URINALYSIS FOR PATIENT COMPLIANCE TO CAM THERAPY

描述(申请人提供)：患者对CAM治疗依从性的尿检：近年来，随着人们对现代药物的日益不满，以及对健康生活的渴望，补充和替代药物(CAM)的使用急剧增加。然而，需要对CAM方法进行严格的测试，以证明其有效性，然后才能在常规实践中接受它。只有患者遵守治疗方案，门诊试验才能奏效。目前，依从性的评估是通过患者报告、药片计数和电子监测设备进行的。据估计，在美国，每年因不遵守条约而造成的财政负担高达1000亿美元。我们在这项应用中的目标是开发一种使用尿液分析来监测患者对CAM技术的依从性的技术。为此，我们建议开发一种快速、实时的检测方法，以确定体液中具有生物活性的CAM制剂成分的有效性。该检测是基于体外生物发光成像系统。最终，我们计划开发一种非侵入性尿液分析试剂盒。一种关键的细胞因子，参与控制许多正常的细胞和组织过程，包括免疫和炎症反应，细胞生长和凋亡，是转录因子NF-kappaB。更重要的是，核因子-kappaB在多种疾病状态下持续活跃，并以潜伏的、不活跃的、与I-kappaB结合的复合体形式存在于细胞质中。I-kappaB是一个由3个亚基组成的复合体。在细胞外信号的刺激下，I-kappaB的α亚基被靶向磷酸化，随后是泛素化和蛋白酶体降解。I-kappaBalpha的磷酸化将其从核因子-kappaB复合体中释放出来，然后未被屏蔽的核因子-kappaB复合体可以进入细胞核激活靶基因的表达。已知的钙调素制剂，如姜黄素，可以抑制I-kappaB的磷酸化，从而抑制NFkappaB的激活，从而抑制病理生理过程。 该项目的主要目标是开发敏感的、非侵入性的试剂盒和分析方法，用于检测生物活性的CAM试剂、它们的成分或体液中的代谢物，如血液或尿液，作为评估遵守良好描述的临床试验方案的一种方式。为了实现这一目标，我们建议验证一种使用I-kappaB稳定化的体外生物检测系统，以确定常见CAM试剂在尿中的代谢物数量(非侵入性程序)，最终开发试剂盒和消耗品。第一阶段的工作将建立检测和测量CAM试剂的原则证明，例如姜黄、黑树莓和西兰花，这些都是有效的抗炎剂，在I-kappaB降解方面的效果。I-kappaB是参与炎症途径的关键因子，对NFkappaB的活性起着至关重要的作用。我们相信，这项技术将增强和补充现有的高效液相/质量分光光度技术，在临床试验前、期间和之后准确评估CAM试剂的使用。预计这种I-kappaB降解和测量系统的推出将促进经验证的CAM试剂的开发，供消费者、患者、行业、监管机构和临床研究人员使用。此外，这种GEM工具将使研究和商业实体能够在体外和体内进行分子和功能研究，并在补充和替代医学领域提供令人兴奋的新机会。

DESCRIPTION (provided by applicant): URINALYSIS FOR PATIENT COMPLIANCE TO CAM THERAPY: In recent years, with increasing dissatisfaction with modern medicines coupled with a desire for healthy living, there has been a dramatic increase in the use of complementary and alternative medicines (CAM). However, rigorous testing of CAM methods is needed to prove efficacy before it can be accepted in regular practice. Outpatient trials can only work if patients adhere to the therapeutic protocol. Currently, assessment of adherence is done by patient reports, pill counts and with electronic monitoring devices. The financial burden of non-adherence is estimated to cost $100 billion each year in the United States. Our goal in this application is to develop a technique to monitor patient adherence to CAM techniques using urinalysis. Towards this, we propose to develop a rapid, real-time assay to determine the effectiveness of constituents of biologically active CAM agents in body fluids. The assay is based on an in vitro bioluminescence imaging system. Ultimately, we plan to develop a non-invasive urinalysis kit. A critical cellular factor that is involved in controlling many normal cellular and organismal processes, including immune and inflammatory responses, cellular growth, and apoptosis is the transcription factor NF-kappaB. Of greater significance is that NF-kappaB is continually active in numerous disease states and is present as a latent, inactive, I-kappaB-bound complex in the cytoplasm. I-kappaB is a complex of 3 subunits. Following stimulation by an extracellular signal, the alpha subunit of I-kappaB is targeted for phosphorylation followed by ubiquitination and proteosomal degradation. Phosphorylation of I-kappaBalpha releases it from the NF-kappaB complex and the unmasked NF-kappaB complex can then enter the nucleus to activate target gene expression. CAM agents, such as curcumin, are known to inhibit I-kappaB phosphorylation, thereby inhibiting NFkappaB activation and suppressing pathophysiological processes. The primary objective of this project is to develop sensitive, non-invasive kits and assays for use in the detection of biologically active CAM agents, their constituents or metabolites in body fluids, such as blood or urine, as a way of assessing adherence to well-described clinical trial protocols. Towards this goal, we propose to validate an in vitro biological assay system using I-kappaB stabilization to determine the amount of metabolites of common CAM agents in urine (non-invasive procedure), to ultimately develop reagent kits and consumables. The Phase I effort will establish proof-of-principle for the detection and measurement of the CAM agents, such as turmeric, black raspberries and broccoli, potent anti-inflammatory agents, in the efficacy of I-kappaB degradation. I-kappaB is a critical factor involved in the inflammatory pathway and plays a vital role in NFkappaB activity. We believe that this technology will enhance and complement existing HPLC/mass spectrophotometry techniques in the accurate assessment of the use of CAM agents-before during and post clinical trials. It is anticipated that the availability of this I-kappaB degradation and measurement system will promote the development of validated CAM agents for use by consumers, patients, industry, regulatory agencies, and clinical researchers. In Addition, this GEM tool will enable both the research and commercial entities to perform molecular and functional in vitro and in vivo studies and offer exciting new opportunities in the field of complementary and alternative medicine.