Activating Effect of Effective Components in Medicinal and Edible Substances on Human Pregnane X Receptor and Cytotoxicity Screening / 中国实验方剂学杂志

ObjectiveTo explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor （PXR）， including Glycyrrhizae Radix et Rhizoma （liquiritin and glycyrrhizic acid）， Houttuyniae Herba （quercetin and houttuyfonate）， Prunellae Spica （rosmarinic acid）， Cassiae Semen （aurantio-obtusin）， Poria （pachymic acid）， Lilii Bulbus （Lilium brownii saponin and colchicine）， and Lycii Fructus （Lycium barbarum polysaccharide） and screen potentially toxic components. MethodCell counting kit-8 （CCK-8） assay was used to investigate the cytotoxic effect of liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， pachymic acid， aurantio-obtusin， and colchicine （10， 20， and 50 μmol·L-1）， and L. brownii saponin and L. barbarum polysaccharide （10， 20， and 50 mg·L-1） on normal human hepatocyte cell line （L02）. The release of lactate dehydrogenase （LDH） in L02 cells after drug treatments was detected by the biochemical analyzer. The apoptosis induced by ten effective components was explored by Hoechst 33342 staining. The secreted luciferase reporter system was used to co-transfect the PXR expression vector and reporter gene vector containing cytochrome P450 3A4 （CYP3A4） transcriptional regulatory region into L02 cells， with 10 μmol·L-1 rifampicin （RIF） as a positive control. After treated with liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， aurantio-obtusin， pachymic acid， and colchicine （5， 10， and 20 μmol·L-1） and L. brownii saponin and L. barbarum polysaccharide （5， 10， and 20 mg·L-1） for 24 h， the cells were tested for secreted luciferase activity. ResultCompared with the control group， colchicine， L. brownii saponin， and quercetin decreased the cell viability （P<0.05， P<0.01）. Compared with the control group， quercetin， rosmarinic acid， glycyrrhizic acid， colchicine， aurantio-obtusin， and pachymic acid increased the release rate of LDH in L02 cells （P<0.05， P<0.01）. The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid， liquiritin， and L. barbarum polysaccharide treatments as compared with the control group （P<0.05， P<0.01）. In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells， compared with the control group， aurantio-obtusin and pachymic acid showed activating effects on PXR （P<0.05）， whereas liquiritin and glycyrrhizic acid showed inhibitory effects （P<0.05）. ConclusionThe findings suggest that when medicinal and edible substances are taken for a long time， attention should be paid to their influence on drug-metabolizing enzymes and possible interactions， so as to improve their safety.

Granatripodins A-B, limonoids featuring a Tricyclo[3.3.1.02,8]nonane motif: Absolute configuration and agonistic effects on human pregnane-X-receptor.

Two unprecedented limonoids incorporating a sterically encumbered cyclopropane ring, named granatripodins A (1) and B (2), featuring the presence of a tricyclo[3.3.1.02,8]nonane motif, were obtained from seeds of the Thai Xylocarpus granatum. The planar structures and absolute configurations of these limonoids were unambiguously established by NMR investigations, TDDFT-ECD and DFT-NMR calculations, and single-crystal X-ray diffraction analysis (Cu Kα). Most notably, granatripodin A (1) exhibited agonistic effects on human pregnane-X-receptor at the concentration of 100.0 nM. The biosynthetic origins of these limonoids via a radical cascade reaction are proposed. This study exemplifies a universal approach for the stereochemical assignment of polycyclic compounds with a cyclopropane-embedded cage scaffold.

Krishnolides E-K: New limonoids from the Krishna mangrove Xylocarpus moluccensis.

Seven new limonoids, named krishnolides E-K (1-7), including three khayanolides, two mexicanolides, a derivative of trangmolin A, and an andirobin, were isolated from seeds of the Indian Krishna mangrove, Xylocarpus moluccensis. The structures of these limonoids were established by HRESIMS, extensive NMR investigations, and X-ray crystallography. Most notably, the absolute configurations of 1, 5, 6, and 7 were unequivocally determined by single-crystal X-ray diffraction analyses (Cu Kα). Krishnolide F (2) exhibited significant agonistic effects on human pregnane-X-receptor (hPXR) at the concentration of 10.0 µM. Molecular docking revealed that 2 could bind a helix near the region of the Helix 12 of hPXR. Polar contribution could be electrostatic effects from the formation of two stable protein-ligand hydrogen bonds between Gln285/1-OH and His407/1-OH, respectively. This is the first report of agonistic effects of a khayanolide-type limonoid on hPXR.

