Heterodimerization of Human UDP-Glucuronosyltransferase 1A9 and UDP-Glucuronosyltransferase 2B7 Alters Their Glucuronidation Activities.

Human UDP-glucuronosyltransferases (UGTs) play a pivotal role as prominent phase II metabolic enzymes, mediating the glucuronidation of both endobiotics and xenobiotics. Dimerization greatly modulates the enzymatic activities of UGTs. In this study, we examined the influence of three mutations (H35A, H268Y, and N68A/N315A) and four truncations (signal peptide, single transmembrane helix, cytosolic tail, and di-lysine motif) in UGT2B7 on its heterodimerization with wild-type UGT1A9, using a Bac-to-Bac expression system. We employed quantitative fluorescence resonance energy transfer (FRET) techniques and co-immunoprecipitation assays to evaluate the formation of heterodimers between UGT1A9 and UGT2B7 allozymes. Furthermore, we evaluated the glucuronidation activities of the heterodimers using zidovudine and propofol as substrates for UGT2B7 and UGT1A9, respectively. Our findings revealed that the histidine residue at codon 35 was involved in the dimeric interaction, as evidenced by the FRET efficiencies and catalytic activities. Interestingly, the signal peptide and single transmembrane helix domain of UGT2B7 had no impact on the protein-protein interaction. These results provide valuable insights for a comprehensive understanding of UGT1A9/UGT2B7 heterodimer formation and its association with glucuronidation activity. SIGNIFICANCE STATEMENT: Our findings revealed that the H35A mutation in UGT2B7 affected the affinity of protein-protein interaction, leading to discernable variations in fluorescence resonance energy transfer efficiencies and catalytic activity. Furthermore, the signal peptide and single transmembrane helix domain of UGT2B7 did not influence heterodimer formation. These results provide valuable insights into the combined effects of polymorphisms and protein-protein interactions on the catalytic activity of UGT1A9 and UGT2B7, enhancing our understanding of UGT dimerization and its impact on metabolite formation.

Oligopeptide/Histidine Transporter PHT1 and PHT2 - Function, Regulation, and Pathophysiological Implications Specifically in Immunoregulation.

The oligopeptide/histidine transporters PHT1 and PHT2, two mammalian solute carrier family 15A proteins, mediate the transmembrane transport of histidine and some di/tripeptides via proton gradient. PHT1 and PHT2 are distributed in a variety of tissues but are preferentially expressed in immune cells and localize to the lysosome-related organelles. Studies have reported the relationships between PHT1/PHT2 and immune diseases. PHT1 and PHT2 participate in the regulation of lysosomal homeostasis and lysosome-associated signaling pathways through their transport and nontransport functions, playing important roles in inflammatory diseases. In this review, we summarize recent research on PHT1 and PHT2, aiming to provide reference for their further biological research and as targets for drug design.

Bilirubin Reduces the Uptake of Estrogen Precursors and the Followed Synthesis of Estradiol in Human Placental Syncytiotrophoblasts via Inhibition and Downregulation of Organic Anion Transporter 4.

Estrogen biosynthesis in human placental trophoblasts requires the human organic anion transporter 4 (hOAT4)-mediated uptake of fetal derived precursors such as dehydroepiandrosterone-3-sulfate (DHEAS) and 16α-hydroxy-DHEA-S (16α-OH-DHEAS). Scant information is available concerning the contribution of fetal metabolites on the impact of placental estrogen precursor transport and the followed estrogen synthesis. This study substantiated the roles of bilirubin as well as bile acids (taurochenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid) on the inhibition of hOAT4-mediated uptake of probe substrate 6-carboxylfluorescein and DHEAS in stably transfected hOAT4-Chinese hamster ovary cells, with the IC50 of 1.53 and 0.98 µM on 6-carboxylfluorescein and DHEAS, respectively, for bilirubin, and 90.2, 129, 16.4, and 12.3 µM on 6-CF for taurochenodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, and chenodeoxycholic acid. Bilirubin (2.5-10 µM) concentration-dependently inhibited the accumulation of estradiol precursor DHEAS in human choriocarcinoma JEG-3 cells (reduced by 60% at 10 µM) and primary human trophoblast cells (reduced by 80% at 10 µM). Further study confirmed that bilirubin (0.625-2.5 µM) concentration-dependently reduced the synthesis and secretion of estradiol in primary human trophoblast cells, among which 2.5 µM of bilirubin reduced the synthesis of estradiol by 30% and secretion by 35%. In addition, immunostaining and Western blot results revealed a distinct downregulation of hOAT4 protein expression in primary human trophoblast cells pretreated with 2.5 µM of bilirubin. In conclusion, this study demonstrated that bilirubin reduced the uptake of estrogen precursors and the followed synthesis of estradiol in human placenta via inhibition and downregulation of organic anion transporter 4. SIGNIFICANCE STATEMENT: Fetal metabolites, especially bilirubin, were first identified with significant inhibitory effects on the hOAT4-mediated uptake of estrogen precursor DHEAS in hOAT4-CHO, JEG-3 and PHTCs. Bilirubin concentration-dependently suppressed the estradiol synthesis and secretion in PHTCs treated with DHEAS, which was synchronized with the decline of hOAT4 protein expression. Additionally, those identified bile acids exhibited a weaker inhibitory effect on the secretion of estradiol.

