Investigation of synergy between Rifampin and SQ109, a new anti-TB drug candidate

描述(申请人提供)：SQ109是一种新的抗结核病候选药物，目前正在进行人体Ia期临床试验。SQ109是从63,238个化合物库中挑选出来的，因为它对感染的巨噬细胞和实验动物中的结核分枝杆菌具有高效的杀灭作用，对培养的哺乳动物细胞具有相对较低的细胞毒性，对药物敏感和耐药的结核分枝杆菌都有活性，并且具有类药物的药代动力学和药效学特征。任何新的抗结核药物都将与其他药物联合使用，以防止出现耐药结核分枝杆菌。SQ109是一种具有独特属性的新药化合物，它如何与现有的一线抗结核药物相互作用对我们来说至关重要，因为这些知识使我们能够设计最有效的治疗组合，并避免新组合疗法的任何拮抗副作用。我们在体外试验和体内小鼠结核病模型中研究了SQ109与一线抗结核药物的不同组合的药物相互作用。在体外生长抑制实验中，我们发现SQ109优先与RIF协同作用，并且当与异烟肼和RIF联合使用时，显示出增强的杀菌活性。这些发现对于优化药物联合疗法在未来的临床试验中进行测试具有重要意义。在这个探索性的R21拨款申请中，我们将：1.评估SQ109是否在结核分枝杆菌体内代谢，以及联合应用RIF如何影响SQ109的代谢。如果存在SQ109代谢物，我们将用质谱仪研究它们的结构，并将它们与人类CYP产生的SQ109代谢物进行比较。如果代谢物相同，我们将制备和纯化足够数量的代谢物，并在体外测试它们对结核分枝杆菌的效力。2.检测RIF诱导的结核分枝杆菌中单个CYP的表达，以确定RIF诱导的候选SQ109激活子。3.构建候选CYP(S)的结核分枝杆菌基因敲除突变体，并在CYP基因敲除突变体中重新考察SQ109与RIF相互作用的协同作用。 公共卫生相关性：标准结核病(TB)治疗最令人头疼的问题之一是疗程长(6-9个月)和药物的毒性。这种长期的治疗方案导致高水平的不依从性，无效的药物浓度导致多药耐药(MDR)结核分枝杆菌的出现。据估计，每年新增耐多药结核病病例40万例，世界卫生组织(WHO)估计，全球有5000万人感染了一种耐药结核病菌株。结核分枝杆菌耐多药菌株导致死亡率增加，未经治疗的耐多药结核病病例的病死率可高达70%。在艾滋病毒和营养不良造成的继发性免疫抑制的背景下，耐多药结核杆菌感染者治愈的可能性变得更小，这些情况在发展中国家非常猖獗。 由于现有疗法的失败，世界迫切需要能够缩短疗程并杀死耐多药结核分枝杆菌的新药。Sequella，Inc.成立于1997年，专注于开发结核病的新诊断工具和新药。我们最成功的项目之一是开发SQ109，这是一种新的抗结核病候选药物，目前正在进行Ia期临床试验。从63,238个化合物库中选择SQ109作为我们的主要候选药物是因为它对感染的巨噬细胞和实验动物中的结核分枝杆菌具有高效的杀灭作用，对培养的哺乳动物细胞具有相对较低的细胞毒性，对药物敏感和耐药的结核分枝杆菌都具有活性，并且具有类药物的药代动力学和药效学特征。重要的是，当与利福平(RIF)+异烟肼(INH)+吡嗪酰胺(PZA)联合使用时，SQ109能够增加细菌杀灭并将时间减少至少25%，以达到与标准药物方案RIF+INH+PZA+乙胺丁醇(EMB)相同的CFU水平。 任何新的抗结核药物都将与其他药物联合使用，以防止出现耐药结核分枝杆菌。SQ109是一种具有独特属性的新药化合物，它如何与现有的一线抗结核药物相互作用对我们来说至关重要，因为这些知识使我们能够设计最有效的治疗组合，并避免新组合疗法的任何拮抗副作用。 在这项R21探索性拨款申请中，我们建议研究可能有助于SQ109和RIF在结核分枝杆菌中协同作用的几个假设之一：(1)SQ109在结核分枝杆菌中易于代谢，(2)RIF能够诱导一个或多个结核分枝杆菌CYP样蛋白的表达，以及(3)RIF诱导的CYP参与SQ109的代谢。任务1和任务2是两个独立的学习领域，它们通过任务3联系在一起。在本应用程序中，我们依次概述了这三个任务的实验设计。此外，如果获得的数据不支持我们的假设，我们将为这一有趣的协同效应的研究提供替代方向。

DESCRIPTION (provided by applicant): SQ109 is a new anti-TB drug candidate that is currently undergoing Phase Ia Clinical Trials in humans. SQ109 was selected out of the library of 63,238 compounds, because it is highly efficacious in killing M. tuberculosis within infected macrophages and in experimental animals, has relatively low cytotoxicity in cultured mammalian cells, has activity against both drug-susceptible and drug-resistant M. tuberculosis, and has drug-like pharmacokinetic and pharmacodynamic profiles. Any new anti-TB drug will be used in combination with other drugs in order to prevent the emergence of drug resistant M. tuberculosis. How SQ109, a new drug compound with unique attributes, interacts with existing first line anti-TB drugs is crucial for us to understand, as this knowledge enables us to design the most efficacious therapeutic combination and to avoid any antagonistic side effects of a new combination therapy. We investigated the drug interaction in various combinations of SQ109 with the first line anti-TB drugs in both in vitro assays and in vivo murine models of TB. We found that SQ109 preferentially synergized with RIF in in vitro growth inhibition assays and demonstrated enhanced bactericidal activity in mice when used in combination with INH and RIF. These findings are significant for optimizing the drug combination therapies to be tested in future clinical trials. In this exploratory R21 grant Application we will: 1. Assess whether SQ109 is metabolized within M. tuberculosis and how co-administration of RIF influences SQ109 metabolism. If SQ109 metabolites exist, we will study their structures by mass spectrometry and will compare them to the SQ109 metabolites generated by human CYP. If the metabolites are the same, we will prepare and purify them in sufficient quantities and test them against M. tuberculosis for potency in vitro. 