Developing a cocktail of enzyme inhibitors to universally treat haemotoxicity caused by snakebite

Background

Snakebite is a neglected tropical disease that kills ~100,000 people each, year and causes long-term morbidity in 3-5 times that number. Venom variation between snake species severely undermines the efficacy of existing snakebite therapies, known as antivenoms. These intravenously delivered treatments work poorly against bites caused by snake species that were not used to make the antivenom. Therefore many different antivenoms have to be manufactured for each region of the world. In addition, existing antivenoms have (i) poor specificity, (ii) poor safety and (iii) high treatment costs. There is therefore an urgent and compelling need to dramatically improve therapy for the rural impoverished people of the tropics suffering the greatest burden of snakebite.

Rationale

The most common lethal snakebite pathology is haemotoxicity, which can present clinically as haemorrhage, coagulopathy and/or hypotension, and is primarily caused by three enzymatic venom toxin families. In this project, we seek to develop a new type of therapy for treating haemotoxicity, consisting of repurposed enzyme inhibitors (small molecules). Our preliminary data shows that various enzyme inhibitors are capable of neutralising the enzymatic activity of haemotoxic venom protein families irrespective of the snake species involved, and at lower therapeutic doses than antivenom. We have also demonstrated that inhibitory molecules are capable of preventing snake venom induced lethality in vivo in pre-clinical animal studies. We therefore anticipate that a rationally selected mixture of different enzyme inhibitors will result in a low-dose therapy that (i) neutralises venom lethality irrespective of the snake species, and thus will exert worldwide efficacy, (ii) exhibits a significantly improved safety profile, and (iii) is substantially more affordable than antivenom.

Approach

We will use a variety of enzyme inhibitors previously approved for human use and test their in vitro and in vivo neutralising capability against ten medically-important, haemotoxic, snake venoms.
1. First, we will use a variety of enzymatic in vitro assays to characterise the haemorrhagic, coagulopathic and hypotensive action of each venom. Subsequently, we will repeat these assays with each venom mixed with individual enzyme inhibitors to identify those inhibitors that confer venom neutralisation.
2. Our preliminary data suggests that combinations of different inhibitors will be required for 'generic' (i.e. across lots of different snake species) neutralisation of venom lethality in vivo, due to the complexity of toxic venom components, and that combinations of enzyme inhibitors can reduce the therapeutic dose required for neutralisation. Consequently, we will repeat our in vitro assays using rational combinations of the different inhibitors identified (those that exhibited neutralising capabilities in 1) at different dose combinations.
3. Lastly, we will demonstrate the superior efficacy of the two inhibitor combinations exhibiting the highest neutralising capability in vitro, in in vivo neutralisation of venom lethality studies. We will use murine pre-clinical efficacy studies (survival and survival times) and subsequent biomarker analyses (e.g. markers of haemorrhage, coagulopathy, hypotension) to determine whether the enzyme inhibitor mixtures exhibit superior venom neutralisation than existing commercial antivenoms.

Implications

We anticipate that our optimal enzyme inhibitory mixture will exert superior venom neutralisation characteristics (lower therapeutic doses, generic cross-species neutralisation) than existing "gold-standard" antivenoms. Consequently, this project has the potential to revolutionise snakebite therapy via the generation of a pre-clinically validated "generic", affordable, treatment for the millions of impoverished snakebite victims who suffer from venom-induced haemotoxicity each year.