Discovery of Thai mangrove tetranortriterpenoids as agonists of human pregnane-X-receptor and inhibitors against human carboxylesterase 2.

Human pregnane-X-receptor (hPXR) is considered to be the key target for the treatment of cholestasis and liver injury. Agonists of hPXR are potential drug leads. Potent and selective inhibitors of human carboxylesterase 2 (hCES2) could be utilized to alleviate the toxicity induced by ester drugs. In this work, fifteen new tetranortriterpenoids with structure diversity, named thaigranatins F-T (1-15), including four limonoids containing a C1-O-C29 bridge (1-4), four mexicanolides (5-8), three phragmalins (9-11), two limonoids belonging to the small group of trichiliton A (12-13), and two apotirucallanes (14-15), were isolated from seeds of the Thai mangrove, Xylocarpus granatum. The structures of these compounds were established by high resolution-electrospray ionization mass spectroscopy, extensive NMR spectroscopic investigations, single-crystal X-ray diffraction analyses, and the comparison of experimental electronic circular dichroism spectra. Most notably, thaigranatins L (7) and P (11) exhibited agonistic effects on hPXR at the concentration of 10.0 µM and 10.0 nM, respectively, whereas thaigranatins J (5), M (8), and T (15) showed inhibitory activities against hCES2 with IC50 values of 6.63, 11.35, and 5.05 µM, respectively. The 8α,30α-epoxy moiety of mexicanolide and the Δ8,14 double bond of phragmalin are pivotal for agonistic effects of these limonoids on hPXR, whereas the 6-OAc group of mexicanolide is crucial for its inhibitory activity against hCES2. Additionally, the flexible C-17-side-chain with appropriate hydroxy groups is considered to be important for the inhibitory activity of apotirucallane against hCES2.

[Effect of six components in Polygoni Multiflori Radix on regulation of CYP3A4 mediated by human pregnane X receptor].

This paper aimed to study the six chemical components of Polygoni Multiflori Radix (gallic acid, quercetin, luteolin, kaempferol, resveratrol, apigenin). By the established pregnane X receptor (human pregnant X receptor, PXR) CYP3A4 mediated drug induced rapid screening technique, the effect of chemical components on the cell activity was detected by MTS cell method, and the value of IC50 was calculated. The dual luciferase reporter system was used to co-transfect PXR reporter gene expression vector containing transcriptional regulation and CYP3A4 with HepG2 cells, with 10 µmol·L⁻¹ rifampicin (RIF) as a positive control, and 10 µmol·L⁻¹ of ketoconazole (TKZ) as negative control. Gallic acid, quercetin, luteolin, kaempferol, apigenin, resveratrol(5, 10, 20 µmol·L⁻¹) were used to incubate for 24 h, and the luciferase activity was detected. The results showed that when plasmid pcDNA3.1 was co-transfected with pGL4.17-CYP3A4, gallic acid and resveratrol had an inhibitory effect on the regulation of CYP3A4, and quercetin, luteolin, kaempferol had an inductive effect on CYP3A4; when pcDNA3.14-PXR was co-transfected with pGL4.17-CYP3A4, quercetin, luteolin, kaempferol, apigenin, resveratrol had an inductive effect. To sum up, the 6 reported liver injury components had inhibitory or activating effects on CYP3A4. After PXR plasmid was involved, 5 components had an inductive effect on CYP3A4, and the inductive effects of 2 components were significantly different. In this experiment, we found that 2 kinds of potential liver injury components in Polygoni Multiflori Radix had been induced by CYP3A4, which was achieved through PXR regulation. It suggested that attention shall be paid to potential drug interactions when combined with Polygoni Multiflori Radix, so as to improve the safety and efficacy.

