Attenuating pregnane X receptor (PXR) activation: a molecular modelling approach

Recent studies have demonstrated that the pregnane X receptor (PXR) is a key regulator of cytochromes P450 3A (e.g. CYP3A4 in human) gene expression. As a result, activation of PXR may lead to CYP3A4 protein over-expression. Because induction of CYP3A4 could result in clinically important drug drug interactions, there has been a great interest in reducing the possibility of PXR activation by drug candidates in drug-discovery programmes. In order to provide structural insight for attenuating drug candidate-mediated PXR activation, we used a docking approach to study the structure activity relationship for PXR activators. Based on our docking models, it is proposed that introducing polar groups to the end of an activator should reduce its human PXR (hPXR) activity via destabilizing interactions in the hydrophobic areas of the PXR ligand-binding pocket. A number of analogues that incorporate these structural features then were designed and synthesized, and they exhibited significantly lower hPXR activation in a transactivation assay and decreased CYP3A4 induction in a human hepatocytes-based assay. In addition, an example in which attenuating hPXR activation was achieved by sterically destabilizing the helices 11 and 12 of the receptor is presented.     

Chlordecone, a mixed pregnane X receptor (PXR) and estrogen receptor alpha (ERalpha) agonist, alters cholesterol homeostasis and lipoprotein metabolism in C57BL/6 mice

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [¹⁴C]CD or [¹⁴C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor α (ERα) in a concentration-dependent manner (0-50 μM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice.     

孕烷X受体(PXR)对药物代谢途径的调控

目的 从孕烷X受体（PXR）对药物代谢途径的调控入手,在代谢性药物相互作用、PXR在CYP3A4调控中的作用及其调控机制等方面作分析和阐述。方法 结合近年来国内外相关文献进行评述。结果 PXR是CYP3A4的主要转录调控因子,药物通过PXR介导的信号通路调节CYP3A4的表达是影响药物体内代谢变化的重要途径。结论 就临床药物而言,由PXR介导的CYP3A4酶蛋白表达的改变可造成合用药物药效的减弱甚至丧失,因此必须引起广大临床药师的足够重视。     

Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene

Metformin is widely used in the treatment of type-2 diabetes. The pleotropic effects of metformin on glucose and lipid metabolism have been proposed to be mediated by the activation of AMP-activated protein kinase (AMPK) and the subsequent up-regulation of small heterodimer partner (SHP). SHP suppresses the functions of several nuclear receptors involved in the regulation of hepatic metabolism, including pregnane X receptor (PXR), which is referred to as a “master regulator” of drug/xenobiotic metabolism.In this study, we hypothesize that metformin suppresses the expression of CYP3A4, a main detoxification enzyme and a target gene of PXR, due to SHP up-regulation.We employed various gene reporter assays in cell lines and qRT-PCR in human hepatocytes and in Pxr^−/− mice.We show that metformin dramatically suppresses PXR-mediated expression of CYP3A4 in hepatocytes. Consistently, metformin significantly suppressed the up-regulation of Cyp3a11 mRNA in the liver and intestine of wild-type mice, but not in Pxr^−/− mice. A mechanistic investigation of the phenomenon showed that metformin does not significantly up-regulate SHP in human hepatocytes. We further demonstrate that AMPK activation is not involved in this process. We show that metformin disrupts PXR's interaction with steroid receptor coactivator-1 (SRC1) in a two-hybrid assay independently of the PXR ligand binding pocket. Metformin also inhibited vitamin D receptor-, glucocorticoid receptor- and constitutive androstane receptor (CAR)-mediated induction of CYP3A4 mRNA in human hepatocytes.We show, therefore, a suppressive effect of metformin on PXR and other ligand-activated nuclear receptors in transactivation of the main detoxification enzyme CYP3A4 in human hepatocytes.     

PXR受体调控的CYP3A诱导及其在药物代谢中的重要意义

机体每日都要接触大量外源性化合物（xenobiotics），包括环境、饮食、药物中的各种成分，其中亲脂性化合物如果不能被及时代谢为极性化合物，就会在肝脏蓄积并影响机体正常生理功能，产生毒性甚至致癌。细胞色素P450（CYPS）属于血红素蛋白基因超家族，编码一系列代谢酶系统，参与各类不同结构亲脂性化合物的生物转化，增强代谢物水溶性，利于排出体外，     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

1. The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR.

2. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells.

3. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401.

4. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb–drug interactions.

