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

毒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，并使生活在世界上最贫困社区的400，000人致残。在全球范围内，所有的蛇咬伤envenoming疗法，现有的和在开发中，使用40岁的，WHO认可的“毒液leathality的中和”模型的疗效进行评估。虽然简单，这种测定不能准确地反映人类envenoming，需要大量的小鼠（n=25/实验/毒液）进行高度痛苦的严重程序。本提案的目的是开发和验证一种新的体内毒液蛰入模型，该模型的最大严重程度限制为“中度”，需要较少的小鼠/实验，并最终提供有关当前和未来毒液蛰入疗法疗效的更多病理学相关数据，这将通过修改毒液蛰入途径以模拟人类毒液蛰入途径来实现。例如，这将通过经常反映人类中毒的局部毒液递送途径来实现。皮内（i.d.），皮下（s.c.）或肌内（i.m.），在人类中毒的典型部位，例如四肢上。将在12小时内观察小鼠是否出现全身性毒液中毒体征。这一模式的一个关键方面是，一旦建立，它将有一个最大的“中等”严重性限制。在开发过程中，将常规采血以监测毒液中毒、aPTT、PT、凝血酶-抗凝血酶水平和特定急性期和炎症标志物的指标。这些生物标志物将与非侵入性鼠生命体征（例如，心率、呼吸率等）以建立合适的、可靠的“适度”人道终点。一旦建立并验证了envenoming模型，我们将进一步开发envenoming疗法的“金标准”比较模型，类似于广泛用于其他疗法临床前测试的模型。将检查的治疗方法与0.5、1进行比较。或2.5倍“金标准”疗法的剂量（即，具有已知临床或临床前功效的抗蛇毒血清或替代物），其提供最低预期生物效应水平（美宝）。该试验将使每次试验所需的小鼠数量减少40%，同时提供直接比较的可扩展剂量数据。一旦建立，我们将在肯尼亚和印度的合作蛇咬伤实验室培训最终用户。这些实验室将独立复制我们的实验，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.