Effect of six components in Polygoni Multiflori Radix on regulation of CYP3A4 mediated by human pregnane X receptor / 中国中药杂志

This paper aimed to study the six chemical components of Polygoni Multiflori Radix (gallic acid, quercetin, luteolin, kaempferol, resveratrol, apigenin). By the established pregnane X receptor (human pregnant X receptor, PXR) CYP3A4 mediated drug induced rapid screening technique, the effect of chemical components on the cell activity was detected by MTS cell method, and the value of IC₅₀ was calculated. The dual luciferase reporter system was used to co-transfect PXR reporter gene expression vector containing transcriptional regulation and CYP3A4 with HepG2 cells, with 10 μmol·L⁻¹ rifampicin (RIF) as a positive control, and 10 μmol·L⁻¹ of ketoconazole (TKZ) as negative control. Gallic acid, quercetin, luteolin, kaempferol, apigenin, resveratrol(5, 10, 20 μmol·L⁻¹) were used to incubate for 24 h, and the luciferase activity was detected. The results showed that when plasmid pcDNA3.1 was co-transfected with pGL4.17-CYP3A4, gallic acid and resveratrol had an inhibitory effect on the regulation of CYP3A4, and quercetin, luteolin, kaempferol had an inductive effect on CYP3A4; when pcDNA3.14-PXR was co-transfected with pGL4.17-CYP3A4, quercetin, luteolin, kaempferol, apigenin, resveratrol had an inductive effect. To sum up, the 6 reported liver injury components had inhibitory or activating effects on CYP3A4. After PXR plasmid was involved, 5 components had an inductive effect on CYP3A4, and the inductive effects of 2 components were significantly different. In this experiment, we found that 2 kinds of potential liver injury components in Polygoni Multiflori Radix had been induced by CYP3A4, which was achieved through PXR regulation. It suggested that attention shall be paid to potential drug interactions when combined with Polygoni Multiflori Radix, so as to improve the safety and efficacy.

Poly- and perfluorinated compounds activate human pregnane X receptor.

Poly- and perfluorinated compounds (PFCs), which have been detected worldwide in human blood, surface water and house dust, are suspected to induce potential endocrine-disrupting hormonal effects. In this study, cell-based reporter gene assays were used to determine the activity of a variety of PFCs against the human pregnane X receptor (hPXR) to identify the critical structural feature of PFCs related to their hPXR activity. Molecular docking studies combined with site-directed mutagenesis were employed to investigate the mechanism by which PFCs interact with and activate hPXR. We found that all tested PFCs can activate hPXR. The hPXR activity of the PFCs correlates with the carbon chain length and the functional group of the chemicals. Hydrogen bonding was characteristic of the interaction between PFCs and hPXR. We also identified the key residues within the hPXR ligand-binding pocket responsible for PFC-hPXR interaction. The outcome of the present study threw a light on the mechanism by which PFCs activate hPXR. PFCs may pose some potential endocrine-disrupting hormonal effects via activation of hPXR.

66034T/C polymorphism of the human pregnane X receptor (hPXR) as potential risk factor for drug resistance in epilepsy - Preliminary study.

INTRODUCTION AND OBJECTIVES: Recent research has suggested that genetic factors may play an important role in the development of drug resistance in epilepsy. It is not clear which gene loci are responsible for the drug-resistant phenotype. Studying certain nuclear receptors may be helpful in predicting drug response, as they regulate drug transporting proteins and enzymes involved in their metabolism. This study focuses on one of these receptors, the human pregnane X receptor (hPXR). The objective was to examine the link between selected single nucleotide polymorphisms (SNPs) 69789A/G rs 7643645 and 66034T/C rs 13059232 hPXR and the lack of response to epilepsy treatment. MATERIALS AND METHODS: 73 patients diagnosed with drug-resistant epilepsy were included in the study. The diagnoses were made according to the criteria published by The International League Against Epilepsy (ILAE) in 2010. The control group was comprised of a group of 122 healthy volunteers. Genetic material isolated from the peripheral blood of the participants was analyzed with TagMan Genotyping Assays in search of the selected hPXR polymorphisms. RESULTS: The distribution of genotypes of the 66034T/C rs 13059232 hPXR polymorphism was significantly different in the group with drug-resistant epilepsy and the control group. In the drug-resistant group the CC genotype was significantly more common compared to the control group (50.7% vs 35.2%) p=0.0339. The distribution of 69789 A/G rs 7643645 hPXR genotypes was comparable in both groups. CONCLUSIONS: There is potential association between hPXR and drug resistance but its relevance for the development of drug-resistant phenotype remains to be studied.

In silico investigation of agonist activity of a structurally diverse set of drugs to hPXR using HM-BSM and HM-PNN.

