Human NeuromuscularJunction Platform to Evaluate Botulinum Toxin Potency

Project Description: Botulinum toxin (Botox) has been proven clinically effective in treating symptoms for conditions including vocal muscle spasticity, strabismus, hyperactive urinary bladder, excessive sweating, and chronic migraine as well as for numerous cosmetic applications. The measurement of potency for commercially available Botox is primarily through a mouse lethality bioassay (MLB). There are over 1 million animals sacrificed each year to establish the potency of clinical dosages of Botox. There are currently few established analytical or in vitro potency testing available and no pharmacodynamic (PD) or traditional pharmacokinetic (PK) studies have been performed on commercially marketed Botox. Hickman from UCF published the first dose response curve for BOTOX® in 2018 utilizing their patented neuromuscular junction (NMJ) model and this has been licensed and commercialized by Hesperos for preclinical disease modeling and toxicity applications. However, it has not been qualified by the FDA yet. Hesperos is a company founded by Hickman and Dr. Michael Shuler, who is considered the founder of the body-on-a-chip field. This proposal seeks to submit validation data to the FDA for qualification to establish a potency assay for Botox and to ensure the capacity to test a significant number of commercial lots on a weekly basis. The Hickman paper described the construction of a functional neuromuscular junction (NMJ) system utilizing human cells that enabled the establishment of dose response curves for BOTOX® from Allergan, as well as curare and bungarotoxin. The system is composed of two chambers that are electrically and fluidically isolated and connected by small tunnels through which the axons grow and innervate the skeletal muscle and each chamber can be separately stimulated and dosed (US Patents 8,835,168B2, 9,267,936B2 and 10,935,541). The first Aim will establish the high throughput NMJ system that will enable 80-100 Botox samples to be evaluated per week. However, once developed it will be straightforward to build additional systems to scale the testing capability to over 400 per week, if demand increases. The second Aim will validate the NMJ system with 10 different commercial Botox samples tested using the mouse lethality bioassay (MLB) for dose response to satisfy the in vivo comparison, and separately for the additional functional parameters. This data will be submitted to the FDA and then the system will be qualified first with blinded commercial samples from the FDA Toxicological Branch and then for in person evaluation by FDA staff in similar fashion to qualification of other facilities. This process, which has been developed in consultation with Drs. Sharma and Fitzpatrick at the FDA and Dr. Tagle from NCATS, will establish a route by which microphysiological systems available through CROs can be validated and qualified for all providers, which is confirmed in an email to support this as the test case. This is a crucial step for commercialization of these MPS models as over half of all preclinical research for pharmaceutical companies is performed by CROs and a regulatory pathway for MPS in CROs is not currently established.