Cathepsin C exacerbates EAE by promoting the expansion of Tfh cells and the formation of TLSs in the CNS.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) mediated by CD4+ T helper (Th) cells, and characterized by immune cell infiltration, demyelination and neurodegeneration, with no definitive cure available. Thus, it is pivotal and imperative to acquire more profound comprehension of the underlying mechanisms implicated in MS. Dysregulated immune responses are widely believed to play a primary role in the pathogenesis of MS. Recently, a plethora of studies have demonstrated the involvement of T follicular helper (Tfh) cells and tertiary lymphoid-like structures (TLSs) in the pathogenesis and progression of MS. Cathepsin C (CatC) is a cysteine exopeptidase which is crucial for the activation of immune-cell-associated serine proteinases in many inflammatory diseases in peripheral system, such as rheumatoid arthritis and septicemia. We have previously demonstrated that CatC is involved in neuroinflammation and exacerbates demyelination in both cuprizone-induced and experimental autoimmune encephalomyelitis (EAE) mouse models. However, the underlying immunopathological mechanism remains elusive. In the present study, we established a recombinant myelin oligodendrocyte glycoprotein 35-55 peptide-induced EAE model using conditional CatC overexpression mice to investigate the effects of CatC on the alteration of CD4+ Th subsets, including Th1, Th2, Th17, Tfh and T regulatory cells. Our findings demonstrated that CatC particularly enhanced the population of Tfh cell in the brain, resulting in the earlier onset and more severe chronic syndrome of EAE. Furthermore, CatC promoted the formation of TLSs in the brain, leading to persistent neuroinflammation and exacerbating the severity of EAE in the chronic phase. Conversely, treatment with AZD7986, a specific inhibitor of CatC, effectively attenuated the syndrome of EAE and its effects caused by CatC both in vivo and in vitro. These findings provide a novel insight into the critical role of CatC in innate and adaptive immunity in EAE, and specific inhibitor of CatC, AZD7986, may contribute to potential therapeutic strategies for MS.

Cathepsin C in health and disease: from structural insights to therapeutic prospects.

Cathepsin C (CTSC) is a lysosomal cysteine protease constitutively expressed at high levels in the lung, kidney, liver, and spleen. It plays a key role in the activation of serine proteases in cytotoxic T cells, natural killer cells (granzymes A and B), mast cells (chymase and tryptase) and neutrophils (cathepsin G, neutrophil elastase, proteinase 3) underscoring its pivotal significance in immune and inflammatory defenses. Here, we comprehensively review the structural attributes, synthesis, and function of CTSC, with a focus on its variants implicated in the etiopathology of several syndromes associated with neutrophil serine proteases, including Papillon-Lefevre syndrome (PLS), Haim-Munk Syndrome (HMS), and aggressive periodontitis (AP). These syndromes are characterized by palmoplantar hyperkeratosis, and early-onset periodontitis (severe gum disease) resulting in premature tooth loss. Due to the critical role played by CTSC in these and several other conditions it is being explored as a potential therapeutic target for autoimmune and inflammatory disorders. The review also discusses in depth the gene variants of CTSC, and in particular their postulated association with chronic obstructive pulmonary disease (COPD), COVID-19, various cancers, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, sudden cardiac death (SCD), atherosclerotic vascular disease, and neuroinflammatory disease. Finally, the therapeutic potential of CTSC across a range of human diseases is discussed.

Cystatin F Depletion in Mycobacterium tuberculosis-Infected Macrophages Improves Cathepsin C/Granzyme B-Driven Cytotoxic Effects on HIV-Infected Cells during Coinfection.

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.

Targeting cathepsin C ameliorates murine acetaminophen-induced liver injury.

Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. Objective: In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. Methods and Results: We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included cxcl2, mmp9, and angpt2. Moreover, ptpn22, a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. Conclusions: This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach.

Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury.

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Toosendanin induces hepatotoxicity by restraining autophagy and lysosomal function through inhibiting STAT3/CTSC axis.

