AZD5904

Myeloperoxidase inhibitor AZD5904 enhances human sperm function in vitro

Study question: Does AZD5904, a myeloperoxidase inhibitor (MPOi), have any effect on human sperm function in vitro? Summary answer: AZD5904 improves sperm function in an in vitro model of oxidative stress (OS) and potentially offers a novel treatment approach for male infertility. What is known already: Male infertility is an underlying or contributory cause in half of all couples experiencing difficulties conceiving, yet there is currently no effective treatment or cure. OS is a common pathology in a significant proportion of infertile men. It can negatively affect sperm motility and the ability to fertilize a mature oocyte, as well as DNA integrity, and therefore represents an attractive target for therapeutic intervention. Study design, size, duration: This study included population-based samples from men (23-50 years) attending Ninewells Assisted Conception Unit, Dundee for diagnostic semen analysis, July 2017-September 2018. Semen samples (n = 47) from 45 patients were used. Participants/materials, setting, methods: Neutrophils activated using zymosan were incubated with prepared human spermatozoa for 2 h (T2) and 24 h (T24) to create an in vitro model of OS. Parallel samples were co-incubated with AZD5904, an MPOi, to examine its effects. Sperm motility was assessed by computer-assisted sperm analysis at T2 and T24. Functional motility was assessed by sperm penetration assay. Statistical analysis was performed using GraphPad Prism. Main results and the role of chance: There was no significant difference in total or progressive sperm motility between any treatment and control groups at T2 or T24. Nonetheless, significant positive effects on sperm function were observed with AZD5904, with 16/45 (35.6%) samples (with both normal and abnormal baseline semen analysis characteristics) displaying a ≥20% increase in sperm penetrated through viscous media (P < 0.003). Limitations, reasons for caution: This was an in vitro study. Wider implications of the findings: Treatment with AZD5904 resulted in significant increased sperm penetration in one of three samples treated, which is likely to represent improvement in sperm function required for fertilization. We are now planning a clinical trial to validate these results and hope that this could represent a new treatment for male infertility. Study funding/competing interest(s): AZD5904 was shared through the AstraZeneca Open Innovation program. The study was funded by AstraZeneca and sponsored by the University of Dundee. Additional funding was provided by Chief Scientist Office/NHS Research Scotland (S.J.M.d.S.). A.W. and H.J.S. are both full time employees of AstraZeneca. A.W. and H.J.S. are inventors on a patent filed by AstraZeneca titled MPOi for use in medicine which includes MPOi for use in the treatment of male infertility (WO 2019/016074 Al). S.J.M.d.S. is Associate Editor of Human Reproduction and Editorial Board member of Reproduction & Fertility. C.L.R.B. is Editor of RBMO and has received lecturing fees from Merck and Ferring and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B. was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016). C.L.R.B. has a patent WO2013054111 A1 issued. The other authors declare no conflict of interest. Trial registration number: N/A.

Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes

Aims: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM.
Methods and results: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy.
Conclusion: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.

Inhibiting myeloperoxidase prevents onset and reverses established high-fat diet-induced microvascular insulin resistance

A high-fat diet (HFD) can rapidly recruit neutrophils to insulin target tissues and within days induce microvascular insulin resistance (IR). Myeloperoxidase (MPO) is highly enriched in neutrophils, can inhibit nitric oxide-mediated vasorelaxation in vitro and is associated with increased cardiovascular disease risk. AZD5904 irreversibly inhibits MPO and in human clinical trials. MPO knockout, or chemical inhibition, blunts HFD-induced metabolic IR in mice. Whether MPO affects microvascular IR or muscle metabolic insulin sensitivity in vivo is unknown. We used contrast-enhanced ultrasound and the euglycemic insulin clamp to test whether inhibiting MPO could prevent the development or reverse established HFD-induced metabolic and/or microvascular IR in Sprague-Dawley rats. Two weeks of HFD feeding blocked insulin-mediated skeletal muscle capillary recruitment, inhibited glucose utilization, and insulin signaling to muscle. Continuous subcutaneous AZD5904 infusion during the 2 wk selectively blocked HFD's microvascular effect. Furthermore, AZD5904 infusion during the last 2 of 4 wk of HFD feeding restored microvascular insulin sensitivity but not metabolic IR. We conclude that inhibiting MPO selectively improves vascular IR. This selective microvascular effect may connote a therapeutic potential for MPO inhibition in the prevention of vascular disease/dysfunction seen in IR humans.

