An acute herb-drug interaction of Magnoliae Officinalis Cortex with methotrexate via inhibiting multidrug resistance-associated protein 2.

Magnoliae Officinalis Cortex (MOC), an herbal drug, contains polyphenolic lignans mainly magnolol (MN) and honokiol (HK). Methotrexate (MTX), a critical drug for cancers and autoimmune deseases, is a substrate of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). This study investigated the effect of coadministration of MOC on the pharmacokinetics of MTX and relevant mechanisms. Sprague-Dawley rats were orally administered MTX alone and with single dose (2.0 and 4.0 g/kg) and repeated seven doses of MOC (2.0 g/kg thrice daily for 2 days, the 7th dose given at 0.5 h before MTX). The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. The results showed that a single dose of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 352% and 308%, and a single dose at 4.0 g/kg significantly enhanced the AUC0-t and MRT by 362% and 291%, respectively. Likewise, repeated seven doses of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 461% and 334%, respectively. Mechanism studies indicated that the function of MRP2 was significantly inhibited by MN, HK and the serum metabolites of MOC (MOCM), whereas BCRP was not inhibited by MOCM. In conclusion, coadministration of MOC markedly enhanced the systemic exposure and mean residence time of MTX through inhibiting the MRP2-mediated excretion of MTX.

Pharmacokinetic interactions between tacrolimus and Wuzhi capsule in liver transplant recipients: Genetic polymorphisms affect the drug interaction.

Wuzhi capsule (WZC), a commonly used Chinese patent medicine to treat various types of liver dysfunction in China, increases the exposure of tacrolimus (TAC) in liver transplant recipients. However, this interaction has inter-individual variability, and the underlying mechanism remains unclear. Current research indicates that CYP3A4/5 and drug transporters influence the disposal of both drugs. This study aims to evaluate the association between TAC dose-adjusted trough concentration (C/D) and specific genetic polymorphisms of CYP3A4/5, drug transporters and pregnane x receptor (PXR), and plasma levels of major WZC components, deoxyschisandrin and γ-schisandrin, in liver transplant patients receiving both TAC and WZC. Liquid chromatography-tandem-mass spectrometry was used to detect the plasma levels of deoxyschisandrin and γ-schisandrin, and nine polymorphisms related to metabolic enzymes, transporters and PXR were genotyped by sequencing. A linear mixed model was utilized to assess the impact of the interaction between genetic variations and WZC components on TAC lnC/D. Our results indicate a significant association of TAC lnC/D with the plasma levels of deoxyschisandrin and γ-schisandrin. Univariate analysis demonstrated three polymorphisms in the genes ABCB1 (rs2032582), ABCC2 (rs2273697), ABCC2 (rs3740066), and PXR (rs3842689) interact with both deoxyschisandrin and γ-schisandrin, influencing the TAC lnC/D. In multiple regression model analysis, the interactions between deoxyschisandrin and both ABCB1 (rs2032582) and ABCC2 (rs3740066), post-operative day (ß < 0.001, p < 0.001), proton pump inhibitor use (ß = -0.152, p = 0.008), body mass index (ß = 0.057, p < 0.001), and ABCC2 (rs717620, ß = -0.563, p = 0.041), were identified as significant factors of TAC lnC/D, accounting for 47.89% of the inter-individual variation. In summary, this study elucidates the influence of the interaction between ABCB1 and ABCC2 polymorphisms with WZC on TAC lnC/D. These findings offer a scientific basis for their clinical interaction, potentially aiding in the individualized management of TAC therapy in liver transplant patients.

Clinical characteristics and follow-up of a newborn with Dubin-Johnson Syndrome: A clinical case report.

