Exploration of mitochondrial-targeted hydrogen sulfide donors as novel therapeutics for fibroproliferative lung disease

Repeated damage to the lungs (caused by pollution, infection or trauma) usually activates normal repair processes with no ill effects. However in some people, it can result in the excessive tissue scarring known as pulmonary fibrosis (PF). Patients with the most common form, idiopathic pulmonary fibrosis (IPF), are generally diagnosed in their 60s or 70s, indicating that ageing plays a major role in susceptibility to the disease.

In Europe and the USA, there are currently in excess of 500,000 patients with IPF, which places a significant clinical and financial burden on healthcare systems. The prognosis is dire, with a median survival of only 3 - 5 years following diagnosis. More than 5,000 people die annually in the UK from IPF alone - 1% of all UK deaths - putting it on a par with mortality rates for leukaemia or brain tumours, and vastly higher than deaths associated with skin cancer, MRSA or HIV/AIDS. For unknown reasons, the incidence is increasing. Two recently licensed drugs (Nintedanib and Pirfenidone) have shown some success at slowing the decline in lung function in some patients, but they are still unable to halt or reverse the disease. Lung transplant remains the most effective treatment strategy, but in the entire period from 1995 to 2009, worldwide there were only 5,740 single or bilateral adult lung transplants for IPF. Identification of better therapeutic interventions is therefore paramount to supplement the extremely limited repertoire of drugs which have shown signs of clinical benefit in PF and other fibrotic disorders.

We have been specifically developing novel drugs which harness the therapeutic potential of the gas, hydrogen sulfide. This gas is normally used by cells in the body to help control inflammation and tissue damage, although it has many other effects which are still being elucidated. We believe there is excellent rationale for assessing our drugs in the context of PF. Our current MRC project grant (MR/S002626/1) is indicating significant efficacy of hydrogen sulfide administration in mouse models of chronic obstructive pulmonary disease - a condition with many parallels to PF. Hydrogen sulfide administration has also shown benefit in neurodegeneration, hypertension and diabetes, amongst others. This project will utilise sophisticated genetically-altered mice to specifically reduce hydrogen sulfide production in the lung, and help us understand its contribution from particular cells involved during the development of PF. This will be supported by lab experiments, to mechanistically understand how hydrogen sulfide is used by cells during the repair process. We will then optimise and therapeutically assess whether (and how) our novel, unique and patented hydrogen sulfide-producing drugs can block the development of fibrosis in our mouse model and in the lab.

As mentioned, many lung diseases, including PF, are associated with older age; incidence, prevalence and mortality all increase with age. Given that the UK has an ageing population (10 million people in the UK are over 65 years old, and the figure is rising), it is becoming increasingly pertinent to perform research using experimental systems which better reflect the human disease. We already have experience in performing lung fibrosis experiments using aged mice; this will form a key component of the current proposal, to help establish why the lung is more susceptible to fibrosis with advancing age. Given the technical and financial limitations, it is perhaps not surprising that there is limited published data on the mechanistic influence of ageing; this study would add significantly to this body of work.

This proposal will therefore begin to identify the mechanisms by which our preliminary observations are connected, and how they contribute to lung scarring, using experimental models of fibrosis (cell-based experiments and mouse studies) and patient samples to confirm our findings.

We wish to elucidate the role of hydrogen sulfide production and supplementation in the lung, and its effects on tissue injury, fibrosis and resolution, in the context of ageing.

An experiment has already been performed using aged mice, demonstrating that they are more susceptible to lung injury. The broad effects of ageing on fibrosis development will be further investigated via comparison of lung samples from old and young mice in this proposal. This will include analysis of target genes of interest using QPCR and immunohistochemical approaches in mouse samples, and human PF samples (to confirm human disease relevance). Immunohistochemistry/fluorescence will be quantified on a Vectra Polaris multispectral imaging platform.