Toosendanin (TSN) is the main active component in the traditional herb Melia toosendan Siebold & Zucc, which exhibits promising potential for development due to its diverse pharmacological properties. However, the hepatotoxicity associated with TSN needs further investigation. Previous research has implicated autophagy dysregulation in TSN-induced hepatotoxicity, yet the underlying mechanisms remain elusive. In this study, the mechanisms of signal transducer and activator of transcription 3 (STAT3) in TSN-induced autophagy inhibition and liver injury were explored using Stat3 knockout C57BL/6 mice and HepG2 cells. TSN decreased cell viability, increased lactate dehydrogenase (LDH) production in vitro, and elevated serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels as well as liver lesions in vivo, suggesting TSN had significant hepatotoxicity. TSN inhibited Janus kinase 2 (JAK2)/STAT3 pathway and the expression of cathepsin C (CTSC). Inhibition of STAT3 exacerbated TSN-induced autophagy inhibition and hepatic injury, whereas activation of STAT3 attenuated these effects of TSN. Mechanistically, STAT3 transcriptionally regulated the level of CTSC gene, which in turn affected autophagy and the process of liver injury. TSN-administered Stat3 knockout mice showed more severe hepatotoxicity, CTSC downregulation, and autophagy blockade than wildtype mice. In summary, TSN caused hepatotoxicity by inhibiting STAT3/CTSC axis-dependent autophagy and lysosomal function.

Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study.

Sickle cell nephropathy (SCN) is a leading cause of morbidity and mortality in sickle cell disease (SCD). Early intervention is crucial for mitigating its effects. However, current diagnostic methods rely on generic tests and may not detect SCN until irreversible renal damage occurs. Therefore, specific biomarkers for early diagnosis of SCN are needed. Urinary exosomes, membrane-bound vesicles secreted by renal podocytes and epithelial cells, contain both common and cell type-specific membrane and cytosolic proteins, reflecting the physiologic and pathophysiologic states of the kidney. Using proteomics, we analyzed the proteomes of urinary exosomes from humanized SCD mice at 2 months (without albuminuria) and 4 months (with albuminuria) of age. Excretion of 164 proteins were significantly increased and 176 proteins was significantly decreased in the exosomes when mice developed albuminuria. Based on the relevance to SCD, chronic kidney disease and Western blot confirmation in mice, we analyzed protein abundance of heparanase, cathepsin C, α2-macroglobulin and sarcoplasmic endoplasmic Ca2+ ATPase-3 (SERCA3) in the urinary exosomes and urine of 18 SCD subjects without albuminuria and 12 subjects with albuminuria using Western blot analyses. Both male and female subjects increased or tended to increase the excretion of these proteins in their urinary exosomes upon developing albuminuria, but female subjects demonstrated stronger correlations between the excretion of these proteins and urine albumin creatinine ratio (UACR) compared to male subjects. In contrast, exosomal excretion of Tamm-Horsfall protein, ß-actin and SHP-1 was independent of albuminuria. These findings provide a foundation for a time-course study to determine whether increases in the levels of these proteins precede the onset of albuminuria in patients, which will help determine the potential of these proteins as biomarkers for early detection of SCN.

Self-reported clinical features and treatment effectiveness of Papillon-Lefèvre syndrome patients from five Latin American countries: A cross-sectional online survey study.

BACKGROUND/OBJECTIVES: Most studies about Papillon-Lefèvre syndrome (PLS) are limited to case reports and patients of the same nationality. This study aimed to determine the self-reported prevalence of signs, symptoms and treatment effectiveness in PLS patients from five Latin American countries. METHODS: An online survey was conducted among adult and paediatric patients from Mexico, Argentina, Colombia and Brazil. Data were collected using multiple-choice, open-ended and image-chooser questions on demographics, signs and symptoms, perceived treatment effectiveness and quality of life. RESULTS: Seventeen patients (10 males and 7 females) aged 4-47 years were surveyed. All had palmoplantar hyperkeratosis. Other affected sites were the feet and hand dorsum (82.35%), Achilles tendon (88.24%), forearms (58.82%), legs (29.41%) and glutes (23.53%). They frequently presented hyperhidrosis and nail pitting. Four had a history of delayed umbilical cord separation. All used topical treatments, with moderate effectiveness; half used oral retinoids, perceived as highly effective. Most reported decreased quality of life and walking difficulties. CONCLUSIONS: The study's results align with prior research on PLS, but reveal new insights, including the impact on patients' quality of life and a history of delayed umbilical cord separation. These findings warrant consideration in future research and patient care.