Snakebite is a neglected tropical disease that kills ~100,000 people each year. Currently, many different antivenoms are used to treat victims, as their efficacy is limited to the snake species used for manufacture. Antivenoms exhibit poor specificity, high incidences of adverse reactions and high treatment costs, and there is therefore an urgent need to dramatically improve therapy for snakebite victims.
The most common lethal snakebite pathology is haemotoxicity, which is the result of enzymatic venom toxins causing haemorrhage, coagulopathy and/or hypotension. Here we propose using small molecules (enzyme inhibitors) to generically inhibit the functional activity of these toxins. Our proof of concept data demonstrates that enzyme inhibitors offer the potential to generically neutralise haemotoxic venoms (i.e. irrespective of snake species, unlike antivenom) both in vitro and in in vivo models of envenoming.
We will apply a variety of in vitro biochemical enzyme/functional assays to characterize the activity of ten medically important snake venoms (both crude and fractionated venom), before assessing their neutralisation by a panel of enzyme inhibitors. Because of venom variation, we will require a combination of inhibitors to effect cure, and thus we will next design mixtures of effective inhibitors before re-assessing venom neutralisation. Finally, the two optimal mixtures will be used in in vivo studies to assess their preclinical efficacy against each of the venoms in comparison with existing commercial antivenoms.
We anticipate that our inhibitory mixtures will exhibit superiority over antivenom by neutralising venom lethality irrespective of the snake species tested and at lower therapeutic doses, resulting in a single, generic, therapy for treating haemotoxic snakebites worldwide. The resulting inhibitory mixture would be eminently translatable into a commercial product by offering economies of scale, improved safety profiles and reduced treatment costs.

The potential beneficiaries of this project are diverse, and are outlined in detail below.

Snakebite victims: We designed this therapeutic project to ultimately save the lives and livelihoods of the medically-neglected snakebite victims of the tropical regions of the world.

Commercial sector: The project will deliver the design of two inhibitory mixtures with worldwide efficacy against haemotoxic snake venoms. We anticipate that these new therapeutics will have unparalleled dose efficacy and will be eminently affordable to impoverished snakebite victims. In combination, these characteristics provide significant, previously missing, incentives for commercial manufacturers. As outputs are delivered during the course of this project we will pursue translation grants with industrial manufacturing companies with a proven record of delivering quality treatments for tropical diseases: e.g. Sanofi Pasteur, Pfizer, etc. We anticipate these therapies being likely to attract significant financial gains through (i) collaborative translation grant funds and (ii) government contracts. This will drive capacity strengthening.

Government Policy Makers: Efficacious and safe snakebite treatments retailed at affordable prices (relative to existing polyspecific antivenoms) will represent the most cost-effective investment in snakebite management ever available to governments in tropical regions of the world - enabling the dislocation of the vicious circle that has limited the commitment of manufacturers and public health authorities to increase the availability of antivenoms to this region. The health benefits are likely to translate to political advances, both nationally and internationally.

International Health Agencies: The current lack of (i) solid government demand/action and (ii) therapeutic tools has limited WHO's ability to interest other agencies in resolving the world's snakebite burden. The tools provided from this project, coupled with new demand for effective and safe snakebite treatments by African and Asian governments, are likely to attract the financial and health-promoting support of agencies such as DFID, USAID, EU and the Bill & Melinda Gates Foundation - providing them with additional evidence of cost-effective success of using public and philanthropic funds to instigate substantial health benefits in communities whose snakebite victims have been neglected for so long.

Clinicians: Snakebite clinicians are currently faced with serious treatment challenges, as existing antivenom therapies are restricted in efficacy to those snake species used for their manufacture. However, for many patients the snake species responsible for the bite will be unknown, and as no clinical diagnostic tools exist, antivenom choice is problematic. Our 'generic' anti-haemotoxicity snakebite treatment will absolve these issues by providing the clinician with confidence to deliver a single efficacious treatment when patients present with haemotoxic snakebite.

Academics: A wide variety of different academics will benefit from the outputs of this project. These include toxinologists, biochemists, haematologists, cardiovascular biologists, etc. Please refer to 'Academic Beneficiaries' for further details.

Project staff and students: The project will enable UK staff to improve their technical skills portfolio and subsequent marketability. The project's translational nature will provide many opportunities for information exchange and scientific discussion in various cultural and scientific environments - all empowerments for young scientists.

Public Sector: The project's potential to have such a positive impact upon the lives of deeply disadvantaged tropical populations will have wide appeal to the lay public and media - by demonstrating how UK government funds benefit human health. We will incorporate our progress within our public engagement activities defined in the 'Communications Plan' and 'Pathways to Impact'.