Ward C. Tucker, Ph.D. - US grants

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are the most toxic substances known to humans causing respiratory failure upon poisoning. BoNT's lethality and ease of production has led to a category "A" bioterrorism agent designation for BoNT by the Department of Defense (DoD). Developing effective, post-exposure antagonists to BoNT is a top priority of the DoD. Despite their lethality, BoNTs have cosmetic and pharmaceutical applications and are currently FDA-approved for the treatment of glabellar lines (wrinkles), cervical dystonia, blepharospasm, cranial nerve VII disorders, and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Development of new BoNT-based therapies is hindered by the lack of assays to measure BoNT activity. Currently, the only widely accepted method of measuring BoNT activity involves injecting BoNT into mice and counting the number of mice that die after one or more days. Because this process takes days to complete, requires special facilities, and is extremely low-throughput, it makes antagonist screening or development of new BoNT-derived therapeutics prohibitively expensive and time-consuming. The overall objective of this Phase I proposal is to develop a unique technology for measuring BoNT activity in living cells. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-a cell-based assay is required to effectively measure BoNT activity. BioSentinel proposes to develop a cell-based assay that measures BoNT activity by using Forster resonance energy transfer (FRET) to monitor BoNT cleavage of SNARE proteins, the target of BoNT. BioSentinel will generate FRET-based reporters for measuring BoNT protease activity, identify cell lines that are highly sensitive to BoNT, generate stable cell lines that express the reporters, and investigate high- throughput instruments for monitoring those reporters in vivo. BioSentinel proposes to develop the assay into a high-throughput platform that will enable researchers and pharmaceutical companies to rapidly quantify BoNT preparations, perform quality control measures, screen for new BoNT-based therapies, and screen for BoNT antagonists. Because of increased pressure for animal-free, high-throughput assays, a cell-based assay for BoNT detection is widely desired by researchers and industry, and would open a gateway to development of new BoNT-based therapies. Public Health Relevance: Botulinum neurotoxins are extremely lethal bacterial toxins that attack the nervous system and are considered a significant bioterrorism threat. Botulinum neurotoxins are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. BioSentinel proposes to develop a high-throughput assay for measuring botulinum neurotoxin activity in living cells, providing a much needed platform for screening antagonists to BoNT and developing new BoNT-based therapies.

This Small Business Innovation Research (SBIR) Phase I project proposes to develop a universal, in vitro assay for determining the enzymatic activity of botulinum neurotoxin (BoNT)-based pharmaceuticals. BoNTs are used in many cosmetic and pharmaceutical applications due to their long lasting effects and specificity for motor neurons. Currently, the strength and quality of BoNT-based pharmaceuticals is assessed using a mouse bioassay where BoNT is injected into mice and a unit of activity depends on the rate or proportion of deaths. Beyond animal testing concerns, the mouse bioassay's inaccuracy and the lack of standardized protocols among manufacturers put patient safety and clinical outcomes at risk. The proposed technology will reduce animal use and provide a method to uniformly assess the activity of BoNT-based pharmaceuticals regardless of manufacturer, thus increasing patient safety and the likelihood of successful treatment.

The broader/commercial impacts of this research are increased patient safety, reduced animal testing, and the potential to enable point-of-use/point-of-care testing. The proposed technology could be used for decontamination testing at manufacturing sites, authenticity (against counterfeit drugs) testing at border control points, and in-office testing for high-dose procedures where toxin concentration is critical for clinical outcomes. The proposed technology would be the first of its kind on the commercial market. Other commercially available assays are not suitable for assessing BoNT-based pharmaceuticals because formulation stabilizers interfere with the assays. The proposed technology could be further adapted for testing BoNT activity in highly complex matrices extending the technology's utility into food, water, biodefense, and environmental testing.