BACKGROUND: Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder, characterized by conjugated hyperbilirubinemia. This case report investigates the clinical characteristics and longitudinal outcomes of a neonate diagnosed with DJS. METHODS: A newborn presented with elevated bilirubin levels and abnormal liver enzyme readings. Comprehensive genetic evaluation was conducted, which included peripheral blood sample collection from the infant and both parents after obtaining informed consent and high-throughput trio exome sequencing was performed. The genetic analysis revealed 2 significant mutations in the ABCC2 gene on chromosome 10: the insertion mutation c.4237(exon30)_c.4238(exon30)ins CT, inherited from the father, and the missense mutation c.517(exon5)G > A, inherited from the mother. Both mutations were classified as pathogenic according to the ACMG 2015 guidelines, indicating a compound heterozygous inheritance pattern. The patient's treatment regimen included phototherapy, which was initiated to address her jaundice upon admission. To support liver function and regulate gut activity, oral ursodeoxycholic acid (20 mg/kg/dose, twice a day) and probiotics were administered. Additionally, a postdischarge medication plan involving a low-dose regimen of phenobarbital (3.5 mg/kg/dose, twice a day) was implemented for 2 weeks. RESULTS: During a 2-year follow-up after discharge, the infant's bilirubin levels significantly decreased, and liver enzymes, including GGT, progressively normalized. CONCLUSION: This case report enhances the understanding of DJS in neonates by emphasizing the clinical ramifications of compound heterozygous mutations within the ABCC2 gene and documenting the evolution of the disease. The gradual normalization of liver function tests suggests potential compensatory mechanisms in response to the genetic abnormalities in neonates with DJS. The correlation between the patient's genetic profile of compound heterozygosity and her milder clinical phenotype warrants attention, suggesting that this specific genetic configuration may be associated with less severe manifestations of the disease. The necessity for long-term follow-up is highlighted, recognizing that intercurrent stress conditions could influence the hepatic profile and potentially exacerbate symptoms. Such sustained observation is crucial to further delineate the genomic and clinical landscape of DJS, offering opportunities to refine prognostic and therapeutic approaches.

Proximal tubular dysfunction related to tenofovir in people living with HIV/AIDS: a pharmacogenetic study.

OBJECTIVES: The purpose of this case-control study was to verify the association between single nucleotide polymorphisms (SNPs) in genes encoding drug transporters related to tenofovir disoproxil fumarate (TDF) and proximal renal tubular dysfunction (PRTD), and the association between PRTD and clinical characteristics. METHODS: The 'cases' met the diagnostic criteria for PRTD, determined by the presence of two or more of the following abnormalities: non-diabetic glycosuria, metabolic acidosis, increased uric acid and phosphorus excretion, decreased tubular phosphorus reabsorption and ß2-microglobulinuria. We analyzed eight SNPs in ABCC2, ABCC4, ABCC10 and SLC28A2 genes. Genotyping was performed using real-time PCR. RESULTS: Of the 204 people living with HIV, 38 (18.6%) met the criteria for diagnosis of PRTD and 131 were male (64.2%), with a mean age of 49 years and a history of previous antiretroviral therapy for an average of 5 years. In the multivariate analysis, older individuals, TDF use, protease inhibitor, antihypertensives and anticonvulsants were associated with a risk of developing PRTD. Increased excretion of ß2microglobulin was associated with the A/G genotype of rsCC8187710 from ABCC2 ( P = 0.003) and the following genotypes of ABCC4 SNPs: A/G from rs1059751 ( P = 0.023), G/G from rs1059751 ( P = 0.030) and C/C of rs3742106 ( P = 0.041). The increase in the fraction of excreted phosphorus was associated with the C/T genotype of SNCC rsP40037 from ABCC2 ( P = 0.0041). CONCLUSIONS: The results indicate an important relationship between SNPs associated with these markers and changes in proximal renal tubule function, and thus support their use as biomarkers for the early detection of PRTD risk.

Deciphering the chemical constituents of Shengjiang Xiexin decoction by ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry and the impact of 20 characteristic components on multidrug resistance-associated protein 2 in the vesicular transport assay.

