Methylophiopogonanone A is a naturally occurring broad-spectrum inhibitor against human UDP-glucuronosyltransferases: Inhibition behaviours and implication in herb-drug interactions.

Methylophiopogonanone A (MOA) is an abundant homoisoflavonoid in the Chinese herb Ophiopogonis Radix. Recent investigations revealed that MOA inhibited several human cytochrome P450 enzymes (CYPs) and stimulated OATP1B1. However, the inhibitory effects of MOA on phase II drug-metabolizing enzymes, such as human UDP-glucuronosyltransferases (hUGTs), have not been well investigated. Herein, the inhibition potentials of MOA on hUGTs were assessed. The results clearly demonstrated that MOA dose-dependently inhibited all tested hUGTs including UGT1A1 (IC50 = 1.23 µM), one of the most important detoxification enzymes in humans. Further investigations showed that MOA strongly inhibited UGT1A1-catalysed NHPH-O-glucuronidation in a range of biological settings including hUGT1A1, human liver microsomes (HLM) and HeLa cells overexpressing UGT1A1. Inhibition kinetic analyses demonstrated that MOA competitively inhibited UGT1A1-catalysed NHPH-O-glucuronidation in both hUGT1A1 and HLM, with Ki values of 0.52 and 1.22 µM, respectively. Collectively, our findings expanded knowledge of the interactions between MOA and human drug-metabolizing enzymes, which would be very helpful for guiding the use of MOA-related herbal products in clinical settings.

A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors.

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drug/herb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived Km values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors.

Evaluation of inhibitors of intestinal UDP-glucuronosyltransferases 1A8 and 1A10 using raloxifene as a substrate in Caco-2 cells: Studies with four flavonoids of Scutellaria baicalensis.

UDP glucuronosyltransferases (UGTs) of the gastrointestinal tract play a crucial role in protection against the toxic effects of xenobiotics in the environment. UGTs such as UGT1A8 and UGT1A10 are predominantly expressed in gastrointestinal tissues. In this study, we examined the phase II metabolism of raloxifene in differentiated Caco-2 monolayers by inducing UGT1A8 and UGT1A10 expression in these cells. The present study evaluated the following four flavonoids of Scutellaria baicalensis as model herbal compounds: scutellarein, salvigenin, baicalein, and wogonin. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The transepithelial electrical resistance values for the individual compounds were comparable regardless of whether they were measured individually. Salvigenin significantly inhibited UGT1A8 and UGT1A10 activities in a concentration-dependent manner. All individual compounds except scutellarein inhibited UGT1A8 and UGT1A10 activity at a concentration of 100 µM. In addition, all individual flavonoids at 100 µM, except wogonin, significantly increased the amount of raloxifene in the basolateral chambers. The positive control, canagliflozin, significantly inhibited both UGT1A8 and UGT1A10 activities. These findings suggest that the Caco-2 assay can be utilized for identifying UGT1A8 and UGT1A10 inhibitors and indicate the potential of salvigenin for enhancing the pharmacological effects of UGT substrate drugs.

Investigation on the metabolic characteristics of isobavachin in Psoralea corylifolia L. (Bu-gu-zhi) and its potential inhibition against human cytochrome P450s and UDP-glucuronosyltransferases.

OBJECTIVES: Isobavachin is a phenolic with anti-osteoporosis activity. This study aimed to explore its metabolic fates in vivo and in vitro, and to investigate the potential drug-drug interactions involving CYPs and UGTs. METHODS: Metabolites of isobavachin in mice were first identified and characterized. Oxidation and glucuronidation study were performed using liver and intestine microsomes. Reaction phenotyping, activity correlation analysis and relative activity factor approaches were employed to identify the main CYPs and UGTs involved in isobavachin metabolism. Through kinetic modelling, inhibition mechanisms towards CYPs and UGTs were also explored. KEY FINDINGS: Two glucuronides (G1 - G2) and three oxidated metabolites (M1 - M3) were identified in mice. Additionally, isobavachin underwent efficient oxidation and glucuronidation by human liver microsomes and HIM with CLint values from 5.53 to 148.79 µl/min per mg. CYP1A2, 2C19 contributed 11.3% and 17.1% to hepatic metabolism of isobavachin, respectively, with CLint values from 8.75 to 77.33 µl/min per mg. UGT1As displayed CLint values from 10.73 to 202.62 µl/min per mg for glucuronidation. Besides, significant correlation analysis also proved that CYP1A2, 2C19 and UGT1A1, 1A9 were main contributors for the metabolism of isobavachin. Furthermore, mice may be the appropriate animal model for predicting its metabolism in human. Moreover, isobavachin exhibited broad inhibition against CYP2B6, 2C9, 2C19, UGT1A1, 1A9, 2B7 with Ki values from 0.05 to 3.05 µm. CONCLUSIONS: CYP1A2, 2C19 and UGT1As play an important role in isobavachin metabolism. Isobavachin demonstrated broad-spectrum inhibition of CYPs and UGTs.

