This study demonstrated that MOD06051, a novel CatC inhibitor, showed potent and specific suppression of CatC activity followed by the functional inhibition of downstream NSPs, including NE and PR3, and NET formation both in vitro and in vivo.
The formation of excess NETs has been implicated in the pathogenesis of a variety of human diseases, including sepsis, gout, diabetes, SLE, RA, and AAV. Upon disease initiating stimuli, including exposure to infectious agents and causative environmental factors, neutrophils are primed by pro-inflammatory cytokines such as TNF-α and/or C5a, which are produced by activation of the complement alternative pathway. Primed neutrophils express MPO on the plasma membrane, to which MPO-ANCA binds, resulting in IC formation on the cellular surface. Consequently, neutrophils are excessively activated and produce ROS, followed by the release of NETs. Several research groups have demonstrated efficacy in suppressing NET formation in in vivo disease models17–19, and these strategies, including ROS elimination and PAD inhibition, are considered as promising clinical therapies for NET-associated diseases4. However, as seen in chronic granulomatous disease, a genetic condition resulting in congenital immunodeficiency whereby neutrophils cannot produce ROS27, ROS inhibition could potentially induce immunosuppression. Furthermore, PAD4-deficient mice are more susceptible to bacterial infection than wild-type mice due to their inability to form NETs12. Another approach is therefore necessary to avoid this serious complication.
Papillon-Lefevre syndrome (PLS) is a human CatC deficiency28. Although PLS patients showed almost no CatC activity in their tissues and cells, including neutrophils, neutrophils isolated from these patients demonstrated no obvious dysfunction other than reduced NSP activity28. In addition, PLS does not result in an increased incidence of serious infectious disease, with periodontitis being the main concern for these patients. In CatC-deficient mice, the effector function of NK cells and CD8+ cytotoxic T cells is equivalent to that of wild-type mice29. Thus, the specific inhibition of CatC could be a safer treatment option for NET-associated diseases, avoiding severe immunosuppression while suppressing excess NET formation.
In the present study, MPO-ANCA-IC-induced NET formation was significantly suppressed in neutrophils derived from MOD06051-administered rats regardless of priming stimuli. Similar findings were observed with the NE inhibitor sivelestat when human peripheral blood neutrophils were stimulated by MPO-ANCA-IC. These collective findings suggest that CatC inhibition, which blocks NE maturation and activation, can suppress NET formation as well as NE inhibition. Interestingly, peripheral blood neutrophils derived from patients with PLS have been shown to fail to generate NETs upon stimulation by phorbol 12-myristate 13-acetate, another NETs inducer30, further supporting our observation.
MPO-AAV is a systemic small-vessel vasculitis31 and is characterized by the development of NCGN and pulmonary hemorrhage due to alveolar septal capillaritis32. MPO-ANCA is known as a pathogenic autoantibody that can activate neutrophils, which had expressed MPO on the plasma membrane upon priming33. As activated neutrophils are critically involved in MPO-AAV pathogenesis34, CatC inhibition could be a potential therapeutic option for AAV. This concept is also supported by a recent report demonstrating that neutrophils derived from patients with PLS failed to be activated by PR3-ANCA and did not injure vascular endothelial cells35. To determine the efficacy of MOD06051 on NET-associated diseases in vivo, an MPO-AAV rat model was adopted in this study.
The results of this study showed that the novel, specific CatC inhibitor MOD06051 could successfully ameliorate MPO-AAV in the rat model. Since activation and maturation of NSPs occurs during neutrophil maturation in the bone marrow, a process which takes weeks, CatC inhibitors would need continuous dosing for several weeks to show significant suppression of downstream NSPs. Thus, it takes about two weeks in order for CatC inhibition to show efficacy in the peripheral neutrophils. This study dosed animals from before MPO-AAV symptoms were exhibited. However, sufficient MPO-ANCA was produced by day 14 in this disease model24, thus inducing tissue injury, and compound treatment resulted in less tissue damage. Therefore, CatC inhibition may be efficacious even after onset of MPO-AAV. Furthermore, MPO-AAV is a recurring disorder with progressively worsening symptoms as constant microinjuries are induced on tissues. Treatment with CatC inhibitors may stop the continuous cycle, even in the presence of MPO-ANCA. This makes MOD06051 a potential therapeutic candidate for the treatment of MPO-AAV in human.
Standard interventions for MPO-AAV include glucocorticoids (GCs) and immunosuppressive drugs such as cyclophosphamide and rituximab36–38. The most critical problem with these drugs is secondary immunodeficiency-based opportunistic infections39. In contrast, CatC inhibition suppresses the maturation of NSPs, but does not affect other antibacterial proteins such as MPO. In addition, there is no effect on acquired immunity. In fact, even in MPO-AAV rats, the amount of MPO-ANCA produced by human MPO immunization was not reduced by MOD06051. Therefore, CatC inhibition appears to have no significant effect on the humoral immune response in vivo.
Recent studies have demonstrated that avacopan, which inhibits the binding of C5a with C5aR and the resulting priming of neutrophils, is a promising drug for MPO-AAV treatment40–42. In this study, avacopan suppressed MPO-ANCA-IC stimulation only in C5a-primed neutrophils but not in TNF-α-primed ones. This finding contrasts with the results demonstrating that MPO-ANCA-IC-induced neutrophil activation was significantly suppressed by MOD06051 regardless of priming stimuli. As a selective antagonist to the C5aR, avacopan acts relatively upstream of neutrophil activation in MPO-AAV pathogenesis34, whereas MOD06051 interferes with the activation of NE and acts further downstream compared with avacopan. Therefore, MOD06051 is expected to be effective against diverse priming factors, including TNF-α and C5a, and is considered to possess high versatility for NET suppression. In addition, because the points of action are different, combination with avacopan could potentially be considered as an MPO-AAV treatment option in the future.
In this study, we demonstrated that the level of NET formation in peripheral blood was quantifiable and correlated with the level of NETs in inflamed tissues such as kidneys. These findings may allow us to use NETs as a biomarker to monitor the disease state of NET-related diseases such as MPO-AAV.
One limitation of this study was the lack of comparison of in vivo efficacy between MOD06051 and the other compounds with a different mechanism of actions. In particular, comparison with NE inhibitors would be useful due to the role NE plays in NET formation. While NE inhibitors suppress mature NE activity specifically in peripheral blood neutrophils, CatC inhibitors inhibit the maturation of not only NE but also other NSPs such as PR3 in the bone marrow. Interestingly, MOD06051 suppressed enzymatic activity and surface expression of PR3 in neutrophils differentiated from bone marrow stem/progenitor cells. Because cell surface PR3 on dead neutrophils interferes with phagocytosis by macrophages and is involved in the persistence of inflammation43, MOD06051 might suppress disease development in MPO-AAV rats through the increase in macrophage-mediated clearance of dead neutrophils in addition to the suppression of NSPs activity and NET formation. Another limitation of this study is the use of a single animal model. Further studies should aim to demonstrate the efficacy of MOD06051 using other NET-associated disease models.
In conclusion, MOD06051 inhibited CatC-mediated NSP activation in neutrophils, NE-mediated NET formation, and ameliorated MPO-AAV in rats. These data taken together suggest that CatC inhibition is a promising therapeutic strategy against a range of activated neutrophil-mediated disorders, including NET-associated diseases.