Shengjiang Xiexin decoction, a traditional Chinese medical formula, has been utilized to alleviate the delayed-onset diarrhea induced by irino tecan. However, the chemical constituents of this formula and the activities of its constituents remain unclear. In this study, ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry was employed to comprehensively analyze the chemical constituents of Shengjiang Xiexin decoction. A total of 270 components, including flavonoids, coumarins, triterpenoids, alkaloids, diarylheptanoids and others, were identified or characterized. Multidrug resistance-associated protein 2 is an efflux transporter responsible for regulating drug absorption. A total of 20 characteristic components from the formula were selected to evaluate their effects on the function of multidrug resistance-associated protein 2 using the vesicular transport assay. Glycyrrhizic acid and glycyrrhetinic acid were identified as potential multidrug resistance-associated protein 2 inhibitors, while 9 flavonoid aglycones increased the uptake of the substrate [3 H]-estradiol 17-ß-glucuronide in the vesicles. This was the first systematic investigation of the chemical constituents from Shengjiang Xiexin decoction and the effect of its characteristic components on the transporter. The results offered a basis for further exploring the detoxification mechanisms of this formula and its interactions with other drugs.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine.

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

An ester derivative of tenacigenin B from Marsdenia tenacissima (Roxb.) Wight et Arn reversed paclitaxel-induced MDR in vitro and in vivo by inhibiting both P-gp and MRP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima is a medicinal plant, used as a raw material for cancer treatment in China. In our previous studies, 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B (MT2), the main steroid aglycone isolated from M. tenacissima, was found to significantly enhance the antitumor activity of paclitaxel (PTX) in vivo. However, it is unclear whether MT2 reverses multidrug resistance (MDR) in tumors. AIM OF THE STUDY: To determine the role and mechanism of MT2 in reversing tumor MDR. MATERIALS AND METHODS: MDR cell line HeLa/Tax was established from the human cervical carcinoma cell line HeLa by long-term exposure to subtoxic concentrations of PTX and was used to evaluate the ability of MT2 to restore chemosensitivity of cells both in vitro and in a nude mouse model. The expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) was determined using western blotting and immunohistochemistry. The substrate transport function was assessed using an MDR function assay kit. The binding modes of MT2 and P-gp were determined using the conformation-sensitive anti-P-gp antibodies. The permeability and transport properties of MT2 were analyzed in Caco-2 cell monolayers. RESULTS: Compared to parental cells, HeLa/Tax cells overexpress P-gp and MRP2 and are approximately 100-360 fold more resistant to the anticancer drugs PTX, docetaxel, and vinblastine. MT2 at 5 or 10 µmol/L significantly increased the sensitivity of HeLa/Tax to these three anticancer drugs (18-56-fold decrease in IC50 value) and suppressed the expression of P-gp and MRP2. Knockdown of P-gp with small interfering RNA partially reversed MT2-induced sensitivity to PTX in HeLa/Tax cells. Moreover, MT2 directly inhibited P-gp-mediated substrate transport while interacting with membrane P-gp in non-substrate ways. MT2 was highly permeable and could not be transported in the Caco-2 cell monolayers. In nude mice bearing HeLa/Tax xenografts, the combination treatment with MT2 and PTX exerted a synergistic inhibitory effect on the growth of tumors and the expression of P-gp and MRP2 without increasing toxicity. CONCLUSION: MT2 is a potential agent for reversing MDR. It impedes membrane drug efflux pumps by suppressing P-gp and MRP2 expression, and directly inhibiting the transport function of P-gp.

Extract from Dioscorea bulbifera L. rhizomes aggravate pirarubicin-induced cardiotoxicity by inhibiting the expression of P-glycoprotein and multidrug resistance-associated protein 2 in the mouse liver.

Chinese herbal medicine is widely used because it has a good safety profile and few side effects. However, the risk of adverse drug reactions caused by herb-drug interactions (HDIs) is often overlooked. Therefore, the task of identifying possible HDIs and elucidating their mechanisms is of great significance for the prevention and treatment of HDI-related adverse reactions. Since extract from Dioscorea bulbifera L. rhizomes (DB) can cause various degrees of liver damage, it is speculated that HDIs may occur between DB extract and chemicals metabolized or excreted by the liver. Our study revealed that the cardiotoxicity of pirarubicin (THP) was increased by co-administration of DB, and the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) in the liver was inhibited by DB extract, which led to the accumulation of THP in heart tissue. In conclusion, there are risks of the co-administration of DB extract and THP. The mechanism of HDIs can be better revealed by targeting the efflux transporters.

