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

蛇咬伤是一种被忽视的热带疾病，每年造成85,000-130,000人死亡，并使生活在世界最贫困社区的1,000,000人致残。在全球范围内，所有毒蛇咬伤疗法，无论是现有的还是正在开发的，都是使用一种已有40年历史、世卫组织认可的“中和毒液致死率”模型来评估疗效的。虽然简单，但该试验不能准确反映人类的毒性，并且需要大量小鼠（n=25/实验/毒液）进行非常痛苦的严格程序。本提案的目标是开发和验证一种新的内毒素体内模型，该模型的最大严重程度限制为“中度”，需要更少的小鼠/实验，并最终提供有关当前和未来内毒素治疗疗效的更多病理相关数据。这将通过修改环境的路径来模拟人类的环境来实现。例如，这将通过经常反映人类毒液的局部毒液输送途径来实现。皮肤内（i.d），皮下（s.c）或肌肉内（i.m），在典型的人体环境部位，如四肢上。在12小时内观察小鼠是否出现全身出血的迹象。该模型的一个关键方面是，一旦建立，它将有一个最大的“中等”严重性限制。在发育过程中，将常规抽血监测内源性指标，aPTT， PT，凝血酶-抗凝血酶水平，以及特定的急性期和炎症标志物。这些生物标志物将与非侵入性小鼠生命体征（如心率、呼吸率等）进行比较评估，以建立合适、可靠的“适度”人道终点。一旦环境模型建立并得到验证，我们将进一步开发环境疗法的“金标准”比较模型，类似于其他疗法临床前测试中广泛使用的模型。要检查的治疗方法将比较0.5,1。或2.5倍的“金标准”疗法（即具有已知临床或临床前疗效的疗法，无论是抗蛇毒血清还是替代疗法），提供最低预期生物效应水平（MABEL）。该试验将使每次试验所需的小鼠数量减少40%，同时提供可直接比较的可扩展剂量数据。一旦建立，我们将培训最终用户在肯尼亚和印度的新模式下与毒蛇咬伤实验室合作。然后，这些实验室将独立地重复我们的实验，以确保模型的可重复性和可靠性。

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.