (r = 0.82, p = 0.01). Conclusions: Our data suggest that assessed tissue parameters increased from ANT to PDACT and that MCs are associated with angiogenesis in PDACT. On this basis inhibition of MCs by tyrosine kinase inhibitors such as masitinib or inhibition of tryptase by gabexate mesylate may be a novel antiangiogenetic approach in pancreatic cancer therapy.

Inhibition of mast cell tryptase attenuates neuroinflammation via PAR-2/p38/NF\u03baB pathway following asphyxial cardiac arrest in rats

BackgroundCardiac arrest survivors suffer from neurological dysfunction including cognitive impairment. Cerebral mast cells, the key regulators of neuroinflammation contribute to neuroinflammation-associated cognitive dysfunction. Mast cell tryptase was demonstrated to have a proinflammatory effect on microglia via the activation of microglial protease-activated receptor-2 (PAR-2). This study investigated the potential anti-neuroinflammatory effect of mast cell tryptase inhibition and the underlying mechanism of PAR-2/p-p38/NFκB signaling following asphyxia-induced cardiac arrest in rats.MethodsAdult male Sprague-Dawley rats resuscitated from 10 min of asphyxia-induced cardiac arrest were randomized to four separate experiments including time-course, short-term outcomes, long-term outcomes and mechanism studies. The effect of mast cell tryptase inhibition on asphyxial cardiac arrest outcomes was examined after intranasal administration of selective mast cell tryptase inhibitor (APC366; 50 μg/rat or 150 μg/rat). AC55541 (selective PAR-2 activator; 30 μg/rat) and SB203580 (selective p38 inhibitor; 300 μg/rat) were used for intervention. Short-term neurocognitive functions were evaluated using the neurological deficit score, number of seizures, adhesive tape removal test, and T-maze test, while long-term cognitive functions were evaluated using the Morris water maze test. Hippocampal neuronal degeneration was evaluated by Fluoro-Jade C staining.ResultsMast cell tryptase and PAR-2 were dramatically increased in the brain following asphyxia-induced cardiac arrest. The inhibition of mast cell tryptase by APC366 improved both short- and long-term neurological outcomes in resuscitated rats. Such behavioral benefits were associated with reduced expressions of PAR-2, p-p38, NFκB, TNF-α, and IL-6 in the brain as well as less hippocampal neuronal degeneration. The anti-neuroinflammatory effect of APC366 was abolished by AC55541, which when used alone, indeed further exacerbated neuroinflammation, hippocampal neuronal degeneration, and neurologic deficits following cardiac arrest. The deleterious effects aggregated by AC55541 were minimized by p38 inhibitor.ConclusionsThe inhibition of mast cell tryptase attenuated neuroinflammation, led to less hippocampal neuronal death and improved neurological deficits following cardiac arrest. This effect was at least partly mediated via inhibiting the PAR-2/p-p38/NFκB signaling pathway. Thus, mast cell tryptase might be a novel therapeutic target in the management of neurological impairment following cardiac arrest.

Novel, Self-Assembling Dimeric Inhibitors of Human β Tryptase.

β-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.

Tıbbi Sülük Terapisi (Hirudoterapi)