Pterostilbene supplements carry the risk of drug interaction via inhibition of UDP-glucuronosyltransferases (UGT) 1A9 enzymes.

Pterostilbene (PT) is a natural stilbene common in small berries and food supplements, possessing numerous pharmacological activities. However, whether PT can affect the activities of UDP-glucuronosyltransferases (UGT) enzymes remains unclear. The aim of the present study was to investigate the effect of PT on UGT activities and to quantitatively evaluate the food-drug interaction potential due to UGT inhibition. Our data indicated that PT exhibited potent inhibition against HLM, UGT1A6, UGT1A9, UGT2B7, and UGT2B15, moderate inhibition against UGT1A1, UGT1A3, UGT1A8, and UGT2B4, negligible inhibition against UGT1A4, UGT1A7, UGT1A10, and UGT2B17. Further kinetic investigation demonstrated that PT exerted potent noncompetitive inhibition 4-MU glucuronidation by UGT1A9, with IC50 and Ki values of 0.92 µM and 0.52 ± 0.04 µM, respectively. Quantitative prediction study suggested that coadministration of PT supplements at 100 mg/day or higher doses may result in at least a 50% increase in the AUC of drugs predominantly cleared by UGT1A9. Thus, the coadministration of PT supplements and drugs primarily cleared by UGT1A9 may result in potential drug interaction, and precautions should be taken when coadministration of PT supplements and drugs metabolized by UGT1A9.

Potential of herb-drug / herb interactions between substrates and inhibitors of UGTs derived from herbal medicines.

Herbal medicines are widely used as alternative or complementary therapies worldwide to treat and prevent chronic diseases. However, herbal medicines coadministration with therapeutic drugs may cause dramatic clinical herb-drug/herb interactions (HDIs/HHIs) that may result in low drug efficacy or serious toxic reactions. Phase II metabolism enzyme UDP-glucuronosyltransferases (UGTs) play a significant detoxification role in vivo. Most drugs and non-drug xenobiotics undergo phase II metabolic transformations to be more polar compounds that are more easily excreted. Herbal medicines are a mixed and chemically varied group that includes flavonoids, stilbenes, coumarins, quinones, and terpenes, which are potential substrates and inhibitors of UGTs. Although increasing studies about glucuronidation metabolism and the inhibition toward UGTs of many herbal medicines have been reported, it is still difficult to determine which compounds from herbal medicines are substrates or inhibitors of UGTs. This article gives an overview of UGTs studies, which mainly focuses on glucuronidation of herbal constituents as substrates catalyzed by UGTs, potential herbal inhibitors for UGTs. We summarize the negative effects of UGT1A polymorphism and single nucleotide polymorphisms (SNPs), relevant clinical situations of HDIs/HHIs induced by inhibition of UGTs, and propose establishing classification criteria for inhibitors. Finally, we also discuss future research and strategic directions to advance the understanding of the potential HDIs/HHIs and suggest some additional studies revealing more information on UGT-mediated HDIs/HHIs.

In vitro investigation of permeability and metabolism of licoricidin.

Aim Licoricidin has multiple pharmacological activities. The present study was designed to investigate the permeability and pharmacokinetic behavior of licoricidin using in vitro models. MATERIAL AND METHODS: First, human liver microsomes and recombinant human supersomes were used to investigate the interactions between metabolic enzymes and licoricidin. In addition, rat, minipig, rabbit, dog, monkey, and human liver microsomes were used to determine metabolic differences among species. The parallel artificial membrane permeability assay (PAMPA) was used to explore licoricidin permeability behavior. KEY FINDINGS: At 100 µM, licoricidin strongly inhibited CYP2C9, CYP2C19, CYP3A4, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Licoricidin metabolism exhibited considerable differences among species; dog, pig, and rat liver microsomes showed higher metabolic capacity than the other species. Seven licoricidin metabolites were identified by liquid chromatography-tandem mass spectrometry, and hydration and hydroxylation were the major metabolic pathways. The PAMPA permeability of licoricidin was moderate at both pH 4.0 and 7.4. SIGNIFICANCE: The present study will support further pharmacological or toxicological research on licoricidin.