     

3,4-methylenedioxymethamphetamine (MDMA) interacts with therapeutic drugs on CYP3A by inhibition of pregnane X receptor (PXR) activation and catalytic enzyme inhibition

Metabolism of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) by the major hepatic drug-metabolizing enzyme cytochrome P450 3A (CYP3A), plays an important role in MDMA-induced liver toxicity. In the present study, we investigated interactions between MDMA and several therapeutic and recreational drugs on CYP3A and its regulator pregnane X receptor (PXR), using a human PXR-mediated CYP3A4-reporter gene assay, rat primary hepatocytes and microsomes. MDMA significantly inhibited hPXR-mediated CYP3A4-reporter gene expression induced by the human PXR activator rifampicin (IC₅₀ 1.26 ± 0.36 mM) or the therapeutic drugs paroxetine, fluoxetine, clozapine, diazepam and risperidone. All these drugs concentration-dependently inhibited CYP3A activity in rat liver microsomes, but in combination with MDMA this inhibition became more efficient for clozapine and risperidone. In rat primary hepatocytes that were pretreated with or without the rodent PXR activator pregnenolone 16alpha-carbonitrile (PCN), MDMA inhibited CYP3A catalytic activity with IC₅₀ values of 0.06 ± 0.12 and 0.09 ± 0.13 mM MDMA, respectively. This decrease appeared to be due to decreased activation of PXR and subsequent decreased CYP3A gene expression, and catalytic inhibition of CYP3A activity. These data suggest that in situations of repeated MDMA use in combination with other (therapeutic) drugs, adverse drug-drug interactions through interactions with PXR and/or CYP3A cannot be excluded.     

Preclinical assessment of the absorption,distribution, metabolism and excretion of GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide), an orally bioavailable systemic Hedgehog signalling pathway inhibitor

1. GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers.

2. The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3?ml min^?1 kg^?1 in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg^?1 in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096–0.649?ml min^?1 kg^?1 and the predicted volume of distribution was 0.766 l kg^?1.

3. Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes.

4. Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1–M3) and three sequential glucuronides (M4–M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3).

5. GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC₅₀ estimates greater than 20?μM. K_i’s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24?μM, respectively. An evaluation with Simcyp® suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15?μM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells.

6. Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.

     

Effect on metabolic enzymes and thyroid receptors induced by BDE-47 by activation the pregnane X receptor in HepG2, a human hepatoma cell line

2,2′,4,4′-Tetra-bromodiphenyl ether (BDE-47), an important congener among polybrominated diphenyl ether (PBDE) compounds, has been predominantly in environmental samples and human tissue. Thyroid disruption is the most sensitive endpoint effect among a number of health effects of exposure to BDE-47 in animals and humans. However, the detailed underlying mechanisms in humans are not well understood. In the present study, human pregnane X receptor (hPXR)-overexpressing HepG2 cell model and a dual-luciferase reporter assay system were constructed to investigate the role of hPXR in BDE-47–induced alterations of expression of metabolic enzymes and TR in vitro. The results showed that hPXR was significantly activated by BDE-47, and expression levels of both mRNA and protein of the thyroid receptor (TR) isoforms TRα1 and TRβ1 were decreased in hPXR-overexpressing HepG2 cells after BDE-47 treatment. However, the increased expression of hepatic microsomal phase I enzyme CYP3A4 and phase II enzymes, UGT1A3 and SULT2A1 were also found. Taken together, the results indicated that BDE-47 was a strong hPXR activator, activation of hPXR played an important role in BDE-47–induced down-regulation of TR, and up-regulations of CYP3A4, UGT1A3, and SULT2A1 participated in the process, which may provide more toxicological evidence on mechanisms of disruption of thyroid hormone induced by BDE-47.     

Indirect activation of pregnane X receptor in the induction of hepatic CYP3A11 by high-dose rifampicin in mice

Rifampicin (RIF), a typical ligand of human pregnane X receptor (PXR), powerfully induces the expression of cytochrome P450 3A4 (CYP3A4) in humans. Although it is thought that RIF is not a ligand of rodent PXR, treatment with high-dose RIF (e.g. more than 20?mg/kg) increases the expression of CYP3A in the mouse liver. In this study, we investigated whether the induction of CYP3A by high-dose RIF in the mouse liver is mediated via indirect activation of mouse PXR (mPXR). The results showed that high-dose RIF increased the expression of CYP3A11 and other PXR-target genes in the liver of wild-type mice but not PXR-knockout mice. However, the results of reporter gene and ligand-dependent assembly assays showed that RIF does not activate mPXR in a ligand-dependent manner. In addition, high-dose RIF stimulated nuclear accumulation of mPXR in the mouse liver, and geldanamycin and okadaic acid attenuated the induction of Cyp3a11 and other PXR-target genes in primary hepatocytes, suggesting that high-dose RIF triggers nuclear translocation of mPXR. In conclusion, the present study suggests that high-dose RIF stimulates nuclear translocation of mPXR in the liver of mice by indirect activation, resulting in the transactivation of Cyp3a11 and other PXR-target genes.     