The relative role of local production of hydrogen sulfide will be determined using transgenic mice where the pivotal enzyme in the hydrogen sulfide biosynthetic pathway, cystathionine gamma-lyase (CSE), is specifically knocked-out in lung fibroblasts or epithelium. We will investigate fibrotic susceptibility (and burden of cellular senescence) using the well-characterised murine model of bleomycin-induced lung injury and fibrosis (compared with littermate controls). Sophisticated imaging (micro-CT) will be used to assess disease development in these mice, in addition to standard molecular (e.g QPCR) and biochemical measures (e.g. HPLC determination of total lung collagen).

In vitro correlates (effects of hydrogen sulfide administration) will be performed using primary human cells - namely fibroblasts and epithelium, to establish a role in senescence and mitochondrial function. Physiologically-relevant air-liquid interface co-cultures will be used, plus three-dimensional fibroblast organoid cultures. In vivo data will further be corroborated using ex vivo precision cut human lung slice cultures.

This combination of in vivo, ex vivo/in vitro and molecular and cell biological approaches will enable us to fully exploit our data.

Our proposal aims to address some of the key features of chronic wound healing responses in the lung, and how this relates to the development of fibrosis. In particular, we wish to establish cogent links between hydrogen sulfide signalling, senescence and ageing. Work by us and others has considered these in isolation; we have an opportunity to bring them together as a cohesive whole. The results will impact on our understanding of aberrant wound healing, which is germane to the pulmonary fibrosis (PF) field, but also COPD, asthma, lung cancer and other organ systems (e.g. liver). Ageing in particular is extremely topical, given the burgeoning age of the UK population and implications for healthcare. This research would be of major benefit to the scientific community, healthcare professionals, the pharmaceutical sector, patients and the general public. We will deliver impact through appropriate interaction with each of these key stakeholders.
Patients will be engaged through our work with local PF patient involvement groups, who have a vested interest in understanding how our research may impact on clinical management of their disease and their quality of life. Updates will be communicated to the public through the University of Exeter (UoE) website, Twitter and Café Scientifique programme. The UoE is committed to public engagement with scientific research (e.g. school outreach activities).
Our proposal will involve advanced transgenic mouse models, imaging approaches and epigenetic analyses, which we hope will inspire the next generation of biomedical scientists. The UoE Press Office will be involved at all stages, to ensure that key findings and results are fully communicated publically.
The UoE has an ethos of research-led education, such that undergrad and postgrad students are provided with the opportunity to share the excitement of research, and extend the boundaries of knowledge. We would enable students to develop valuable skills for critical and independent inquiry, to ultimately sustain the UK's intellectual and creative resources. This rationale would also apply to the staff appointed in this proposal - developing their transferable research skills to have a positive impact on their future careers.
Our work is designed to be translational in nature, through identification of key disease mechanisms which may be targets for therapeutic intervention using our novel H2S compounds. Our proposal will also investigate aged mice as a more representative and robust pre-clinical model for lung disease research. Through conference presentation, publication and on-going academic-industrial liaison, this may impact on the approach taken by the pharmaceutical industry for drug development and pre-clinical validation, where animal models need to be fit for purpose. Feedback from healthcare professionals (through local/regional meetings) will be important for steering our research towards clinical applicability in the longer-term, whether in management of PF, or other diseases such as COPD or asthma. Commercial exploitation will be actively pursued via the UoE Innovation, Impact and Business office, to ensure appropriate IP protection and engagement with the pharmaceutical sector.
Aside from the obvious impact of sharing knowledge with the scientific community (through presentation and publication etc), our work would also entail expansion of our lung tissue biobank (in partnership with the NIHR Exeter Clinical Research Facility), and deposition of aged mouse tissue samples (multi-organ, not just lung) with the Shared Ageing Research Model (ShARM) biorepository. This approach will maximise the impact of our research beyond our immediate professional circle, imbuing UK-based biomedical researchers with access to key clinical or murine samples, necessary for the progression of their own research plans. The long term knock-on effects for translational medicine, drug discovery and economic/health improvement are considerable.