Inhibition of UGT1A1 by natural and synthetic flavonoids.

Flavonoids are widely distributed phytochemicals in vegetables, fruits and medicinal plants. Recent studies demonstrate that some natural flavonoids are potent inhibitors of the human UDP-glucuronosyltransferase 1A1 (UGT1A1), a key enzyme in detoxification of endogenous harmful compounds such as bilirubin. In this study, the inhibitory effects of 56 natural and synthetic flavonoids on UGT1A1 were assayed, while the structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated. The results demonstrated that the C-3 and C-7 hydroxyl groups on the flavone skeleton would enhance UGT1A1 inhibition, while flavonoid glycosides displayed weaker inhibitory effects than their corresponding aglycones. Further investigation on inhibition kinetics of two strong flavonoid-type UGT1A1 inhibitors, acacetin and kaempferol, yielded interesting results. Both flavonoids were competitive inhibitors against UGT1A1-mediated NHPN-O-glucuronidation, but were mixed and competitive inhibitors toward UGT1A1-mediated NCHN-O-glucuronidation, respectively. Furthermore, docking simulations showed that the binding areas of NHPN, kaempferol and acacetin on UGT1A1 were highly overlapping, and convergence with the binding area of bilirubin within UGT1A1. In summary, detailed structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated carefully and the findings shed new light on the interactions between flavonoids and UGT1A1, and will contribute considerably to the development of flavonoid-type drugs without strong UGT1A1 inhibition.

Effects of Ganoderma, Rhodiola and grape seed extracts on the glucuronidation and oral bioavailability of resveratrol in rats.

Herb extracts were shown to inhibit the activity of UDP-glucuronosyltransferases (UGTs) in vitro. However, the actual in vivo effect of the inhibitory ability on oral bioavailability is yet verified. In this study, resveratrol (RES) was used as a model drug to study the effect of three Chinese herb extracts, Ganoderma, Rhodiola and grape seed, on the in vitro and in vivo inhibition of glucuronidation and the in vivo bioavailability of RES. Overall, although herb extracts might show inhibition on glucuronidation of RES in vitro and in vivo, the inhibition of glucuronidation did not necessarily mean to improve the in vivo bioavailability of RES.

Inhibitory and Inductive Effects of Opuntia ficus indica Extract and Its Flavonoid Constituents on Cytochrome P450s and UDP-Glucuronosyltransferases.

Opuntia ficus indica (OFI) is grown abundantly in arid areas and its fruits are regarded as an important food and nutrient source owing to the presence of flavonoids, minerals, and proteins. The previous report that OFI exerts phytoestrogenic activity makes it plausible for OFI-containing supplements to be used as alternative estrogen replacement therapy. In the case of polypharmacy with the consumption of OFI-containing botanicals in post- or peri-menopausal women, it is critical to determine the potential drug-OFI interaction due to the modulation of drug metabolism. In the present study, the modulating effects on the hepatic drug metabolizing enzymes (DMEs) by OFI and its flavonoid constituents (kaempferol, quercetin, isorhamnetin, and their glycosidic forms) were investigated using the liver microsomal fractions prepared from ovariectomized (OVX) rats, human liver microsomes, and human hepatocarcinoma cell line (HepG2). As a result, the oral administration of extracts of OFI (OFIE) in OVX rats induced hepatic CYP2B1, CYP3A1, and UGT2B1. OFIE, hydrolyzed (hdl) OFIE, and several flavonols induced the transcriptional activities of both CYP2B6 and CYP3A4 genes in HepG2 cells. Finally, OFIE did not inhibit activities of cytochrome P450 (CYPs) or uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), whereas hdl OFIE or flavonol treatment inhibited CYP1A2 and CYP3A1/3A4 in rat and human liver microsomes. Our data demonstrate that OFIE may induce or inhibit certain types of DMEs and indicate that drug-OFI interaction may occur when the substrate or inhibitor drugs of specific CYPs or UGTs are taken concomitantly with OFI-containing products.

Effects of Herbal Formulas Bojungikgi-tang and Palmijihwang-hwan on Inflammation in RAW 264.7 Cells and the Activities of Drug-Metabolizing Enzymes in Human Hepatic Microsomes.

