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，每年导致8.5万至13万人死亡，40万人致残，生活在世界上最贫困的社区。在全球范围内，所有毒蛇咬伤的毒液疗法，无论是现有的还是正在开发的，都是使用一个已有40年历史的世界卫生组织认可的“中和毒液致命性”的模型来评估疗效的。虽然简单，但这种测试并不能准确地反映人类的毒液作用，并且需要大量的小鼠(n=25/实验/毒液)接受非常令人痛苦的严格程序。这项建议的目的是开发和验证一种新的体内致毒模型，该模型的最大严重程度限制为“中等”，需要较少的小鼠/实验，并最终提供更多关于当前和未来致毒治疗效果的病理学相关数据。这将通过修改致毒途径以模拟人类致病的途径来实现。例如，这将通过当地的毒液输送路线实现，这些路线经常反映出人类的毒液。皮内(I.D.)、皮下(S.C.)或肌肉内(I.M.)，在典型的人类毒化部位，例如在四肢上。将观察小鼠在12小时内是否出现全身毒液的迹象。这个模型的一个关键方面是，一旦建立，它将有一个最大的“中等”严重程度限制。在开发过程中，将常规抽取血液以监测蛇毒指标、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。这些生物标记物将与非侵入性小鼠生命体征(例如心率、呼吸频率等)进行比较，以建立合适、可靠的“适度”人性化终点。一旦毒化模型建立和验证，我们将进一步开发一种类似于其他疗法临床前试验中广泛使用的模型的毒化疗法的“黄金标准”比较器模型。将接受检查的疗法的剂量将与提供最低预期生物效应水平(Mabel)的“黄金标准”疗法(即，具有已知临床或临床前疗效的抗蛇毒血清或替代疗法)的0.5、1.或2.5倍剂量进行比较。这项测试将减少40%所需的小鼠数量，同时提供可直接比较的可扩展剂量数据。一旦建立，我们将培训终端用户在肯尼亚和印度合作的蛇咬伤毒液实验室的新模式。然后，这些实验室将独立复制我们的实验，以确保模型的重复性和可靠性。

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.