Comprehensive Identification of Astilbin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-Q-Exactive Mass Spectrometry.

BACKGROUND: Astilbin, a dihydroflavonoid compound widely found in plants, exhibits a variety of pharmacological activities and biological effects. However, little is known about the metabolism of this active compound in vivo, which is very helpful for elucidating the pharmacodynamic material basis and application of astilbin. OBJECTIVE: To establish a rapid profiling and identification method for metabolites in rat urine, faeces and plasma using a UHPLC-Q-Exactive mass spectrometer in negative ion mode. METHODS: In this study, a simple and rapid systematic strategy and 7 metabolite templates, which were established based on previous reports, were utilized to screen and identify astilbin metabolites. RESULTS: As a result, a total of 71 metabolites were detected and characterized, among which 32 metabolites were found in rat urine, while 27 and 38 metabolites were characterized from rat plasma and faeces, respectively. These metabolites were presumed to be generated through ring cleavage, sulfation, dehydrogenation, methylation, hydroxylation, glucuronidation, dehydroxylation and their composite reactions. CONCLUSION: This study illustrated the capacity of the sensitive UHPLC-Q-Exactive mass spectrometer analytical system combined with the data-mining methods to rapidly elucidate the unknown metabolism. Moreover, the comprehensive metabolism study of astilbin provided an overall metabolic profile, which will be of great help in predicting the in vivo pharmacokinetic profiles and understanding the action mechanism of this active ingredient.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Regulation of organic anion transporters: Role in physiology, pathophysiology, and drug elimination.

The members of the organic anion transporter (OAT) family are mainly expressed in kidney, liver, placenta, intestine, and brain. These transporters play important roles in the disposition of clinical drugs, pesticides, signaling molecules, heavy metal conjugates, components of phytomedicines, and toxins, and therefore critical for maintaining systemic homeostasis. Alterations in the expression and function of OATs contribute to the intra- and inter-individual variability of the therapeutic efficacy and the toxicity of many drugs, and to many pathophysiological conditions. Consequently, the activity of these transporters must be highly regulated to carry out their normal functions. This review will present an update on the recent advance in understanding the cellular and molecular mechanisms underlying the regulation of renal OATs, emphasizing on the post-translational modification (PTM), the crosstalk among these PTMs, and the remote sensing and signaling network of OATs. Such knowledge will provide significant insights into the roles of these transporters in health and disease.

Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored. AIM OF STUDY: To clarify the metabolism of liquiritigenin and liquiritin in mice. MATERIALS AND METHODS: In this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching. RESULTS: A total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation. CONCLUSION: This study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice.

In Vivo-to-In Vitro Extrapolation of Transporter-Mediated Renal Clearance: Relative Expression Factor Versus Relative Activity Factor Approach.

About 30% of approved drugs are cleared predominantly by renal clearance (CLr). Of these, many are secreted by transporters. For these drugs, in vitro-to-in vivo extrapolation of transporter-mediated renal secretory clearance (CLsec,plasma) is important to prospectively predict their renal clearance and to assess the impact of drug-drug interactions and pharmacogenetics on their pharmacokinetics. Here we compared the ability of the relative expression factor (REF) and the relative activity factor (RAF) approaches to quantitatively predict the in vivo CLsec,plasma of 26 organic anion transporter (OAT) substrates assuming that OAT-mediated uptake is the rate-determining step in the CLsec,plasma of the drugs. The REF approach requires protein quantification of each transporter in the tissue (e.g., kidney) and transporter-expressing cells, whereas the RAF approach requires the use of a transporter-selective probe substrate (both in vitro and in vivo) for each transporter of interest. For the REF approach, 50% and 69% of the CLsec,plasma predictions were within 2- and 3-fold of the observed values, respectively; the corresponding values for the RAF approach were 65% and 81%. We found no significant difference between the two approaches in their predictive capability (as measured by accuracy and bias) of the CLsec,plasma or CLr of OAT drugs. We recommend that the REF and RAF approaches can be used interchangeably to predict OAT-mediated CLsec,plasma Further research is warranted to evaluate the ability of the REF or RAF approach to predict CLsec,plasma of drugs when uptake is not the rate-determining step. SIGNIFICANCE STATEMENT: This is the first direct comparison of the relative expression factor (REF) and relative activity factor (RAF) approaches to predict transporter-mediated renal clearance (CLr). The RAF, but not REF, approach requires transporter-selective probes and that the basolateral uptake is the rate-determining step in the CLr of drugs. Given that there is no difference in predictive capability of the REF and RAF approach for organic anion transporter-mediated CLr, the REF approach should be explored further to assess its ability to predict CLr when basolateral uptake is not the sole rate-determining step.