In the present study, Bojungikgi-tang (BJIKT: Buzhongyiqi-tang, Hochuekki-to) and Palmijihwang-hwan (PMJHH: Baweidìhuang-wan, Hachimijio-gan), traditional herbal formulas, investigated anti-inflammatory efficacies in murine macrophage cell line and the influence on the activities of drug-metabolizing enzymes (DMEs). The anti-inflammatory potentials of the herbal formulas were evaluated to inhibit the production of the inflammatory mediators and cytokines and the protein expression of inducible nitric oxide and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The activities of the major human DMEs, cytochrome P450 isozymes (CYP450s) and UDP-glucuronosyltransferase isozymes (UGTs), were measured by in vitro enzyme assay systems. BJIKT and PMJHH significantly suppressed the prostaglandin E2 (PGE2) production (IC50 = 317.3 and 282.2 µg/mL, respectively) and the protein expression of COX-2 in LPS-treated RAW264.7 cells. On the human microsomal DMEs, BJIKT inhibited the activities of CYP1A2 (IC50 = 535.05 µg/mL), CYP2B6 (IC50 > 1000 µg/mL), CYP2C9 (IC50 = 800.78 µg/mL), CYP2C19 (IC50 = 563.11 µg/mL), CYP2D6 (IC50 > 1000 µg/mL), CYP2E1 (IC50 > 1000 µg/mL), CYP3A4 (IC50 = 879.60 µg/mL), UGT1A1 (IC50 > 1000 µg/mL), and UGT1A4 (IC50 > 1000 µg/mL), but it showed no inhibition of the UGT2B7 activity at doses less than 1000 µg/mL. PMJHH inhibited the CYP2D6 activity (IC50 = 280.89 µg/mL), but IC50 values of PMJHH exceeded 1000 µg/mL on the activities of CYP1A2, CYP2C19, CYP2E1, and CYP3A4. At concentrations less than 1000 µg/mL, PMJHH did not affect the activities of CYP2B6, CYP2C9, UGT1A1, UGT1A4, and UGT2B7. The results indicate that both BJIKT and PMJHH may be potential candidates to prevent and treat PGE2- and COX-2-mediated inflammatory diseases. In addition, this study will expand current knowledge about herb-drug interactions by BJIKT and PMJHH.

Inhibition of UGT2B7 Enzyme Activity in Human and Rat Liver Microsomes by Herbal Constituents.

The co-use of conventional drug and herbal medicines may lead to herb-drug interaction via modulation of drug-metabolizing enzymes (DMEs) by herbal constituents. UDP-glucuronosyltransferases (UGTs) catalyzing glucuronidation are the major metabolic enzymes of Phase II DMEs. The in vitro inhibitory effect of several herbal constituents on one of the most important UGT isoforms, UGT2B7, in human liver microsomes (HLM) and rat liver microsomes (RLM) was investigated. Zidovudine (ZDV) was used as the probe substrate to determine UGT2B7 activity. The intrinsic clearance (Vmax/Km) of ZDV in HLM is 1.65 µL/mg/min which is ten times greater than in RLM, which is 0.16 µL/mg/min. Andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone inhibited ZDV glucuronidation in HLM with IC50 values of 6.18 ± 1.27, 18.56 ± 8.62, 8.11 ± 4.48 and 4.57 ± 0.23 µM, respectively, hence, herb-drug interactions are possible if andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone are taken together with drugs that are highly metabolized by UGT2B7. Meanwhile, only mitragynine and zerumbone inhibited ZDV glucuronidation in RLM with IC50 values of 51.20 ± 5.95 µM and 8.14 ± 2.12 µM, respectively, indicating a difference between the human and rat microsomal model so caution must be exercised when extrapolating inhibitory metabolic data from rats to humans.

Identification and characterization of in vitro inhibitors against UDP-glucuronosyltransferase 1A1 in uva-ursi extracts and evaluation of in vivo uva-ursi-drug interactions.