Cathepsin C regulates tumor progression via the Yes-associated protein signaling pathway in non-small cell lung cancer.

Cathepsin C (CTSC), also known as dipeptidyl peptidase I, is a cathepsin with lysosomal exocysteine protease activity and a central coordinator for the activation of neutrophil-derived serine proteases in the lysosomes of neutrophils. Although the role of CTSC in various cancers, including liver and breast cancers, has recently been reported, its role in non-small cell lung cancer (NSCLC) is largely unknown. This study aimed to investigate the functional role of CTSC in NSCLC and the molecular mechanisms underlying CTSC involvement in disease progression. CTSC overexpression markedly enhanced the growth, motility, and invasiveness of NSCLC cells in vitro and in vivo. CTSC knockdown using shRNA in NSCLC cells reversed the migratory and invasive behavior of NSCLC cells. CTSC also induced epithelial-mesenchymal transition through the Yes-associated protein signaling pathway. In addition, our analyses of clinical samples confirmed that high CTSC expression was associated with lymph node metastasis and recurrence in lung adenocarcinoma. In conclusion, CTSC plays an important role in the progression of NSCLC. Thus, targeting CTSC may be a promising treatment option for patients with NSCLC.

Characterization and function analysis of cathepsin C in Marsupenaeusjaponicus.

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.

The oral microbiome of a family including Papillon-Lefèvre-syndrome patients and clinically healthy members.

AIMS: The oral microbiota composition of patients diagnosed with Papillon-Lefèvre-syndrome and treated for several years were compared to those existing in the oral cavity of the clinically healthy family members and a cohort of patients having various stages of chronic periodontitis. MATERIALS AND METHODS: A family with two sisters affected with severe periodontitis and with the typical skin symptoms of Papillon-Lefèvre-syndrome, and symptomless parents and third sibling were investigated. The Patients received periodontal treatment for several years and their oral microbiome was analysed by amplicon sequencing. Data were evaluated by microbial cluster analysis. RESULTS: The microbiome of the patients with Papillon-Lefèvre-syndrome was predominated with Aggregatibacter actinomycetemcomitans and associated oral periodontopathogens. Although the clinically healthy family members showed no oral disorder, their microbiome resembled that of subjects having mild periodontitis. CONCLUSIONS: Predominance of A. actinomycetemcomitans in the subgingival microbiome of patients with Papillon-Lefèvre-syndrome suggests that specific treatment strategies directed against this pathobiont may improve the oral health status of the affected individuals. TRIAL REGISTRATION: The study was conducted in accordance with the Declaration of Helsinki and the ethical permission has been issued by the Human Investigation Review Board of the University of Szeged, Albert Szent-Györgyi Clinical Centre (Permission No. 63/2017-SZTE). September 19, 2017.  https://u-szeged.hu/klinikaikutatas/rkeb-altal-jovahagyott/rkeb-2017 .

Cathepsin C role in inflammatory gastroenterological, renal, rheumatic, and pulmonary disorders.

Cathepsin C (CatC, syn. Dipeptidyl peptidase I) is a lysosomal cysteine proteinase expressed in several tissues including inflammatory cells. This enzyme is important for maintaining multiple cellular functions and for processing immune cell-derived proteases. While mutations in the CatC gene were reported in Papillon-Lefèvre syndrome, a rare autosomal recessive disorder featuring hyperkeratosis and periodontitis, evidence from clinical and preclinical studies points toward pro-inflammatory effects of CatC in various disease processes that are mainly mediated by the activation of neutrophil serine proteinases. Moreover, tumor-promoting effects were ascribed to CatC. The aim of this review is to highlight current knowledge of the CatC as a potential therapeutic target in inflammatory disorders.

CTSC promoted the migration and invasion of glioma cells via activation of STAT3/SERPINA3 axis.

