Regulation of BCRP expression and sulfasalazine pharmacokinetics by the nuclear receptor REV-ERBα.

BCRP (breast cancer resistance protein) is a crucial efflux transporter involved in the regulation of the pharmacokinetics and pharmacodynamics of a wide range of drugs. Herein, we aimed to investigate a potential role for the nuclear receptor REV-ERBα in the regulation of BCRP expression and sulfasalazine (a BCRP probe substrate) pharmacokinetics.Regulation of BCRP expression by REV-ERBα was assessed using Rev-erbα-/- mice and AML12 and CT26 cells. Pharmacokinetic analysis was performed with Rev-erbα-/- and wild-type mice after sulfasalazine administration.We found that the expression levels of BCRP mRNA and protein were downregulated in the liver and small intestine of Rev-erbα-dificient mice. In line with this, Rev-erbα ablation increased the systemic exposures of oral sulfasalazine.Positive regulation of BCRP expression and function by REV-ERBα was furtherly confirmed in AML12 and CT26 cells. Moreover, indirect regulation of Bcrp expression by REV-ERBα was potentially mediated by a negative transcription factor DEC2, which is a downstream target of REV-ERBα.In conclusion, REV-ERBα positively regulates BCRP expression in mice, thereby affecting sulfasalazine pharmacokinetics.

Visuoauditory Associative Memory Established with Cholecystokinin Under Anesthesia Is Retrieved in Behavioral Contexts.

Plastic change in neuronal connectivity is the foundation of memory encoding. It is not clear whether the changes during anesthesia can alter subsequent behavior. Here, we demonstrated that in male rodents under anesthesia, a visual stimulus (VS) was associated with electrical stimulation of the auditory cortex or natural auditory stimulus in the presence of cholecystokinin (CCK), which guided the animals' behavior in a two-choice auditory task. Auditory neurons became responsive to the VS after the pairings. Moreover, high-frequency stimulation of axon terminals of entorhinal CCK neurons in the auditory cortex enabled LTP of the visual response in the auditory cortex. Such pairing during anesthesia also generated VS-induced freezing in an auditory fear conditioning task. Finally, we verified that direct inputs from the entorhinal CCK neurons and the visual cortex enabled the above neural plasticity in the auditory cortex. Our findings suggest that CCK-enabled visuoauditory association during anesthesia can be translated to the subsequent behavior action.SIGNIFICANCE STATEMENT Our study provides strong evidence for the hypothesis that cholecystokinin plays an essential role in the formation of cross-modal associative memory. Moreover, we demonstrated that an entorhinal-neocortical circuit underlies such neural plasticity, which will be helpful to understand the mechanisms of memory formation and retrieval in the brain.

A novel pyrrole-imidazole polyamide targets Aurora kinase A and suppresses tumor growth in vivo.

Aurora kinase A (Aurora A) plays a critical role in regulating cell mitotic progression and has been considered as a promising drug target for cancer therapy. To develop a novel molecule targeting Aurora A with high selectivity and efficacy, we designed and synthesized a pyrrole-imidazole polyamide (PIP) Hoechst conjugate, PIP-Ht, targeting to a cell-cycle regulated DNA sequence locating at the promoter of human Aurora A gene (AURKA). PIP-Ht potently suppressed AURKA promoter activities, mRNA expression and protein level, induced tumor cell cycle delay and inhibited tumor cell proliferation in vitro. Furthermore, subcutaneous injection of PIP-Ht into mice bearing human cancer xenografts induced significant tumor growth suppression and cell apoptosis. Collectively, PIP-Ht exhibits the potential as an effective therapeutic candidate for the tumor treatment.

Coibamide A kills cancer cells through inhibiting autophagy.

Natural products are useful tools for biological mechanism research and drug discovery. Due to the excellent tumor cell growth inhibitory profile and sub-nanomolar potency, Coibamide A (CA), an N-methyl-stabilized depsipeptide isolated from marine cyanobacterium, has been considered as a promising lead compound for cancer treatment. However, the molecular anti-cancer mechanism of the action of CA remains unclear. Here, we showed that CA treatment induced caspase-independent cell death in breast cancer cells. CA treatment also led to severe lysosome defects, which was ascribed to the impaired glycosylation of lysosome membrane protein LAMP1 and LAMP2. As a consequence, the autophagosome-lysosome fusion was blocked upon CA treatment. In addition, we presented evidence that this autophagy defect partially contributed to the CA treatment-induced tumor cell death. Together, our work uncovers a novel mechanism underlying the anti-cancer action of CA, which will promote its further application for cancer therapy.