Uva-ursi leaf is widely used to treat symptoms of lower urinary tract infections. Here, we evaluated the in vitro inhibitory effects of uva-ursi extracts on 10 major human UDP-glucuronosyltransferases (UGT) isoforms. Of the 10 tested UGT isoforms, uva-ursi extracts exerted the strongest inhibitory effect on UGT1A1-mediated ß-estradiol 3-glucuronidation with the lowest IC50 value of 8.45 ±â€¯1.56 µg/mL. To identify the components of uva-ursi extracts showing strong inhibitory effects against UGT1A1, the inhibitory effects of nine major constituents of the extracts were assessed. Among the tested compounds, gallotannin exerted the most potent inhibition on UGT1A1, followed by 1,2,3,6-tetragalloylglucose; both demonstrated competitive inhibition, with Ki values of 1.68 ±â€¯0.150 µM and 3.55 ±â€¯0.418 µM. We found that gallotannin and 1,2,3,6-tetragalloylglucose also inhibited another UGT1A1-specific biotransformation, SN-38-glucuronidation, showing the same order of inhibition. Thus, in vitro UGT1A1 inhibitory potentials of uva-ursi extracts might primarily result from the inhibitory activities of gallotannin and 1,2,3,6-tetragalloylglucose present in the extracts. However, in rats, co-administration with uva-ursi extracts did not alter the in vivo marker for UGT1A1 activity, expressed as the molar ratio of AUCSN-38 glucuronide/AUCSN-38, because plasma concentrations of gallotannin and 1,2,3,6-tetragalloylglucose may be too low to inhibit the UGT1A1-mediated metabolism of SN-38 in vivo. The poor oral absorption of gallotannin and 1,2,3,6-tetragalloylglucose in uva-ursi extracts might cause the poor in vitro-in vivo correlation. These findings will be helpful for the safe and effective use of uva-ursi extracts in clinical practice.

Simultaneous determination of intestinal permeability and potential drug interactions of complex mixtures using Caco-2 cells and high-resolution mass spectrometry: Studies with Rauwolfia serpentina extract.

Caco-2 cells are a commonly used model for estimating the intestinal bioavailability of single chemical entity pharmaceuticals. Caco-2 cells, when induced with calcitriol, also express other biological functions such as phase I (CYP) and phase II (glucuronosyltransferases) drug metabolizing enzymes which are relevant to drug-supplement interactions. Intestinal bioavailability is an important factor in the overall safety assessment of products consumed orally. Foods, including herbal dietary supplements, are complex substances with multiple chemical components. Because of potential interactions between components of complex mixtures, more reliable safety assessments can be obtained by studying the commercial products "as consumed" rather than by testing individual chemical components one at a time. The present study evaluated the apparent intestinal permeability (Papp) of a model herbal extract, Rauwolfia serpentina, using both whole plant extracts and the individual purified Rauwolfia alkaloids. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The Papp values for individual Rauwolfia alkaloids were comparable whether measured individually or as components of the complete extract. Both Rauwolfia extract and all individual Rauwolfia alkaloids except yohimbine inhibited CYP3A4 activity (midazolam 1'-hydroxylation). Both Rauwolfia extract and all individual Rauwolfia alkaloids except corynanthine and reserpic acid significantly increased glucuronosyltransferase activity (glucuronidation of 4-methylumbelliferone). The positive control, ketoconazole, significantly inhibited both CYP3A4 and glucuronosyltransferase activities. These findings suggest that the Caco-2 assay is capable of simultaneously identifying both bioavailability and potentially hazardous intestinal drug-supplement interactions in complex mixtures.

Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation.

Green tea (Camellia sinensis) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 µg/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC50 values (105 and 59 µM, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 µM, respectively). Using the clinical intestinal UGT substrate raloxifene, the Ki values were ∼1.0 and 2.0 µM, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing.

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases.

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 µM to 16.81 µM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 µM to 11.51 µM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

Inhibitory Effect of Sauchinone on UDP-Glucuronosyltransferase (UGT) 2B7 Activity.

Herb-drug interaction (HDI) limits clinical application of herbs and drugs, and inhibition of herbs towards uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) has gained attention as one of the important reasons to cause HDIs. Sauchinone, an active lignan isolated from aerial parts of Saururus chinensis (Saururacease), possesses anti-oxidant, anti-inflammatory, and anti-viral activities. In pharmacokinetics of sauchinone, sauchinone is highly distributed to the liver, forming extensive metabolites of sauchinone via UGTs in the liver. Thus, we investigated whether sauchinone inhibited UGTs to explore potential of sauchinone-drug interactions. In human liver microsomes (HLMs), sauchinone inhibited activities of UGT1A1, 1A3, 1A6, and 2B7 with IC50 values of 8.83, 43.9, 0.758, and 0.279 µM, respectively. Sauchinone also noncompetitively inhibited UGT1A6 and 2B7 with Ki values of 1.08 and 0.524 µM, respectively. In in vivo interaction study using mice, sauchinone inhibited UGT2B7-mediated zidovudine metabolism, resulting in increased systemic exposure of zidovudine when sauchinone and zidovudine were co-administered together. Our results indicated that there is potential HDI between sauchinone and drugs undergoing UGT2B7-mediated metabolism, possibly contributing to the safe use of sauchinone and drug combinations.