Cathepsin C (CTSC) has been reported to be upregulated in several cancers, however, there are still many missing links about the role of CTSC in glioma. To address this knowledge gap, the present study employed bioinformatics analysis, Transwell assay, RT-qPCR and Western blot assays to investigate the expression level of CTSC in glioma tissues, its relationship with survival period, and its effect on the migration and invasion ability of glioma cells. The findings revealed that CTSC was upregulated in glioma and was associated with poor prognosis. Moreover, CTSC was found to promote cell migration and invasion abilities as well as epithelial-mesenchymal transition (EMT). A further study found that CTSC induced SERPINA3 and STAT3 expression in glioma cells. Additionally, we demonstrated that STAT3 signaling mediated upregulation of SERPINA3 expression by CTSC. In sum, our findings suggest that CTSC activates the STAT3/SERPINA3 axis to promote migration and invasion of glioma cells, which may lead to new potential therapeutic approaches for humans with cancer.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats.

BACKGROUND: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. METHODS: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. RESULTS: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. CONCLUSIONS: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Expression and significance of cathepsin C and cathepsin D during pregnancy and Preeclampsia.

BACKGROUND: Cathepsin C (Cat C) is involved in the inflammatory-immune system and can be degraded by cathepsin D (Cat D). Preeclampsia (PE) and the inflammation-immunity relationship is currently a hot research topic, but there are still few studies. The aim was to investigate the expression and significance of Cat C and D in the serum of nonpregnant women, patients in various stages of pregnancy and patients with PE, and in the placenta of patients with normal pregnancy and PE. METHODS: Sixty young healthy nonpregnant women were selected: 180 normal pregnant women, including 60 each in the first, second, and third trimesters, and 100 women with PE, including 39 women with severe preeclampsia. The levels of Cat C and D in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the expression levels of Cat C and D in placentas were detected by immunohistochemistry (IHC). RESULTS: The serum of Cat C in the first trimester was significantly lower than that in the nonpregnant group (P < 0.001), whereas Cat D was significantly higher than that in the nonpregnant group (P < 0.01). The levels of Cat C and D in the second trimester and third trimester were significantly higher than those in the first trimester (P < 0.05), but there was no significant difference in Cat C and D between the second trimester and third trimester. The levels of Cat C in the serum and placentas of patients with PE were significantly higher than those in the third trimester (P < 0.001) and positively correlated with the severity of PE (P < 0.001), whereas the levels of Cat D in the serum and placentas of patients with PE were significantly lower than those in the third trimester (P < 0.001) and negatively correlated with the severity of PE (P < 0.001). Age, primigravida proportion, and body mass index were significantly higher in the PE group than in the control group (P < 0.05), which were high-risk factors for PE. CONCLUSIONS: Cat C and D are associated with the maintenance of normal pregnancy. In patients with preeclampsia, a significant increase in Cat C and a significant decrease in Cat D levels may lead to the occurrence and development of preeclampsia.

Diagnosis and Management of Papillon-Lefevre Syndrome: A Rare Case Report and a Brief Review of Literature.

Papillon-Lefevre syndrome (PLS) manifests as an autosomal recessive disorder caused by a mutation in the cathepsin C (CTSC) gene. This genetic alteration results in palmoplantar hyperkeratosis, rapid onset of periodontitis, and premature shedding of both primary and permanent teeth. The major etiological factor responsible for the development of this disorder appears to be variations in the CTSC gene, which is responsible for the production of the cathepsin C enzyme in the body. The multifactorial aetiology of the syndrome is influenced by immunologic, genetic, or microbial factors. This case report presents a clinical picture of a 21-year-old Indian male patient with oligodontia and mobile teeth accompanied by palmoplantar keratosis and a history of recurrent infection. The detailed family history of the patient revealed genetic relevance with PLS. This article will discuss in detail the diagnosis, evaluation and treatment modalities involved in the management of the case.

Update on Mast Cell Proteases as Drug Targets.

