Rapid in vitro substrate assay for the multi-drug resistance p-glycoprotein

多药耐药 p-糖蛋白的快速体外底物测定

• 批准号: 7547045
• 负责人: DONALD Lee MELCHIOR
• 金额: $ 50.46万
• 依托单位: GLSYNTHESIS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-19 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7547045
• 关键词: ABCB1 gene; ATP phosphohydrolase; American; Award; Binding; Biological Assay; Biological Models; Biotechnology; Bisbenzimide; Blood - brain barrier anatomy; Carrier Proteins; Cells; Computer software; Contractor; Data Analyses; Development; Diffusion; Direct Costs; Disease; Drug Efflux; Drug Industry; Drug Transport; Educational workshop; Epithelial Cells; Fee-for-Service Plans; Foundations; Glycoproteins; Goals; Grant; Guidelines; Housing; Human; In Vitro; Intestines; Knowledge; Link; Lipid Bilayers; Lipids; Liposomes; Marketing; Measures; Membrane; Membrane Lipids; Membrane Proteins; Metabolic Pump; Methodology; Methods; Micelles; Modification; Molecular Biology; Multi-Drug Resistance; Oral; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phase II Clinical Trials; Placenta; Predisposition; Production; Property; Protein Chemistry; Proteins; Pump; Reagent; Research; Research Contracts; Robotics; Science; Scientist; Staging; System; Techniques; Technology; Testing; Time; Tissues; Validation; Verapamil; Vinblastine; absorption; base; chemotherapy; commercialization; design; drug candidate; drug discovery; efflux pump; high throughput screening; in vitro Assay; inhibitor/antagonist; interest; nanoparticle; novel; reconstitution; scale up; small molecule; success; tool; uptake

DESCRIPTION (provided by applicant): The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, drug efflux proteins such as p-glycoprotein (pgp) actively pump hydrophobic drugs away from target tissues and are linked to low oral absorption and multidrug resistance in chemotherapy. Protein pumps are of increasing interest to the pharmaceutical industry, most importantly based on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate or inhibitor of pgp. Current pgp assays are cumbersome, expensive and unreliable. Our overall goal is to prepare and validate a novel assay reagent - Fluorosome-trans-pgp - which capitalizes on the Fluorosome Technology that we have developed for measuring passive permeability of small molecules through lipid membranes. Fluorosome-trans-pgp will provide a direct, reliable, simple, rapid and inexpensive assay to determine if a drug is a pgp substrate or inhibitor. Toward this goal, in phase I of this project we have: cloned and expressed the human pgp construct containing a 10His tag in HEK293 cells. isolated and purified the protein "pgp 10His" in lipid micelles. demonstrated verapamil-stimulated ATPase activity in the purified pgp 10His lipid micelles. reconstituted the purified pgp 10His into liposome membranes. developed the methodology for the manufacture and assay of Fluorosome-trans-pgp. validated the test systems that will be used to screen potential pgp substrates and inhibitors. Phase II of this project will bring the Fluorosome-trans-pgp assay to the state of an important commercial product. To complete this development, the Specific Aims of phase II are to: 1. complete Fluorosome-trans-pgp production and validation. 2. scale up Fluorosome-trans-pgp production to commercial levels. 3. demonstrate the utility of Fluorosome-trans-pgp with representative pgp substrates and inhibitors. 4. optimize the properties of the Fluorosome-trans-pgp system. 5. develop data analysis software. Our overriding goal in Phase II is to bring Fluorosome-trans-pgp and accompanying software to market. Successful completion of phase II will satisfyi the increasing need for a reliable and economical method to measure a drug's susceptibility to efflux by the pgp pump or to act as a pgp inhibitor. This project also lays the foundation for the design of Fluorosome-based systems to test for the susceptibility of drug candidates to other drug transport proteins. Markets include pharmaceutical and biotechnology companies, contract research organizations, and in-house fee-for service assays. This project completes the development of a test which determines if drugs will be extruded from their target tissue by biological pumps. The test thereby allows the pharmaceutical industry to evaluate, at an early stage, the suitability of drug candidates for continued development. The test is reliable, simple, rapid, inexpensive and amenable to robotics.

描述（由申请人提供）： 药物到达和穿透体内预期靶点的能力对其治疗疾病的成功至关重要。然而，药物外排蛋白，如p-糖蛋白（pgp），积极泵疏水性药物远离靶组织，并与低口服吸收和化疗的多药耐药性。蛋白质泵越来越受到制药行业的关注，最重要的是基于FDA指南的新草案，该指南要求了解候选药物是pgp的底物还是抑制剂。目前的pgp检测方法繁琐、昂贵且不可靠。我们的总体目标是制备和验证一种新型检测试剂-荧光体-反式-pgp -其利用了我们开发的用于测量小分子通过脂质膜的被动渗透性的荧光体技术。荧光体-反式-pgp将提供一种直接、可靠、简单、快速和廉价的检测方法来确定药物是否为pgp底物或抑制剂。为了实现这一目标，在本项目的第一阶段，我们已经：克隆并表达人pgp构建体含有10 His标签在HEK 293细胞。 在脂质胶束中分离并纯化了“pgp 10 His”蛋白。 证明维拉帕米刺激ATP酶活性的纯化的Pgp 10 His脂质胶束。 将纯化的Pgp 10 His重组到脂质体膜中。 开发了生产和测定荧光体-反式-Pgp的方法。 验证了将用于筛选潜在的pgp底物和抑制剂的测试系统。该项目的第二阶段将使荧光体-反式-Pgp测定达到重要商业产品的状态。为了完成这一发展，第二阶段的具体目标是：1。完成Fluorosome-trans-pgp的生产和验证。2.将荧光体-反式-Pgp生产规模扩大到商业水平。3.证明荧光体-反式-Pgp与代表性Pgp底物和抑制剂的效用。4.优化荧光体-反式-Pgp系统的性质。5.开发数据分析软件。我们在第二阶段的首要目标是将Fluorosome-trans-pgp和配套软件推向市场。II期的成功完成将满足日益增长的对可靠和经济的方法的需求，以测量药物对Pgp泵外排的敏感性或作为Pgp抑制剂。该项目还为基于荧光体的系统的设计奠定了基础，以测试候选药物对其他药物转运蛋白的敏感性。市场包括制药和生物技术公司、合同研究组织和内部收费服务分析。该项目完成了一项测试的开发，该测试确定药物是否会通过生物泵从其靶组织中挤出。因此，该测试允许制药行业在早期阶段评估候选药物是否适合继续开发。该测试是可靠的，简单的，快速的，廉价的，适合机器人。