Modulation of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) by 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives, ligands of peripheral benzodiazepine receptor (PBR)

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate xenobiotic sensing and metabolism through interactions with multiple exogenous and endogenous chemicals. Compounds that activate CAR are often ligands of PXR; attention is therefore given to discovery of new, receptor-specific chemical entities that may be exploited for therapeutic and basic research purposes. Recently, ligands of the peripheral benzodiazepine receptor (PBR), PK11195 and FGIN-1-27, were shown to modulate both CAR and PXR. PBR is a mitochondrial transport protein responsible for multiple regulatory functions, including heme biosynthesis, a major component in cytochrome P450 (CYP) enzymes. To investigate possible new roles for PBR involvement in metabolic regulation, expression of the CAR and PXR target genes, CYP2B6 and CYP3A4, was measured in human hepatocytes following treatment with a targeted PBR ligand set. Luciferase reporter assays with transiently expressed wild-type CAR (CAR1), splice variant CAR3, or PXR in HuH-7 cells were used to further study activation of these receptors. Four structurally related PBR ligands (benzothiazepines) differentially modulate CAR1, CAR3 and PXR activity. Benzothiazepine NF49 is an agonist ligand of CAR3, a partial agonist of PXR, exhibits greater inverse agonist activity on CAR1 than does PK11195, and is a new tool for studying these closely related nuclear receptors.     

A physiologically based kinetic (PBK) model describing plasma concentrations of quercetin and its metabolites in rats

Biological activities of flavonoids in vivo are ultimately dependent on the systemic bioavailability of the aglycones as well as their metabolites. In the present study, a physiologically based kinetic (PBK) model was developed to predict plasma concentrations of the flavonoid quercetin and its metabolites and to tentatively identify the regiospecificity of the major circulating metabolites. The model was developed based on in vitro metabolic parameters and by fitting kinetic parameters to literature available in vivo data. Both exposure to quercetin aglycone and to quercetin-4′-O-glucoside, for which in vivo data were available, were simulated. The predicted plasma concentrations of different metabolites adequately matched literature reported plasma concentrations of these metabolites in rats exposed to 4′-O-glucoside. The bioavailability of aglycone was predicted to be very low ranging from 0.004%-0.1% at different oral doses of quercetin or quercetin-4′-O-glucoside. Glucuronidation was a crucial pathway that limited the bioavailability of the aglycone, with 95–99% of the dose being converted to monoglucuronides within 1.5–2.5 h at different dose levels ranging from 0.1 to 50 mg/kg bw quercetin or quercetin-4′-O-glucoside. The fast metabolic conversion to monoglucuronides allowed these metabolites to further conjugate to di- and tri-conjugates. The regiospecificity of major circulating metabolites was observed to be dose-dependent. As we still lack in vivo kinetic data for many flavonoids, the developed model has a great potential to be used as a platform to build PBK models for other flavonoids as well as to predict the kinetics of flavonoids in humans.     

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

1. The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).

2. The treatment of WT rats for 7?d with 500?ppm NaPB in the diet and 100?mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.

3. Male C57BL/6?J (WT) and CAR KO/PXR KO mice were given 1000?ppm NaPB in the diet for 7?d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.

4. These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

     

The interaction potential of herbal medicinal products: a luminescence-based screening platform assessing effects on cytochrome P450 and its use with devil's claw (Harpagophyti radix) preparations

Objectives Potential interactions between herbal medicinal products and the cytochrome (CYP) P450 system are an important safety concern. We set out to develop a screening panel for assessing such interactions and use it to evaluate the interaction potential of devil's claw. Methods The panel consisted of luminescence‐based inhibition assays for CYP1A2, 2C9, 2C19, 2D6 and 3A4, and a reporter gene (luciferase) assay for pregnane X receptor (PXR) activation and CYP3A4 induction. Caftaric acid and chlorogenic acid, two compounds with strong fluorescence quenching properties, were used to demonstrate the assay's resistance to interference. We tested 10 commercial devil's claw preparations as well as harpagoside and harpagide, two important constituents of devil's claw. Key findings Five preparations were found to weakly inhibit CYP3A4 (IC50 124.2–327.6 µg/ml) and five were found to weakly activate PXR (EC50 10.21–169.3 µg/ml). Harpagoside and harpagide did not inhibit CYP3A4. In agreement with published data, bergamottin, a natural product known to interact with CYP3A4, was shown to inhibit CYP3A4 with an IC50 of 13.63 µm and activate PXR with an EC50 of 6.7 µm . Conclusions Devil's claw preparations are unlikely to have a clinically relevant effect on CYP function. The assay panel proved effective in screening devil's claw preparations, demonstrating its suitability for use with plant extracts. It showed superior sensitivity and resistance to interference.