Replacement in vivo preclinical models to substantially refine and reduce severe protocols used in snakebite envenoming research

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Snakebite envenoming is a Neglected Tropical Disease that annually kills 85,000-130,000 and maims >400,000 people living in the world's most disadvantaged communities. Globally, all snakebite envenoming therapies, both existing and in development, are assessed for efficacy using a 40-year-old, WHO-endorsed model of "neutralisation of venom leathality".Whilst simple, this assay does not accurately reflect human envenoming and requires large numbers of mice (n=25/experiment/venom) to be subjected to highly distressing severe procedures. The objective of this proposal is to develop and validate a new in vivo model of envenoming which will have a maximum severity limit of 'moderate', require fewer mice/experiment and ultimately provide more pathologically relevant data on the efficacy of current and future envenoming therapies.This will be achieved through modifying the route of envenoming to mimic that of human envenoming. This will be achieved through local venom delivery routes frequently reflective of envenoming in humans, for example. Intradermally (i.d.), subcutaneously (s.c.) or intramuscularly (i.m.), in typical sites of human envenoming, for example on the limbs. Mice will be observed for development of signs of systemic envenoming over 12 hours. One of the key aspects of this model is that once established it will have a maximum "moderate' severity limit. During development, blood will be drawn routinely to monitor indicators of envenoming, aPTT, PT, thrombin-antithrombin levels, and specific acute phase and inflammatory markers. These biomarkers will be assessed in comparison to non-invasive murine vital signs (e.g., heart rate, respiration rate, etc) to establish suitable, reliable 'moderate' humane endpoints. Once the envenoming model has been established and validated, we will further develop a 'gold standard' comparator model of envenoming therapies, similar to models widely used in the preclinical testing of other therapeutics. Therapies to be examined will be compared to 0.5, 1. Or 2.5 x the dose of a "gold standard' therapy (i.e., one with known clinical or preclinical efficacy, either antivenom or alternative) which provides the minimum anticipated biological effect level (MABEL). This assay will reduce the numbers of mice required/assay by 40%, whilst providing directly comparative scalable dose data. Once established, we will train end users in collaborating snakebite envenoming laboratories in Kenya and India in the new model. These laboratories will then independently replicate our experiments to ensure reproducibility and reliability of the model.