Validation of preclinical assays with clinical data to evaluate the safety of estrogenic botanical dietary supplements

摘要 超过一半最畅销的植物性膳食补充剂(BDS)是销售给围产期和产后的 更年期女性。约70%的这一年龄段的女性报告使用过BDS来管理 症状或治疗疾病。本项目的重点是解决这些流行的BDS的安全性。我们 假设大多数女性用来治疗更年期症状的BDS是安全的，但一些植物性药物 可能通过调节雌激素的生物合成和代谢而带来风险。通过对以下项目进行事后分析 来自我们完成的黑升麻(总状花序Actaea)I期和II期临床试验的尿液和血清， 啤酒花(Humulus Lupulus)和红三叶草(Trifolium Pratense)，我们将确认可能对激素的调节 级别。两种体外细胞测试将被验证以模拟雌激素的生物合成和雌激素代谢。我们的 三个具体目标包括：1)发展快速液相色谱-串联质谱仪 提高雌激素及其含量测定灵敏度和选择性的分析方法 代谢物。目标是最大限度地减少样品准备和分析的时间，并且只利用商业上可用的 衍生化试剂和标准仪器。这些方法将被用于血清的分析， 尿液和细胞培养液样本。2)建立植物对雌激素调节的体外检测方法 生物合成和雌激素代谢。两种广泛用于研究实验室调控的细胞系 将根据它们的生物学相关性和可用性来评估它们的用途。3)对雌激素及其 服用BDS的妇女临床样本中的代谢物。这将使我们能够确定显著的调制 激素水平，并用于验证Aim 2的体外分析。 我们关于雌激素调节在BDS产品安全性中的重要性及其作用机制的假设， 而经过验证的体外试验将提供一种快速而准确的临床前机制来评估BDS的安全性 成本适中。

ABSTRACT More than half of the top selling botanicals dietary supplements (BDS) are marketed to peri- and post- menopausal women. Approximately 70% of women in this age group reported having consumed BDS to manage symptoms or treat disease. The focus of this project is to address the safety of these popular BDS. We hypothesize that most BDS used by women to manage menopausal symptoms are safe, but some botanicals might pose risk through modulation of estrogen biosynthesis and metabolism. By carrying out post-analyses of urine and serum from our completed Phase I and Phase II clinical trials of black cohosh (Actaea racemosa), hops (Humulus lupulus) and red clover (Trifolium pratense), we will confirm possible modulation to hormone levels. Two in vitro cell assays will be validated to model estrogen biosynthesis and estrogen metabolism. Our three Specific Aims will include the following: 1) Develop fast liquid chromatography-tandem mass spectrometry analytical methods with improved sensitivity and selectivity for the quantitation of estrogens and their metabolites. The goal is to minimize sample preparation and analysis time and utilize only commercially available derivatization reagents and standard instrumentation. The methods will be validated for the analysis of serum, urine, and cell media samples. 2) Establish in vitro assays for identifying botanical modulations to estrogen biosynthesis and estrogen metabolism. Two cell lines widely used in research laboratories for regulatory purposes will be evaluated for their biological relevance and availability. 3) Quantify estrogens and their metabolites in clinical samples of women taking BDS. This will enable us to determine significant modulations to hormone levels and be used to validate the in vitro assays from Aim 2. Together, the clinical data will answer our hypothesis regarding the importance of estrogen modulation in BDS product safety and mechanism of action, and the validated in vitro assays will provide a fast and accurate preclinical mechanism to assess BDS safety at modest cost.