The human pregnane X receptor (hPXR) plays a critical role in the metabolism, transport and clearance of xenobiotics in the liver and intestine. The hPXR can be activated by a structurally diverse of drugs to initiate clinically relevant drug-drug interactions. In this article, in silico investigation was performed on a structurally diverse set of drugs to identify critical structural features greatly related to their agonist activity towards hPXR. Heuristic method (HM)-Best Subset Modeling (BSM) and HM-Polynomial Neural Networks (PNN) were utilized to develop the linear and non-linear quantitative structure-activity relationship models. The applicability domain (AD) of the models was assessed by Williams plot. Statistically reliable models with good predictive power and explain were achieved (for HM-BSM, r (2)=0.881, q LOO (2) =0.797, q EXT (2) =0.674; for HM-PNN, r (2)=0.882, q LOO (2) =0.856, q EXT (2) =0.655). The developed models indicated that molecular aromatic and electric property, molecular weight and complexity may govern agonist activity of a structurally diverse set of drugs to hPXR.

Accessing the molecular interactions of phthalates and their primary metabolites with the human pregnane X receptor using in silico profiling.

Phthalates are known to cause endocrine disruption in humans and animals. Being lipophilic xenobiotic chemicals, phthalates from the surrounding environments can easily be absorbed into the biological system, thereby causing various health dysfunctions. This molecular docking study evaluates a variety of molecular interactions of 12 commonly used diphthalates and respective monophthalates onto the ligand binding domain (LBD) of the human pregnane X receptor (hPXR), a xenosensor, which would be beneficial for further in vitro and in vivo studies on hazardous phthalates. Out of 12 diphthalates and their monophthalates tested, diisodecyl phthalate (-9.16 kcal mol-1 ) showed more affinity toward hPXR whereas diisononyl phthalate (-8.77) and di(2-ethyhexyl)phthalate (-8.56), the predominant plasticizers found in a variety of plastics and allied products, showed comparable binding scores with that of the control ligands such as hyperforine (-9.99) and dexamethasone (-7.36). In addition to the above diphthalates, some of their monophthalates (monoisodecyl phthalate, mono-2-etheylhexyl phthalate, etc.) also established similar interactions with certain crucial amino acids in the LBD, which led to higher G scores. In fact, bisphenol A, a well-studied and proven endocrine disruptor, showed lesser G scores (-6.69) than certain phthalates. Copyright © 2016 John Wiley & Sons, Ltd.

In silico investigation of agonist activity of a structurally diverse set of drugs to hPXR using HM-BSM and HM-PNN / 华中科技大学学报（医学）（英德文版）

The human pregnane X receptor (hPXR) plays a critical role in the metabolism, transport and clearance of xenobiotics in the liver and intestine. The hPXR can be activated by a structurally diverse of drugs to initiate clinically relevant drug-drug interactions. In this article, in silico investigation was performed on a structurally diverse set of drugs to identify critical structural features greatly related to their agonist activity towards hPXR. Heuristic method (HM)-Best Subset Modeling (BSM) and HM-Polynomial Neural Networks (PNN) were utilized to develop the linear and non-linear quantitative structure-activity relationship models. The applicability domain (AD) of the models was assessed by Williams plot. Statistically reliable models with good predictive power and explain were achieved (for HM-BSM, r (2)=0.881, q LOO (2) =0.797, q EXT (2) =0.674; for HM-PNN, r (2)=0.882, q LOO (2) =0.856, q EXT (2) =0.655). The developed models indicated that molecular aromatic and electric property, molecular weight and complexity may govern agonist activity of a structurally diverse set of drugs to hPXR.

Molecular insights into the promiscuous interaction of human pregnane X receptor (hPXR) with diverse environmental chemicals and drug compounds.