Mast cell granules are packed with proteases, which are released with other mediators by degranulating stimuli. Several of these proteases are targets of potentially therapeutic inhibitors based on hypothesized contributions to diseases, notably asthma and ulcerative colitis for ß-tryptases, heart and kidney scarring for chymases, and airway infection for dipeptidyl peptidase-I. Small-molecule and antibody-based ß-tryptase inhibitors showing preclinical promise were tested in early-phase human trials with some evidence of benefit. Chymase inhibitors were given safely in Phase II trials without demonstrating benefits, whereas dipeptidyl peptidase-I inhibitor improved bronchiectasis, in effects likely related to inactivation of the enzyme in neutrophils.

Brensocatib (an oral, reversible inhibitor of dipeptidyl peptidase-1) attenuates disease progression in two animal models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a painful and incurable disease characterized by chronic joint inflammation and a progressive destruction of cartilage and bone. Although current treatments have improved clinical outcomes for some patients, the high relapse rates and sizeable proportion of non-responders emphasize the need for further research. Arthritic joints are massively infiltrated by neutrophils, which influence inflammatory and immune processes by releasing cytokines, chemokines, eicosanoids, and neutrophil serine proteases (NSPs) - all of which are known to contribute to RA initiation and progression. Active NSPs are generated from zymogens at the promyelocytic stage of neutrophil differentiation under the action of dipeptidyl peptidase 1 (DPP-1) and DPP-1 knockout mice are resistant to the development of arthritis. Thus, DPP-1 inhibition represents a promising therapeutic approach in RA. In this study, we assessed the efficacy of a potent and highly selective DPP-1 inhibitor, brensocatib, in two well established RA models - rat collagen-induced arthritis (CIA) and mouse collagen antibody-induced arthritis (CAIA). In both models, brensocatib at 3 and 30 mg/kg/day significantly reduced bone marrow NSP levels, in keeping with prior pharmacodynamic studies in rodents. More importantly, brensocatib treatment significantly improved disease score at both dosages in both rodent models. In the mouse CAIA model, brensocatib even proved at least as potent as anti-TNF antibodies in diminishing both the histopathological score and neutrophil infiltration into arthritic joints. Together, these results show that brensocatib alters RA disease progression in rodents and supports the need for its further evaluation as a potential therapeutic option, or to complement existing RA treatments.

The pharmacokinetic profile of brensocatib and its effect on pharmacodynamic biomarkers including NE, PR3, and CatG in various rodent species.

Brensocatib is a novel, oral, selective, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), which activates several neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) in the bone marrow during the early stage of neutrophil maturation. These NSPs are associated with pathogen destruction and inflammatory mediation; their dysregulated activation can result in excess secretion of active NSPs causing damaging inflammation and contributing to neutrophil-mediated inflammatory and autoimmune diseases. Pharmacological inhibition of DPP1 in the bone marrow could therefore represent an attractive strategy for these neutrophil-driven diseases. A completed Phase 2 trial in non-cystic fibrosis bronchiectasis patients (ClinicalTrials.gov number NCT03218917; EudraCT number: 2017-002533-32) indeed demonstrated that administration of brensocatib attenuated the damaging effects of chronic inflammation by inhibiting the downstream activation of NSPs. To support a range of preclinical programs and further understand how rodent species and strains may affect brensocatib's pharmacokinetic (PK) profile and its pharmacodynamic (PD) effects on NE, PR3, and CatG, an extensive naïve dosing study with brensocatib at different dosing levels, frequencies, and durations was undertaken. Dose-dependent PK exposure responses (AUC and Cmax) were observed regardless of the rodent species and strain. Overall, mice showed greater reduction in NSP activities compared to rats. Both mice and rats dosed once daily (QD) had equivalent NSP activity reduction compared to BID (twice a day) dosing when the QD dose was 1.5-times the BID daily dose. For both mouse strains, CatG activity was reduced the most, followed by NE, then PR3; whereas, for both rat strains, PR3 activity was reduced the most, followed by CatG, and then NE. Maximum reduction in NSP activities was observed after ∼7 days and recoveries were nearly symmetrical. These results may facilitate future in vivo brensocatib study dosing considerations, such as the timing of prophylactic or therapeutic administration, choice of species, dosage and dosing frequency.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis.

BACKGROUND: Airway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583. METHODS: Binding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model. RESULTS: BI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma. CONCLUSION: BI 1291583-mediated inhibition of CatC is expected to restore the protease-antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.