This Small Business Innovation Research (SBIR) Phase II project proposes to complete development of a universal, in vitro assay for determining the enzymatic activity of botulinum neurotoxin (BoNT)-based pharmaceuticals. This assay, called BoTest Matrix A Assay, will enable standardized comparisons of different BoNT preparations, regardless of manufacturer. BoNTs are used in many different cosmetic and pharmaceutical applications due to their exquisite specificity for motor neurons and their long-lasting effects. Currently, the strength and quality of BoNT-based pharmaceuticals is assessed using a mouse bioassay where BoNT is injected into dozens of mice and a unit of activity depends on the rate or proportion of mouse deaths. Beyond the obvious concerns about animal testing, the variability of the mouse bioassay and the lack of suitable standards and uniform protocols among manufacturers put patient safety and clinical outcomes at risk. The proposed assay(s) will reduce the use of animals and will provide a method to uniformly assess the activity of BoNT-based pharmaceuticals, thus increasing patient safety and the likelihood of successful treatment.

The broader/commercial impacts of this research are improved procedures and protocols that would greatly increase patient safety while reducing animal testing. The BoTest Matrix Assay would be applicable to all stages of BoNT-based drug manufacturing, including quality control, quantification, stability testing, and decontamination testing at manufacturing sites?all applications that are currently performed with animal assays. In addition, the assay would be applicable for authenticity testing (against counterfeit drugs) at border control points and inoffice testing for high-dose procedures where toxin activity concentration is critical for clinical outcomes. The proposed technology would be also the first of its kind on the commercial market. Other commercially available assays for detecting BoNT activity are not suitable for assessing BoNT-based pharmaceuticals, because the stabilizers added to the pharmaceutical formulations interfere with those assays. The proposed technology could be further adapted for testing BoNT activity in highly complex matrices (e.g., blood, food, water). Thus, the assay's utility would extend into food, biodefense, and environmental testing.

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are the most toxic substances known to humans, causing respiratory failure upon intoxication. Despite their lethality, BoNTs have clinical health and cosmetic applications and are currently FDA-approved for treating cervical dystonia, blepharospasm, cranial nerve VII disorders, glabellar lines (wrinkles), and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long-lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Quality control and quantification of BoNT-based therapies are currently achieved using the mouse bioassay which involves injecting samples into mice and counting the number of mice that die one or more days post-injection. Each manufacturer of a BoNT-based pharmaceutical uses a unique protocol for the mouse bioassay;the result is non-standardized units of activity. This non-uniformity has led to confusion about manufacturer-to-manufacturer activity and equivalent dosing, thus putting patients at risk. In addition, the mouse assay is low-throughput, time- and labor-intensive, and sacrifices large numbers of animals. The overall objective of this Phase I proposal is to validate BioSentinel's unique cell-based assay for BoNT/A detection against the mouse bioassay. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-only a cell-based assay can effectively measure BoNT activity with the same fidelity as the mouse bioassay. BioSentinel, in collaboration with Metabiologics, proposes to optimize BioSentinel's cell-based assay for BoNT/A detection, generate a BoNT/A reference standard, determine whether the assay offers improved precision compared to the mouse bioassay, and, using thermally stressed BoNT/A samples, determine whether the cell-based assay accurately measures the activity of BoNT/A samples with the same fidelity as the mouse bioassay. BioSentinel's validated cell-based assay will enable researchers and manufacturers to quantitate BoNT/A preparations, perform high-throughput screens for BoNT/A inhibitors, and develop new BoNT-based therapies using an animal-free, cost-effective system. This assay will also satisfy the requirements of SBIR solicitation PA-09-179, "Development of In-Vitro Assays to Assess the Potency of Botulinum Neurotoxin Type A." PUBLIC HEALTH RELEVANCE: Botulinum neurotoxins are extremely lethal bacterial toxins that are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. Currently, botulinum neurotoxin-based therapies are quantified using the mouse bioassay where animal death is the assay endpoint. BioSentinel proposes to validate its animal-free, cell-based assay for detecting botulinum neurotoxin against the mouse bioassay with the goal of replacing the mouse bioassay for quality control and quantification of botulinum neurotoxin-based products.