Sülükler halkalı omurgasızlar sınıfındadır, kan ile beslenirler ve antik dönemden beri çeşitli hastalıkları tedavi etmek için flebotomide kullanılırlar. Hirudoterapi, tedavi için tıbbi sülüklerin uygulanmasıdır. Sülük tedavisinin terapötik kullanımı köklü bir yöntemdir ve eski Mısır'a, medeniyetin başlangıcına dayanır. 20. yüzyılın sonunda yeni terapi kavramları sülük tedavisinin yerini almıştır. Mikrovasküler cerrahi tekniklerle birlikte son yıllarda tıbbi sülüklerin kullanımında bir “yeniden doğuş” olmuştur. Hirudo medicinalis, sülükler arasında en geniş terapotik kullanıma sahiptir ancak dünya çapında birçok farklı tür test edilmiş ve incelenmiştir. Sülük tedavisi genellikle ağrısız olan bir ilk ısırığı, ardından 5 ila 15 ml kan emmeyi içerir. Başlıca terapötik faydaları sadece ısırma sırasında emilen kandan değil, aynı zamanda çeşitli biyoaktif maddelerden de kaynaklanmaktadır. Sülük tükürüğü; eglin, bdellin, hirudin, hyaluronidaz, calin, destabilas, apiraz, dekorin, hirustatin, triptaz inhibitörleri ve histamin benzeri maddeler, kompleman inhibitörleri, karboksipeptidaz A-inhibitörleri ve asetilkolin gibi çeşitli biyoaktif bileşikler içerir. Bu bileşikler; koagülasyonun inhibisyonundan, analjezik etkiden, kas gevşetici etkiden ve bağışıklık sistemi üzerindeki düzenlemeden sorumludur. Hirudoterapi; kardiyoloji, ortopedi, jinekoloji, oftalmoloji, plastik ve rekonstrüktif cerrahi gibi alanlarda geniş bir terapötik uygulama yelpazesine sahiptir. Bu derleme sülükler hakkında; tarihçesi, genel özellikleri, etki mekanizmaları ve tıbbi bir cihaz olarak uygulanmasını içeren genel bir bakış sunar.

Dengue virus-elicited tryptase induces endothelial permeability and shock.

Dengue virus (DENV) infection causes a characteristic pathology in humans involving dysregulation of the vascular system. In some patients with dengue hemorrhagic fever (DHF), vascular pathology can become severe, resulting in extensive microvascular permeability and plasma leakage into tissues and organs. Mast cells (MCs), which line blood vessels and regulate vascular function, are able to detect DENV in vivo and promote vascular leakage. Here, we identified that a MC-derived protease, tryptase, is consequential for promoting vascular permeability during DENV infection, through inducing breakdown of endothelial cell tight junctions. Injected tryptase alone was sufficient to induce plasma loss from the circulation and hypovolemic shock in animals. A potent tryptase inhibitor, nafamostat mesylate, blocked DENV-induced vascular leakage in vivo. Importantly, in two independent human dengue cohorts, tryptase levels correlated with the grade of DHF severity. This study defines an immune mechanism by which DENV can induce vascular pathology and shock.

Dengue vascular leak syndrome: insights into potentially new treatment modalities.

Dengue viruses (DENV) are the most common cause of mosquito-borne viral illness in the world, affecting approximately 400 million people annually. Symptomatic illness ranges from a mild, self-limiting febrile illness to one manifested by plasma leakage that can lead to vascular collapse and death. In this issue of the JCI, Rathore et al. report that DENV can cause mast cell degranulation independently of mast cell infection, resulting in the release of the vasoactive mediators chymase and tryptase. The authors showed that recombinant chymase and tryptase increased endothelial permeability in a dose-dependent manner in human microvascular endothelial cells. They went on to evaluate the tryptase inhibitor nafamostat mesylate in a mouse model for severe DENV viremia. Strikingly, the potential therapeutic prevented and reversed the tryptase-induced vascular permeability. As there are currently no licensed drugs for the treatment of dengue, these findings present a possible treatment modality for severe disease.

Nafamostat Mesilate Is an Extremely Potent Inhibitor of Human Tryptase

Previously, nafamostat mesilate was found to be a potent inhibitor of human tryptase. In present study, we performed a kinetic study to determine its Ki value for tryptase and compared it with that of gabexate mesilate. Nafamostat mesilate inhibited human tryptase in a competitive manner. The apparent Ki value was estimated to be 95.3 pM, which was 1,000 times lower than that of gabexate mesilate (95.1 nM). These results strongly indicated that nafamostat mesilate is an extremely potent inhibitor of tryptase and suggested that some of its beneficial effects in the treatment of clinical status may be due to tryptase inhibition.