Diurnal hepatic CYP3A11 contributes to chronotoxicity of the pyrrolizidine alkaloid retrorsine in mice.

1. Retrorsine (RTS) is a pyrrolizidine alkaloid (distributed in many medicinal plants) that has significant hepatotoxicity. Here, we aimed to determine the daily variations in RTS hepatotoxicity (chronotoxicity) in mice, and to investigate the role of metabolism in generating RTS chronotoxicity.2. Acute toxicity and pharmacokinetic studies were performed with mice after RTS administration at different times of the day. Hepatotoxicity was assessed by measuring plasma ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels. mRNA and proteins were determined by qPCR and Western blotting, respectively. Time-dependent in vitro metabolism of RTS was assessed by using mouse liver microsomes.3. We found that RTS toxicity was more severe in the dark phase (zeitgeber time 14 or ZT14 and ZT18) than in the light phase (ZT2 and ZT6). This chronotoxicity was associated with a dosing time difference in the systemic exposures of RTS and a pyrrolic ester metabolite (a cause of hepatotoxicity, measured by the levels of pyrrole-GSH conjugate and pyrrole-protein adducts due to a high chemical reactivity). Moreover, the CYP3A11 (a major enzyme for RTS bioactivation) inhibitor ketoconazole decreased the production of pyrrole-GSH conjugate and abrogated diurnal rhythm in RTS metabolism. In addition, E4bp4 (a circadian regulator of Cyp3a11) ablation abolished the rhythm of CYP3A11 expression and abrogated the dosing time-dependency of RTS toxicity.4. In conclusion, RTS chronotoxicity in mice was attributed to time-varying hepatic metabolism regulated by the circadian clock. Our findings have implications for reducing pyrrolizidine alkaloid-induced toxicity via a chronotherapeutic approach.

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.

Disposition of Mianserin and Cyclizine in UGT2B10-Overexpressing Human Embryonic Kidney 293 Cells: Identification of UGT2B10 as a Novel N-Glucosidation Enzyme and Breast Cancer Resistance Protein as an N-Glucoside Transporter.

UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and detoxification of amine-containing chemicals; however, the disposition mechanisms for amines via UGT metabolism are not fully clear. We aimed to investigate a potential role of UGT2B10 in N-glucosidation and to determine the transporters for the excretion of N-glucosides. We first established a human embryonic kidney cell line 293 (HEK293) that stably overexpressed UGT2B10. Incubation of mianserin or cyclizine with the cells generated one N-glucuronide and one N-glucoside. Chemical inhibition (using specific chemical inhibitor Ko143) and biologic inhibition [using specific short hairpin RNA of breast cancer resistance protein (BCRP)] resulted in a significant reduction in efflux of N-glucuronide. Similar results were observed when multidrug resistance-associated protein (MRP4) was inhibited. Furthermore, inhibition of BCRP led to increased intracellular N-glucoside, whereas inhibition of MRP4 caused no changes in disposition of N-glucoside. Overall, the data indicated that BCRP, not MRP4, was responsible for the excretion of N-glucosides, whereas both BCRP and MRP4 contributed to excretion of N-glucuronides. Interestingly, downregulation of N-glucuronidation led to increased N-glucosidation in the cells, supporting the glucosidation as a potential complementary pathway for conventional glucuronidation. In conclusion, UGT2B10 was for the first time identified as an N-glucosidation enzyme. Generated N-glucosides were excreted primarily by the BCRP transporter.

REV-ERBα Regulates CYP7A1 Through Repression of Liver Receptor Homolog-1.

Nuclear heme receptor reverse erythroblastosis virus (REV-ERB) α (a transcriptional repressor) is known to regulate cholesterol 7α-hydroxylase (CYP7A1) and bile acid synthesis. However, the mechanism for REV-ERBα regulation of CYP7A1 remains elusive. Here, we investigate the role of LRH-1 in REV-ERBα regulation of CYP7A1 and cholesterol metabolism. We first characterized the tertiary amine N-(4-chloro-2-methylbenzyl)-N-(4-chlorobenzyl)-1-(5-nitrothiophen-2-yl)methanamine (GSK2945) as a highly specific Rev-erbα/REV-ERBα antagonist using cell-based assays and confirmed expression of Rev-erbα in mouse liver. GSK2945 treatment increased hepatic mouse cholesterol 7α-hydroxylase (Cyp7a1) level and lowered plasma cholesterol in wild-type mice. Likewise, the compound increased the expression and microsomal activity of Cyp7a1 in hypercholesterolemic mice. This coincided with reduced plasma and liver cholesterol and enhanced production of bile acids. Increased levels of Cyp7a1/CYP7A1 were also found in mouse and human primary hepatocytes after GSK2945 treatment. In these experiments, we observed parallel increases in Lrh-1/LRH-1 (a known hepatic activator of Cyp7a1/CYP7A1) mRNA and protein. Luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Lrh-1/LRH-1 was a direct Rev-erbα/REV-ERBα target gene. Furthermore, conditional deletion of Lrh-1 in the liver abrogated the regulatory effects of Rev-erbα on Cyp7a1 and cholesterol metabolism in mice. In conclusion, Rev-erbα regulates Cyp7a1 and cholesterol metabolism through its repression of the Lrh-1 receptor. Targeting the REV-ERBα/LRH-1 axis may represent a novel approach for management of cholesterol-related diseases.