CD206+CD68+ mono-macrophages and serum soluble CD206 level are increased in antineutrophil cytoplasmic antibodies associated glomerulonephritis

Background: Antineutrophil Cytoplasmic Antibodies (ANCA) associated glomerulonephritis (AGN) is a group of autoimmune diseases and mono-macrophages are involved in its glomerular injuries. In this study, we aim to investigate the role of CD206⁺ mono-macrophages in AGN.
Methods: 27 AGN patients (14 active AGN, 13 remissive AGN) together with healthy controls (n = 9), disease controls (n = 6) and kidney function adjusted controls (n = 9) from Department of Nephrology, Ruijin hospital were recruited. Flow cytometry was used to study proportion of CD206⁺ cells in peripheral blood. Immunohistochemistry for CD206 staining was performed and CD206 expression was scored in different kidney regions. Serum soluble CD206 (sCD206) was measured by enzyme-linked immunosorbent assay (ELISA). We also generated murine myeloperoxidase (MPO) (muMPO) ANCA by immunizing Mpo^-/- mice. Mouse bone marrow-derived macrophages (BMDMs) from wild C57BL/6 mice and peripheral blood mononuclear cell (PBMC) derived macrophages from healthy donors were treated with MPO ANCA with or without its inhibitor AZD5904 to investigate the effects of MPO-ANCA on CD206 expression.
Results: The proportion of peripheral CD206⁺CD68⁺ cells in active AGN patients were significantly higher than that in remissive patients (p < 0.001), healthy controls (p < 0.001) and kidney function adjusted controls (p < 0.001). Serum sCD206 level in active AGN patients was higher than that in healthy controls (p < 0.05) and remissive patients (p < 0.01). Immunohistochemistry showed CD206 was highly expressed in different kidney regions including fibrinoid necrosis or crescent formation, glomeruli, periglomerular and tubulointerstitial compartment in active AGN patients in comparison with disease controls. Further studies showed MPO ANCA could induce CD206 expression in BMDMs and PBMC derived macrophages and such effects could be reversed by its inhibitor AZD5904.
Conclusion: ANCA could induce CD206 expression on mono-macrophages and CD206⁺ mono-macrophages are activated in AGN. CD206 might be involved in the pathogenesis of AAV and may be a potential target for the disease.

Inhibition of myeloperoxidase enhances immune checkpoint therapy for melanoma

Background: The presence of a highly immunosuppressive tumor microenvironment has limited the success of immune checkpoint therapy (ICT). Immune suppressing myeloid cells with increased production of reactive oxygen species are critical drivers of this immunosuppressive tumor microenvironment. Strategies to limit these immune suppressing myeloid cells are needed to enhance response to ICT.
Methods: To evaluate the contribution of myeloperoxidase (MPO), a myeloid lineage-restricted enzyme and a major source of reactive oxygen species, to mediating ICT response, we compared treatment outcome and immune composition in wild-type, MPO-deficient (MPO ^-/- ), and MPO inhibitor-treated wild-type mice using established primary melanoma models.
Results: Tumor growth and survival studies demonstrated that either host deficiency (MPO ^-/- ) or pharmacological inhibition of MPO enhanced ICT response in two preclinical models of established primary melanoma in aged animals. The tumor microenvironment and systemic immune landscape underwent striking changes in infiltration of myeloid cells, T cells, B cells, and dendritic cells in MPO ^-/- mice; furthermore, a significant increase in myeloid cells was observed in ICT non-responders. The contribution of CD4⁺ T cells and NK cells during ICT response also changed in MPO ^-/- mice. Interestingly, MPO enzymatic activity, but not protein, was increased in CD11b⁺Ly6G⁺ myeloid cells isolated from marrow, spleen, and peritoneal cavities of mice bearing untreated melanoma, indicating systemic activation of innate immunity. Notably, repurposing MPO-specific inhibitors (verdiperstat, AZD5904) in combination with ICT pointedly enhanced response rates above ICT alone. Indeed, long-term survival was 100% in the YUMM3.3 melanoma model on treatment with verdiperstat plus ICT.
Conclusion: MPO contributes to ICT resistance in established melanoma. Repurposing MPO-specific inhibitors may provide a promising therapeutic strategy to enhance ICT response.