Biliary excretion of arsenic by human HepaRG cells is stimulated by selenide and mediated by the multidrug resistance protein 2 (MRP2/ABCC2).

Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.

Artichoke leaf extract supplementation lowers hepatic oxidative stress and inflammation and increases multidrug resistance-associated protein 2 in mice fed a high-fat and high-cholesterol diet.

Artichoke (Cynara scolymus) leaf extract (ALE) contains many phytonutrients that may have antioxidant and anti-inflammation activities against many diseases including liver damage. To investigate the protective effects of ALE on high-fat and high-cholesterol (HFHC) diet-induced steatohepatitis and liver damage in mice, twenty-four female mice were fed an HFHC diet without or with 0.5% and 1% ALE supplementation for 6 weeks. The antioxidant and anti-inflammation activities and histological changes in the liver after ALE treatment were evaluated. The results show that ALE treatment reduced the HFHC diet-induced elevation of liver damage, as indicated by an increased alanine aminotransferase activity in plasma and perivenular inflammatory infiltrates in the liver. In addition, ALE ameliorated HFHC diet-induced depletion of hepatic glutathione (GSH) and elevations of plasma total cholesterol, triglyceride and hepatic triglyceride. ALE suppressed HFHC diet-induced accumulation of cholesterol precursors, including squalene and desmosterol in the liver. Higher hepatic GSH contents and activities of GSH-related enzymes were observed after ALE treatment. Higher expressions of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 (HO-1) were induced by the HFHC diet; however, ALE treatment reduced HO-1 expression. The NOD-like receptor protein 3, caspase-1, and interleukin-1ß protein and mRNA levels were reduced in the liver by ALE. A higher multidrug resistance-associated protein 2 expression in the liver was found after ALE treatment. These results suggest that ALE may ameliorate oxidative stress, inflammation and lipid metabolism disorder in HFHC diet-induced steatohepatitis and liver damage.

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza.

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Exploration for the real causative agents of licorice-induced pseudoaldosteronism.

I investigated the causative agents of licorice-induced pseudoaldosteronism, which is a frequent side effect of Japanese traditional Kampo medicines. Glycyrrhizin (GL), the main ingredient of licorice, is absorbed after being metabolized to glycyrrhetinic acid (GA) by intestinal bacteria, and then metabolized in liver to 3-monoglucuronyl-glycyrrhetinic acid (3MGA). In normal condition, 3MGA is excreted into bile via a multidrug resistance-related protein (Mrp) 2, therefore, 3MGA does not appear in blood circulation. However, under the dysfunction of Mrp2, 3MGA appears in the blood circulation and is excreted into the urine by not glomerular filtration but tubular secretion via organic anion transporter (OAT) 1 and 3. At this time, 3MGA inhibits type 2 11ß-hydroxysteroid dehydrogenase (11ßHSD2) in tubular cells to cause pseudoaldosteronism. Since GA is not the substrates of these transporters, GA cannot inhibit 11ßHSD2 in tubular cells. Therefore, it was considered that 3MGA was the causative agents of licorice-induced pseudoaldosteronism. After that, I isolated and identified three other GL metabolites, 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2), and 18ß-glycyrrhetyl-3-O-sulfate (3) from the urine of Mrp2-deficient rats orally treated with GA, and found that their blood and urinary concentrations were much higher than 3MGA and that their pharmacokinetic behaviors were similar to 3MGA. 3MGA was not detected in the blood of patients with pseudoaldosteronism who developed rhabdomyolysis due to licorice, and compound 3 was detected at a high concentration. In addition, a multicenter retrospective study was conducted using the serum and urine of 97 patients who took Kampo medicines containing licorice. Of a total of 97 patients, 67 detected GA in the serum (median 122 nM, 5 nM-1.8 µM) and 68 detected compound 3 (median 239 nM, 2 nM-4.2 µM), and there were no cases of detection of GL, 3MGA, compounds 1, and 2. High blood concentrations of compound 3 were associated with low plasma renin activity, plasma aldosterone levels, and serum potassium levels. It is highly probable that compound 3 is the true causative agent of pseudoaldosteronism.

Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell.

BACKGROUD: Dihydromyricetin (DMY), a natural flavonoid compound from the leaves of the Chinese medicinal herb Vitis heyneana, has been shown to have the potential to combat chemoresistance by inhibiting Nrf2/MRP2 signaling in colorectal cancer (CRC) cells. However, the precise underlying molecular mechanism and its therapeutic target are not well understood. PURPOSE: Our study aims to investigate the effects of DMY on multidrug resistance (MDR), and elucidate the underlying mechanisms. STUDY DESIGN: In vitro, HCT116/OXA and HCT8/VCR cells were employed as our MDR models. The cells were treated with DMY (50 µM) or MK-571 (50 µM) plus oxaliplatin (OXA) (10 µM) or vincristine (VCR) (10 µM) for 48 h. In vivo, we used BALB/c mice as a CRC xenograft mouse model. BALB/c mice were given DMY (100 mg/kg), OXA (5 mg/kg) and DMY (100 mg/kg) combined with OXA (5 mg/kg) via intraperitoneal route every 2 days per week for 4 weeks. METHODS: We used MTT and colony forming assays to detect DMY's ability to reverse MDR. Flow cytometric analysis was used to detect apoptosis. Immunocytochemistry was used to detect the localization of Nrf2 and NF-κB/p65. Western blot, qRT-PCR and reporter gene assays were employed to measure the protein and gene transcriptional levels (MRP2, Nrf2, NF-κB/p65). Moreover, chromatin immunoprecipitation (ChIP) assay was used to investigate the endogenous promoter occupancy of NF-κB/p65. Finally, immunohistochemistry and TUNEL staining were used to detect protein expression and apoptosis in vivo. RESULTS: DMY restored chemosensitivity (OXA and VCR) by inhibiting both MRP2 expression and its promoter activity in HCT116/OXA and HCT8/VCR cell lines. Furthermore, DMY could inhibit NF-κB/p65 expression, reducing NF-κB/p65 translocation to the nucleus to silence Nrf2 signaling, which is necessary for MRP2 expression. Overexpressing NF-κB/p65 expression reduced the reversal effect of DMY. In addition, NF-κB/p65 regulated Nrf2 expression by directly binding to its specific promoter region and activating its transcription. Finally, we proved that the combination of OXA and DMY has a synergistic tumor suppression effect in vivo. CONCLUSION: Our study provided a novel mechanism of DMY boosted chemosensitivity in human CRC. The downstream signals of DMY, NF-κB or Nrf2 could also be potential targets for the treatment of CRC.

Polysaccharides of vinegar-baked radix bupleuri promote the hepatic targeting effect of oxymatrine by regulating the protein expression of HNF4α, Mrp2, and OCT1.

ETHNOPHARMACOLOGICAL RELEVANCE: Vinegar-baked Radix Bupleuri (VBRB) is a processed form of Bupleurum chinense DC. As a well-known meridian-guiding drug, it is traditionally used as a component of traditional Chinese medicine formulations indicated for the treatment of liver diseases. However, the liver targeting component in VBRB remains unclear. Therefore, this study aims to explore the efficacy and mechanism of PSS (polysaccharides in Vinegar-baked Radix Bupleuri) in enhancing liver targeting. MATERIALS AND METHODS: Drug distribution of OM alone or combined with PSS was investigated in vivo. Relative uptake efficiency (RUE) and relative targeting efficiency (RTE) were calculated to evaluate liver targeting efficiency. The mRNA and protein expression of organic cation transporter 1 (OCT1), multi-drug resistance protein 2 (Mrp2), and hepatocyte nuclear factor 4α (HNF4α) in the liver were determined by q-PCR and Western blot. Then, AZT, the inhibitor of OCT1 and BI6015, the inhibitor of HNF4α were used to investigate regulatory mechanisms involved in the uptake of OM in the cell. At last, the role of PSS in the anti-hepatitis B virus (HBV) was explored on HepG2.2.15. RESULTS: PSS increased the AUC of OM in the liver and increase the RUE and RTE in the liver which indicated a liver targeting enhancing effect. The mRNA and protein expression of OCT1 was increased while Mrp2 and HNF4α decreased. PSS could increase the uptake of OM in HepG2 by increasing the protein expression of HNF4α and OCT1, while inhibited Mrp2. Moreover, PSS combined with OM could enhance the anti-HBV effect of OM. CONCLUSION: PSS enhanced the liver targeting efficiency and the underlying mechanism related to up-regulating the expression of OCT1 and HNF4α, while down-regulating of Mrp2. These results suggest that PSS may become a potential excipient and provide a new direction for new targeted research.