The nuclear receptor member human pregnane X receptor (hPXR) regulates enzymes and transporters involved in xenobiotic detoxification as well as maintains homeostatic balance of bile acids, thyroid and steroid hormones. hPXR can be recognized and activated by a structurally diverse array of environmental chemicals and drug compounds to initiate adverse biological effects, such as perturbing normal physiological functions and causing dangerous drug-drug interactions and exhibiting a high promiscuity in its ligand spectrum. Understanding of the molecular mechanism and biological implication underlying the promiscuous interaction of hPXR with its diverse ligands is fundamentally important for toxicological and pharmaceutical researches. In the current study, molecular docking and hybrid quantum mechanics/molecular mechanics (QM/MM) were employed to investigate the binding mode, structural basis and energetic property of hPXR interactions with various activators and non-activators. It was found that, as compared to non-activators, the activators adopt few dominant modes to tightly interact with hPXR, which are specified by few polar spots located on the hydrophobic surface of hPXR active pocket. Based on the findings, a novel method called multiple binding mode-based quantitative structure-activity relationship (MBMB-QSAR) that characterizes the nonbonded interaction profile of hPXR with its ligand in multiple binding modes was proposed to model and predict the activating potency of small-molecule compounds on hPXR. Several partial least square (PLS) predictors derived from the MBMB-QSAR modeling were demonstrated to be effective for quantitative characterization of the biological behavior of experimentally confirmed activators, and for qualitatively differentiating the activators from a large number of non-activators. From the predictor models it is suggested that the hydrophobic force and electrostatic interaction play an important role in hPXR-ligand binding, while steric factor contributes moderately to the binding.

Effect of Chinese herbs on CYP3A4 activity and expression in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) has become more popular among cancer patients in the Western world, who often use Chinese herbs as adjuvant therapy to reduce the adverse effects of conventional chemotherapy. However, pharmacokinetic (PK) interactions between Chinese herbs and anticancer drugs can occur and have dramatic consequences for these patients. Currently, only a few possible PK interactions between Chinese herbs and conventional Western drugs have been documented. AIM OF THE STUDY: Since the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) contributes to most of the PK interactions with (anticancer) drugs, the effect of four Chinese herbs (Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus) on the activity and expression of CYP3A4 was investigated in vitro. MATERIALS AND METHODS: Ethanol and water-ethanol extracts of the four Chinese herbs were prepared from raw material. CYP3A4 inhibition was assessed by the use of Supersomes™ in a fluorescence assay. Furthermore, CYP3A4 induction was evaluated in a human pregnane X receptor (hPXR)-mediated CYP3A4 reporter gene assay and a quantitative real time PCR assay, both in human colon adenocarcinoma-derived LS180 cells (LS180). RESULTS: Extracts of Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus inhibited CYP3A4 in human CYP3A4 Supersomes™ (IC50 values: 17-83 µg/mL). Oldenlandia diffusa and Rehmannia glutinosa significantly induced PXR-mediated CYP3A4 (p<0.001). Oldenlandia diffusa also significantly induced CYP3A4 mRNA levels (p<0.001 at 250 µg/mL). CONCLUSIONS: Concomitant use of Oldenlandia diffusa and Rehmannia glutinosa could result in induction of CYP3A4, leading to a reduced efficacy of drugs that are CYP3A4 substrates and have a narrow therapeutic window. Because of the possible enhanced toxicity caused by CYP3A4 inhibition, clinical effects of CYP3A4 inhibition by Astragalus propinquus and Codonopsis tangshen must also be taken into account. In conclusion, herb-drug interactions between Chinese herbs and various CYP3A4 substrates can occur. Further research to investigate the clinical relevance of the interactions caused by Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus is required.

Establishment of hPXR mediated reporter gene model for UGT1A1 and its application / 中国药学杂志

OBJECTIVE: To develop hPXR mediated reporter gene model for UGT1A1, and use it as an in vitro model for determination of induction activity toward UGT1A1 of various commonly used traditional Chinese medicines (TCMs) extracts. METHODS: The distal and proximal promoters of UGT1A1 were amplified from human genetic DNA, and were cloned into pGL3 vector as pGL3-PXRE plasmid, which was then cotransfected into HepG2 cells with hPXR expression plasmid to establish the reporter gene model. Then the model was applied to determine the induction activity toward UGT1 A1 of various commonly used TCMs extracts. RESULTS: The promoters of UGT1A1 were successfully cloned into pGL3 vector to form pGL3-PXRE recombinant plasmid, and the reporter gene model was successfully established. Nine kinds of TCMs extracts were investigated, and three were found to activate hPXR and therefore have the potential to induce UGT1A1. CONCLUSION: The model established is an effective method for determination of activators of hPXR and potential inducers of UGT1A1 in vitro. Copyright 2012 by the Chinese Pharmaceutical Association.