Transcriptional Regulation of Human UDP-Glucuronosyltransferase 2B10 by Farnesoid X Receptor in Human Hepatoma HepG2 Cells.

Little is known about transcriptional regulators of UDP-glucuronosyltransferase 2B10 (UGT2B10), an enzyme known to glucuronidate many chemicals and drugs such as nicotine and tricyclic antidepressants. Here, we uncovered that UGT2B10 was transcriptionally regulated by farnesoid X receptor (FXR), the bile acid sensing nuclear receptor. GW4064 and chenodeoxycholic acid (two specific FXR agonists) treatment of HepG2 cells led to a significant increase in the mRNA level of UGT2B10. The treated cells also showed enhanced glucuronidation activities toward amitriptyline (an UGT2B10 probe substrate). In reporter gene assays, the extent of UGT2B10 activation by the FXR agonists was positively correlated with the amount of cotransfected FXR. Consistently, knockdown of FXR by shRNA attenuated the induction effect on UGT2B10 expression. Furthermore, a combination of electrophoretic mobility shift assay and chromatin immunoprecipitation showed that the FXR receptor trans-activated UGT2B10 through its specific binding to the -209- to -197-bp region (an IR1 element) of the UGT2B10 promoter. In summary, our results for the first time established FXR as a transcriptional regulator of human UGT2B10.

Structure- and isoform-specific glucuronidation of six curcumin analogs.

1. In the present study, we aimed to characterize the glucuronidation of six curcumin analogs (i.e. RAO-3, RAO-8, RAO-9, RAO-18, RAO-19, and RAO-23) derived from galangal using human liver microsomes (HLM) and twelve expressed UGT enzymes. 2. Formation of glucuronide was confirmed using high-resolution mass spectrometry. Single glucuronide metabolite was generated from each of six curcumin analogs. The fragmentation patterns were analyzed and were found to differ significantly between alcoholic and phenolic glucuronides. 3. All six curcumin analogs except one (RAO-23) underwent significant glucuronidation in HLM and expressed UGT enzymes. In general, the methoxy group (close to the phenolic hydroxyl group) enhanced the glucuronidation liability of the curcumin analogs. 4. UGT1A9 and UGT2B7 were primarily responsible for the glucuronidation of two alcoholic analogs (RAO-3 and RAO-18). By contrast, UGT1A9 and four UGT2Bs (UGT2B4, 2B7, 2B15 and 2B17) played important roles in conjugating three phenolic analogs (RAO-8, RAO-9, and RAO-19). Interestingly, the conjugated double bonds system (in the aliphatic chain) was crucial to the substrate selectivity of gastrointestinal UGTs (i.e. UGT1A7, 1A8 and 1A10). 5. In conclusion, glucuronidation of six curcumin analogs from galangal were structure- and isoform-specific. The knowledge should be useful in identifying a curcumin analog with improved metabolic property.

In vitro characterization of belinostat glucuronidation: demonstration of both UGT1A1 and UGT2B7 as the main contributing isozymes.