High folic acid intake increases methylation-dependent expression of Lsr and dysregulates hepatic cholesterol homeostasis.

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/- mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

Decreased drug resistance of bladder cancer using phytochemicals treatment.

The aim of the study is to investigate the ability of phytochemicals to overcome the multiple drug resistance (MDR) of bladder cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic sensitivity of T24-GCB cells, a GCB resistant cell line, to different phytochemicals, including capsaicin, quercetin, curcumin, and resveratrol, and their combination with gemcitabine. Western blot analysis was used to detect the expression of membranous ABCC2 and metabolic proteins, DCK, TK1, and TK2 in tumor cells. Animal models were used to confirm the treatment efficacy of phytochemicals in combination with gemcitabine to bladder cancer. The observed/expected ratio of cytotoxicity analysis revealed that capsaicin has synergistic effect with gemcitabine to T24-GCB cells in a dose-dependent pattern. Quercetin, curcumin, and resveratrol have additive effect with gemcitabine to T24-GCB cells. Capsaicin and quercetin alone and combination with gemcitabine decreased the expression of ABCC2 and DCK and TKs, in T24-GCB cells. On the contrary, resveratrol and curcumin alone and combination with gemcitabine increased the expression of ABCC2 but decreased cytoplasmic kinases simultaneously. In xenografted subcutaneous tumor model on nude mice, combination treatment of capsaicin and gemcitabine demonstrated the highest tumor suppression effect when compared to capsaicin or gemcitabine treatment alone. The MDR of bladder cancer is closely related to membranous ABCC2, cytoplasmic DCK, and TKs expression. Capsaicin owns the strongest synergistic cytotoxic effect of gemcitabine to T24-GCB cells. This combination regimen may provide as an adjunctive treatment for overcoming MDR in bladder cancer.

A Two-Tiered In Vitro Approach to De-Risk Drug Candidates for Potential Bile Salt Export Pump Inhibition Liabilities in Drug Discovery.

Hepatocellular accumulation of bile salts by inhibition of bile salt export pump (BSEP/ABCB11) may result in cholestasis and is one proposed mechanism of drug-induced liver injury (DILI). To understand the relationship between BSEP inhibition and DILI, we evaluated 64 DILI-positive and 57 DILI-negative compounds in BSEP, multidrug resistance protein (MRP) 2, MRP3, and MRP4 vesicular inhibition assays. An empirical cutoff (5 µM) for BSEP inhibition was established based on a relationship between BSEP IC50 values and the calculated maximal unbound concentration at the inlet of the human liver (fu*Iin,max, assay specificity = 98%). Including inhibition of MRP2-4 did not increase DILI predictivity. To further understand the potential to inhibit bile salt transport, a selected subset of 30 compounds were tested for inhibition of taurocholate (TCA) transport in a long-term human hepatocyte micropatterned co-culture (MPCC) system. The resulting IC50 for TCA in vitro biliary clearance and biliary excretion index (BEI) in MPCCs were compared with the compound's fu*Iin,max to assess potential risk for bile salt transport perturbation. The data show high specificity (89%). Nine out of 15 compounds showed an IC50 value in the BSEP vesicular assay of <5µM, but the BEI IC50 was more than 10-fold the fu*Iin,max, suggesting that inhibition of BSEP in vivo is unlikely. The data indicate that although BSEP inhibition measured in membrane vesicles correlates with DILI risk, that measurement of this assay activity is insufficient. A two-tiered strategy incorporating MPCCs is presented to reduce BSEP inhibition potential and improve DILI risk. SIGNIFICANCE STATEMENT: This work describes a two-tiered in vitro approach to de-risk compounds for potential bile salt export pump inhibition liabilities in drug discovery utilizing membrane vesicles and a long-term human hepatocyte micropatterned co-culture system. Cutoffs to maximize specificity were established based on in vitro data from a set of 121 DILI-positive and -negative compounds and associated calculated maximal unbound concentration at the inlet of the human liver based on the highest clinical dose.

Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters.

Chrysin is an abundant flavonoid in nature, and it is also contained by several dietary supplements. Chrysin is highly biotransformed in the body, during which conjugated metabolites chrysin-7-sulfate and chrysin-7-glucuronide are formed. These conjugates appear at considerably higher concentrations in the circulation than the parent compound. Based on previous studies, chrysin can interact with biotransformation enzymes and transporters; however, the interactions of its metabolites have been barely examined. In this in vitro study, the effects of chrysin, chrysin-7-sulfate, and chrysin-7-glucuronide on cytochrome P450 enzymes (2C9, 2C19, 3A4, and 2D6) as well as on organic anion-transporting polypeptides (OATPs; 1A2, 1B1, 1B3, and 2B1) and ATP binding cassette [P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance protein (BCRP)] transporters were investigated. Our observations revealed that chrysin conjugates are strong inhibitors of certain biotransformation enzymes (e.g., CYP2C9) and transporters (e.g., OATP1B1, OATP1B3, OATP2B1, and BCRP) examined. Therefore, the simultaneous administration of chrysin-containing dietary supplements with medications needs to be carefully considered due to the possible development of pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Chrysin-7-sulfate and chrysin-7-glucuronide are the major metabolites of flavonoid chrysin. In this study, we examined the effects of chrysin and its conjugates on cytochrome P450 enzymes and on organic anion-transporting polypeptides and ATP binding cassette transporters (P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2). Our results demonstrate that chrysin and/or its conjugates can significantly inhibit some of these proteins. Since chrysin is also contained by dietary supplements, high intake of chrysin may interrupt the transport and/or the biotransformation of drugs.

Circadian Clock-Controlled Drug Metabolism: Implications for Chronotherapeutics.

Dependence of drug metabolism on dosing time has long been recognized. However, only recently are the underlying mechanisms for circadian drug metabolism being clarified. Diurnal rhythmicity in expression of drug-metabolizing enzymes is believed to be a key factor determining circadian metabolism. Supporting the notion that biological rhythms are generated and maintained by the circadian clock, a number of diurnal enzymes are under the control of the circadian clock. In general, circadian clock genes generate and regulate diurnal rhythmicity in drug-metabolizing enzymes via transcriptional actions on one or two of three cis-elements (i.e., E-box, D-box, and Rev-erb response element or RAR-related orphan receptor response element). Additionally, cycling or clock-controlled nuclear receptors such as hepatocyte nuclear factor 4α and peroxisome proliferator-activated receptor γ are contributors to diurnal enzyme expression. These newly discovered mechanisms for each of the rhythmic enzymes are reviewed in this article. We also discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics. Our discussion is also extended to two diurnal transporters (P-glycoprotein and multidrug resistance-associated protein 2) that have an important role in drug absorption. Although the experimental evidence is lacking in metabolism-based chronoefficacy, circadian genes (e.g., Rev-erbα) as drug targets are shown to account for diurnal variability in drug efficacy. SIGNIFICANCE STATEMENT: Significant progress has been made in understanding the molecular mechanisms for generation of diurnal rhythmicity in drug-metabolizing enzymes. In this article, we review the newly discovered mechanisms for each of the rhythmic enzymes and discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics.