Validation of a cell-based assay for BoNT/A detection against the mouse bioassay

针对小鼠生物测定验证基于细胞的 BoNT/A 检测测定

• 批准号: 8061867
• 负责人: WARD C TUCKER
• 金额: $ 21.23万
• 依托单位: BIOSENTINEL, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8061867
• 关键词: Accounting; Adverse event; Animals; Bacteria; Bacterial Toxins; Binding; Biological; Biological Assay; Blepharospasm; Bontoxilysin; Botulinum Toxin Type A; Botulism; Cells; Cervical Dystonia; Cessation of life; Cleaved cell; Clinical; Clostridium; Collaborations; Confusion; Cosmetics; Cyclic GMP; Cytosol; Detection; Development; Disease; Dose; Drug Industry; End Point Assay; Ensure; Evaluation; FDA approved; Facial Nerve Diseases; Failure; Funding; Future; Gangliosides; Generations; Goals; Government; Health; Health Personnel; Human; Infection; Injection of therapeutic agent; Intoxication; Lead; Manufacturer Name; Measures; Mus; Muscle Contraction; Muscle Tension; Myalgia; Myopathy; Neurons; Pain; Paralysed; Patients; Persons; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Poison; Preparation; Procedures; Proteins; Protocols documentation; Quality Control; Reader; Reference Standards; Reporter; Research; Research Personnel; Respiratory Failure; Risk; Sampling; Serotyping; Small Business Innovation Research Grant; Specificity; Stress; System; Testing; Therapeutic Equivalency; Time; TimeLine; Toxin; Validation; Variant; Vesicle; Work; base; conventional therapy; cost effective; density; high throughput screening; improved; in vitro Assay; inhibitor/antagonist; meetings; nervous system disorder; neurotransmitter release; next generation; novel; receptor; receptor binding; reconstitution; research study; thermal stress; uptake; validation studies

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.

描述（由申请人提供）：肉毒杆菌神经毒素（BoNT）是人类已知的毒性最大的物质，中毒后引起呼吸衰竭。尽管bont具有致命性，但它在临床健康和美容方面仍有应用，目前fda已批准用于治疗颈肌张力障碍、眼睑痉挛、颅神经紊乱、眉间纹（皱纹）和皱纹。BoNT通过抑制引起肌肉收缩的神经元来缓解肌肉紧张。对于许多疾病，基于bont的治疗提供了显著和持久的疼痛减轻。BoNT对神经元的精细特异性和长时间的作用使其成为传统治疗失败的神经和肌肉疾病的主要候选药物。目前，基于bont的治疗的质量控制和定量是通过小鼠生物测定来实现的，该方法包括将样品注射到小鼠体内，并计算注射后一天或多天死亡的小鼠数量。每个基于bont的药物制造商都使用独特的小鼠生物测定方案；其结果是活动的非标准化单位。这种不一致性导致了对制造商与制造商之间的活动和等效剂量的混淆，从而使患者处于危险之中。此外，小鼠实验是低通量，时间和劳动密集型，并牺牲了大量的动物。该I期提案的总体目标是验证BioSentinel独特的基于细胞的BoNT/A检测方法与小鼠生物测定方法的对比。因为BoNT有三种活性——细胞受体结合和摄取、囊泡易位和靶标切割——只有基于细胞的测定才能有效地测量BoNT的活性，并且具有与小鼠生物测定相同的保真度。BioSentinel与metabolologics合作，提出优化BioSentinel基于细胞的BoNT/A检测方法，生成BoNT/A参考标准，确定该方法与小鼠生物测定法相比是否提供更高的精度，并使用热应激BoNT/A样品，确定基于细胞的测定方法是否准确地测量BoNT/A样品的活性，具有与小鼠生物测定法相同的保真度。BioSentinel经过验证的基于细胞的分析将使研究人员和制造商能够定量BoNT/A制剂，对BoNT/A抑制剂进行高通量筛选，并使用无动物、经济高效的系统开发新的基于BoNT/A的疗法。该试验也将满足SBIR招标文件PA-09-179的要求，“开发a型肉毒毒素神经毒素效力的体外试验”。