2. Determine the expression of individual CYP in RIF-treated M. tuberculosis in order to identify candidate SQ109 activators that are induced by RIF. 3. Generate M. tuberculosis knock out mutants of the candidate CYP(s) and re-examine the synergy of interaction between SQ109 and RIF in the CYP knockout mutants. Public Health Relevance: One of the most vexing problems with standard tuberculosis (TB) therapy is the lengthy treatment course (6-9 months) and toxicity of the drugs in the regiment. This prolonged therapeutic regimen results in a high level of noncompliance, and ineffective concentrations of drugs lead to the emergence of multidrug resistant (MDR) M. tuberculosis. There are an estimated 400,000 new cases of MDR TB each year, and the World Health Organization (WHO) estimates there are 50 million individuals worldwide infected with a resistant strain of TB. MDR strains of M. tuberculosis cause increased mortality, and the fatality rate of untreated cases of MDR TB can be as high as 70%. The likelihood of cure for MDR M. tuberculosis-infected individuals becomes even less in the setting of secondary immunosuppression caused by HIV and malnutrition, conditions that are rampant in developing countries. Because of the failures of the existing therapies, the world desperately needs new drugs that can shorten treatment duration and kill MDR M. tuberculosis. Sequella, Inc., founded in 1997, focuses its R&D on developing new diagnostic tools and new drugs for TB disease. One of our most successful projects is the development of SQ109, a new anti-TB drug candidate that is currently undergoing Phase Ia Clinical Trials. SQ109 was selected as our lead drug candidate out of the library of 63,238 compounds, because it is highly efficacious in killing M. tuberculosis within infected macrophages and in experimental animals, has relatively low cytotoxicity in cultured mammalian cells, has activity against both drug-susceptible and drug-resistant M. tuberculosis, and has drug-like pharmacokinetic and pharmacodynamic profiles. Importantly, when used in combination with rifampin (RIF) + isoniazid (INH) + pyrazinamide (PZA), SQ109 is able to increase bacterial killing and reduce the time by at least 25% to achieve the same level of CFU as the standard drug regimen, RIF + INH + PZA + ethambutol (EMB) in a mouse model of TB. Any new anti-TB drug will be used in combination with other drugs in order to prevent the emergence of drug resistant M. tuberculosis. How SQ109, a new drug compound with unique attributes, interacts with existing first line anti-TB drugs is crucial for us to understand, as this knowledge enables us to design the most efficacious therapeutic combination and to avoid any antagonistic side effects of a new combination therapy. In this R21 exploratory grant application, we propose to investigate one of the several hypotheses that may contribute to the synergistic interaction between SQ109 and RIF in M. tuberculosis: (1) SQ109 is readily metabolized in M. tuberculosis, (2) RIF is able to induce the expression of one or more M. tuberculosis CYP- like proteins, and (3) CYP induced by RIF is involved in SQ109 metabolism. Tasks 1 and 2 are two independent areas of study, which are linked by task 3. In this application, we outline the experiment design for all three tasks in sequence. Further, we provide alternative directions for the study of this interesting synergy if the data obtained do not support our hypothesis.