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

摘要

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.