Development of a LC-MS/MS method to investigate the interference of pharmacokinetics of the main constituents in Saxifraga stolonifera: Involvement of drug metabolism enzymes.

A specific and sensitive LC-MS/MS method was established for the simultaneous determination of bergenin, protocatechuic acid and gallic acid, the main active constituents of Saxifraga stolonifera (L.) Meerb. herb, in rat plasma. After fully validated, the method was applied to the comparative pharmacokinetic studies of the three compounds orally administered alone and in combination in the S. stolonifera extract, respectively. The results showed that the pharmacokinetic parameters, including Cmax, Tmax, AUC, CLz/F, MRT0-∞, were significantly different for both bergenin and protocatechuic acid in the extract as compared to the corresponding compounds administered alone. However, the pharmacokinetic behavior of gallic acid in the extract did not differ from that administered alone. Further studies found that quercetin, coexisting in the herb extract, significantly decreased the glucuronidation of bergenin through inhibiting the activities of UGT1A1 and UGT1A3, and reduced the metabolism of protocatechuic acid by inhibiting the activity of catechol-O-methyltransferase. Quercetin and other flavonoids occurring in the S. stolonifera extract might increase the absorption and improve the bioavailability of bergenin and protocatechuic acid by slowing down the liver metabolism. The findings provide a good guidance for the development and clinical application of S. stolonifera.

Everolimus-inhibited multiple isoforms of UDP-glucuronosyltransferases (UGTs).

1. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and has been clinically utilized to prevent the rejection of organ transplants. This study aims to determine the inhibition of everolimus on the activity of phase-II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs). 2. The results showed that 100 µM of everolimus exerted more than 80% inhibition toward UGT1A1, UGT-1A3 and UGT-2B7. UGT1A3 and UGT2B7 were selected to elucidate the inhibition mechanism, and in silico docking showed that hydrogen bonds and hydrophobic interactions mainly contributed to the strong binding of everolimus toward the activity cavity of UGT1A3 and UGT2B7. Inhibition kinetic-type analysis using Lineweaver-Burk plot showed competitive inhibition toward all these UGT isoforms. The inhibition kinetic parameters (Ki) were calculated to be 2.3, 0.07 and 4.4 µM for the inhibition of everolimus toward UGT1A1, UGT-1A3 and UGT-2B7, respectively. 3. In vitro-in vivo extrapolation (IVIVE) showed that [I]/Ki value was calculated to be 0.004, 0.14 and 0.002 for UGT1A1, UGT-1A3 and UGT-2B7, respectively. Therefore, high DDI potential existed between everolimus and clinical drugs mainly undergoing UGT1A3-catalyzed glucuronidation.

Identification and characterization of the structure-activity relationships involved in UGT1A1 inhibition by anthraquinone and dianthrone constituents of Polygonum multiflorum.

The adverse effects of Polygonum (P.) multiflorum, including abnormal bilirubin metabolism, are a serious public health issue. As uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) is the only enzyme responsible for bilirubin metabolism, we investigated the inhibitory effect of a P. multiflorum extract and 10 anthraquinone and dianthrone compounds on UGT1A1 in rat liver microsomes in vitro. The P. multiflorum extract exhibited the strongest inhibitory effect on UGT1A1 activity (inhibition constant [Ki] = 0.3257 µM, 1422 µg of material/mL), followed by cis-emodin dianthrones (Ki = 0.8630 µM), trans-emodin dianthrones (Ki = 1.083 µM), emodin-8-O-glc (Ki = 3.425 µM), and polygonumnolide C2 (Ki = 4.291 µM). Analysis of the structure-activity relationships of these compounds suggested that the spatial orientation of the molecules and the presence of particular functional groups affect UGT1A1 inhibition. A mechanistic analysis showed that all the tested compounds docked into two of the nine active sites of UGT1A1 and suggested that hydrophobic interactions and hydrogen bonds are important for the affinity of the tested compounds for UGT1A1; moreover, their interaction energies were generally in agreement with the Ki values. These findings provide insight into adverse reactions to P. multiflorum and identify the pharmacophores involved in inhibition of UGT1A1.