1. Belinostat is a histone deacetylase inhibitor that has been approved for the treatment of peripheral T-cell lymphoma. This study aimed to identify the UDP-glucuronosyltransferase (UGT) enzymes responsible for belinostat glucuronidation through kinetic determination using recombinant enzymes with determined enzyme concentrations. 2. The rate of glucuronidation was determined by incubation of belinostat with enzyme preparations. Kinetic parameters such as Km and Vmax were derived by fitting an appropriate model to the glucuronidation data. The role of active UGT enzymes to belinostat metabolism was evaluated using inhibition experiments and activity correlation analyses. 3. Human liver microsomes generated a glucuronide metabolite (i.e. belinostat glucuronide) from belinostat. The glucuronide structure was confirmed by high-resolution mass spectrometry as well as the fragmentation pattern. Of 12 test UGT enzymes, only four (UGT1A1, 1A3, 2B4, and 2B7) showed metabolic activities toward belinostat. UGT1A1 was the most active enzyme, followed by UGT2B7, 1A3, and 2B4. Kinetic profiles for UGT1A1, 1A3, 2B4, and 2B7 were well described by Michaelis-Menten, Michaelis-Menten, Hill equation, and substrate inhibition equation, respectively. 4. Glucuronidation of belinostat was markedly inhibited by emodin and apigenin (two potent inhibitors of UGT1A1), and by quinidine and diclofenac sodium (two selective inhibitors of UGT2B7). Belinostat glucuronidation was found to be significantly correlated with ß-estradiol 3-O-glucuronidation and zidovudine glucuronidation. 5. It was concluded that in addition to UGT1A1, UGT2B7 was also an important contributor to belinostat glucuronidation.

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound.

1. Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes. 2. The glucuronide of bakuchiol was confirmed by liquid chromatography-mass spectrometry (LC-MS) and ß-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol. 3. Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CLint value of 100 µl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r = 0.933; p < 0.001), 3-O-glucuronidation of ß-estradiol (r = 0.719; p < 0.01) and significantly correlated with 24-O-glucuronidation of CDCA (r = 0.594; p < 0.05). In addition, a marked species difference existed in hepatic glucuronidation of bakuchiol. 4. In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.

Glucose-Based Mesoporous Carbon Nanospheres as Functional Carriers for Oral Delivery of Amphiphobic Raloxifene: Insights into the Bioavailability Enhancement and Lymphatic Transport.

PURPOSE: Oral therapy with raloxifene (RXF), an amphiphobic drug for remedy of the postmenopausal osteoporosis and estrogen-dependent breast cancer, is less effective due to its poor bioavailability (2% or so). This work aimed to devise mesoporous carbon nanospheres (MCNs) for oral delivery of RXF and evaluate their performance in bioavailability enhancement and lymphatic transport. METHODS: Glucose-based MCNs were fabricated by hydrothermal reaction followed by high-temperature activation. RXF-loaded MCNs (RXF-MCNs) were prepared by solvent-diffusion/high-pressure homogenization and stabilized by phospholipid. RXF-MCNs were fully characterized by particle size, morphology, in vitro drug release and metabolism, in vivo pharmacokinetics and lymphatic transport, and ex vivo fluorescent imaging. RESULTS: The prepared RXF-MCNs were 230 nm around in particle size, showing high entrapment efficiency (95.35%) and satisfactory physical stability. The oral bioavailability of RXF was enhanced by 2.07 folds through MCNs compared with RXF suspensions in rats. It was shown that reduced intestinal metabolism due to entrapment into MCNs, active transcellular uptake and increased lymphatic transport were responsible for enhanced bioavailability as a result of transport improvement. CONCLUSIONS: The results suggest that MCNs are suitable nanocarriers for oral delivery of poorly bioavailable RXF.

Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1.

1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes. 2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC-MS/MS and/or NMR analyses. 3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors. 4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible. 5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.

Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells.

Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 µM, and Ksi= 58.1 µM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 µM, and Ksi= 49.1 µM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 µM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-3'-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

Identification of glucuronidation and biliary excretion as the main mechanisms for gossypol clearance: in vivo and in vitro evidence.

1. The natural polyphenol gossypol possesses many therapeutic benefits. Here we aim to determine the elimination pathways of gossypol in vivo and in vitro. 2. Metabolite elucidation of gossypol was performed using UPLC-QTOF/MS coupled with Metabolynx analysis. Clearance of gossypol was evaluated in bile duct cannulated rats and in the single-pass perfused rat intestine model. In vitro glucuronidation of gossypol was characterized using liver and intestine microsomes as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes. 3. Analysis of rat plasma, urine, and feces revealed glucuronidation as the only metabolic pathway for gossypol. In bile duct cannulated rats, considerable amounts of glucuronides (G1, G2 and G3; 58.8-83.2% of dose) and parent compound (5.0-20%) were excreted into bile after IV administration. In the perfused rat intestine model, gossypol was well absorbed with a [Formula: see text] (the dimensionless effective permeability) value of 4.4. Significant amounts of glucuronides (G1, G2 and G3) were excreted into the gut lumen (2.5%) and into the bile (4.8%). Biliary excretion of unchanged gossypol (6.0%) was comparable to that of glucuronides. Further, gossypol was subjected to rapid glucuronidation by liver and intestine microsomes. Reaction phenotyping showed that multiple UGT1A enzymes (including UGT1A1, 1A3, 1A7 and 1A8) are mainly responsible for gossypol metabolism. 4. In conclusion, glucuronidation was the only metabolic pathway for gossypol in rats. Excretion of unchanged gossypol into bile was also an important clearance mechanism.

