Sarathy Jansy P, Liang Hsin-Pin Ho, Weiner Danielle, Gonzales Jacqueline, Via Laura E, Dartois Véronique
Public Health Research Institute Centre, New Jersey Medical School, Rutgers;
Public Health Research Institute Centre, New Jersey Medical School, Rutgers.
J Vis Exp. 2017 May 8(123):55559. doi: 10.3791/55559.
The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.
结核病的根除需要能够穿透复杂肺部病变多层结构的药物治疗方案。药物在空洞和病变的干酪样核心中的分布尤为关键，因为这些部位存在耐药物细菌亚群，通常也被称为持留菌。现有的测量药物在结核病变中渗透的方法涉及成本高昂且耗时的体内药代动力学研究，并结合生物分析或成像技术。由于药物与干酪样大分子的结合会阻碍药物分子通过干酪样物质的被动扩散，因此有人提出体外测量药物与干酪样大分子的结合作为此类技术的替代方法。快速平衡透析是进行血浆蛋白和组织结合研究的快速可靠系统。在本方案中，我们使用快速平衡透析（RED）装置来测量药物与从结核感染兔子的病变和空洞中切除的干酪样物质匀浆的结合。该方案还描述了如何从负载脂质的THP-1巨噬细胞生成替代基质以替代干酪样物质。这种干酪样物质/替代物结合测定是结核病药物发现中的一项重要工具，并且可以进行调整以帮助研究其他疾病引起的病变或脓肿中的药物分布。