Kinetic Characterization of Estradiol Glucuronidation by Liver Microsomes and Expressed UGT Enzymes: The Effects of Organic Solvents.

BACKGROUND AND OBJECTIVE: In vitro glucuronidation of 17ß-estradiol (estradiol) is often performed to assess the role of uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) in xenobiotic/drug metabolism. The objective of this study was to determine the effects of four commonly used organic solvents [i.e., dimethyl sulfoxide (DMSO), methanol, ethanol, and acetonitrile] on the glucuronidation kinetics of estradiol, which can be glucuronidated at C3 and C17 positions. METHODS: The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. RESULTS: In human liver microsomes (HLM), methanol, ethanol, and acetonitrile significantly altered estradiol glucuronidation kinetics with increased Vmax (up to 2.6-fold) and CLmax (up to 2.8-fold) values. Altered estradiol glucuronidation in HLM was deduced to be attributed to the enhanced metabolic activities of UGT1A1 and UGT2B7, whose activities differ at the two glucuronidation positions. The effects of organic solvents on estradiol glucuronidation were glucuronidation position-, isozyme-, and solvent-specific. Furthermore, both ethanol and acetonitrile have a greater tendency to modify the glucuronidation activity of estradiol in animal liver microsomes. CONCLUSION: Organic solvents such as methanol, ethanol, and acetonitrile showed great potential in adjusting the glucuronidation of estradiol. DMSO is the most suitable solvent due to its minimal influence on estradiol glucuronidation. Researchers should be cautious in selecting appropriate solvents to get accurate results when assessing the metabolism of a new chemical entity.

Quantitative analysis of Cistanches Herba using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry combined with chemometric methods.

We aim to determine the chemical constituents of three species of Cistanches Herba using HPLC coupled with diode array detection and high-resolution MS. Ten phenylethanoid glycosides were identified and further quantified as marker substances by HPLC coupled with diode array detection method. The separation was conducted using an Agilent TC-C18 column with 0.1% formic acid and methanol as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of 36 batches of Cistanche plants. The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were used to compare different species of Cistanches Herba, leading to successful classification of the Cistanche samples in accordance with their origins. In conclusion, this study provides a chemical basis for quality control of Cistanches Herba.

Pharmacokinetics-based Chronotherapy.

Dosing time-dependency of pharmacokinetics (or chronopharmacokinetics) has been long recognized. Studies in recent years have revealed that daily rhythmicity in expression of drug-metabolizing enzymes and transporters (DMETs) are key factors determining chronopharmacokinetics. In this article, we briefly summarize current knowledge with respect to circadian mechanisms of DMETs and discuss how rhythmic DMETs are translated to drug chronoeffects. More importantly, we present our perspectives on pharmacokinetics-based chronotherapy.

Puerariae lobatae radix protects against UVB-induced skin aging via antagonism of REV-ERBα in mice.

Puerariae lobatae radix (PLR) is a wildly used herbal medicine. Here we aimed to assess the PLR efficacy against UVB (ultraviolet-B)-induced skin aging and to determine the mechanisms thereof. We found a significant protective effect of PLR (topical application) on UVB-induced skin aging in mice, as evidenced by reduced skin wrinkles, epidermal thickness, and MDA (malondialdehyde) content as well as increased levels of HYP (hydroxyproline) and SOD (superoxide dismutase) in the skin. In the meantime, Mmp-1, p21 and p53 levels were decreased in the skin of PLR-treated mice. Anti-aging effects of PLR were also confirmed in L929 cells. Furthermore, PLR up-regulated skin expression of BMAL1, which is a known regulator of aging by promoting Nrf2 and antioxidant enzymes. Consistently, Nrf2 and several genes (i.e., Prdx6, Sod1, and Sod2) encoding antioxidant enzymes in the skin were increased in PLR-treated mice. Moreover, based on Gal4 chimeric assay, Bmal1 reporter gene and expression assays, we identified PLR as an antagonist of REV-ERBα that can increase Bmal1 expression. Intriguingly, loss of Rev-erbα protected mice against UVB-induced skin aging and abrogated the protective effect of PLR. In conclusion, PLR acts as an antagonist of REV-ERBα and promotes the expression of BMAL1 to protect against skin aging in mice.