Downregulation of glucose-6-phosphatase expression contributes to fluoxetine-induced hepatic steatosis.

Fluoxetine is a first-line selective serotonin reuptake inhibitor widely applied for the treatment of depression; however, it induces abnormal hepatic lipid metabolism. Considering decreased expression or function of glucose-6-phosphatase (G6Pase), a key enzyme in gluconeogenesis, or the upregulation of fatty acid uptake, causes hepatic lipid accumulation. The aim of this study was to elucidate whether G6Pase regulation and fatty acid uptake alteration contribute to fluoxetine-induced abnormal hepatic lipid metabolism. Our study revealed that 8-week oral administration of fluoxetine dose-dependently increased hepatic triglyceride, causing hepatic steatosis. Concomitantly, the expression of G6Pase in mouse livers and primary mouse hepatocytes (PMHs) was downregulated in a concentration-dependent manner. Furthermore, fluoxetine increased the concentrations of glucose-6-phosphate (G6Pase substrate) and acetyl CoA (the substrate for de novo lipogenesis) in mouse livers. Additionally, fluoxetine also induced lipid accumulation and downregulated G6Pase expression in HepG2 cells. However, the uptake of green fluorescent fatty acid (BODIPY™ FL C16) in PMHs was not changed after fluoxetine treatment, indicating that fluoxetine-induced hepatic steatosis was not associated with fatty acid uptake alteration. In conclusion, fluoxetine downregulated hepatic G6Pase expression, subsequently enhanced the transformation of glucose to lipid, and ultimately resulted in hepatic steatosis, but with no impact on fatty acid uptake.

Epigenetic Regulation of Differentially Expressed Drug-Metabolizing Enzymes in Cancer.

Drug metabolism is a biotransformation process of drugs, catalyzed by drug-metabolizing enzymes (DMEs), including phase I DMEs and phase II DMEs. The aberrant expression of DMEs occurs in the different stages of cancer. It can contribute to the development of cancer and lead to individual variations in drug response by affecting the metabolic process of carcinogen and anticancer drugs. Apart from genetic polymorphisms, which we know the most about, current evidence indicates that epigenetic regulation is also central to the expression of DMEs. This review summarizes differentially expressed DMEs in cancer and related epigenetic changes, including DNA methylation, histone modification, and noncoding RNAs. Exploring the epigenetic regulation of differentially expressed DMEs can provide a basis for implementing individualized and rationalized medication. Meanwhile, it can promote the development of new biomarkers and targets for the diagnosis, treatment, and prognosis of cancer. SIGNIFICANCE STATEMENT: This review summarizes the aberrant expression of DMEs in cancer and the related epigenetic regulation of differentially expressed DMEs. Exploring the epigenetic regulatory mechanism of DMEs in cancer can help us to understand the role of DMEs in cancer progression and chemoresistance. Also, it provides a basis for developing new biomarkers and targets for the diagnosis, treatment, and prognosis of cancer.

Organic Cation Transporter 1 and 3 Contribute to the High Accumulation of Dehydrocorydaline in the Heart.

Dehydrocorydaline (DHC), one of the main active components of Corydalis yanhusuo, is an important remedy for the treatment of coronary heart disease. Our previous study revealed a higher unbound concentration of DHC in the heart than plasma of mice after oral administration of C. yanhusuo extract or DHC, but the underlying uptake mechanism remains unelucidated. In our investigations, we studied the transport mechanism of DHC in transgenic cells, primary neonatal rat cardiomyocytes, and animal experiments. Using quantitative real-time polymerase chain reaction and Western blotting, we found that uptake transporters expressed in the mouse heart include organic cation transporter 1/3 (OCT1/3) and carnitine/organic cation transporter 1/2 (OCTN1/2). The accumulation experiments in transfected cells showed that DHC was a substrate of OCT1 and OCT3, with K m of 11.29 ± 3.3 and 8.96 ± 3.7 µM, respectively, but not a substrate of OCTN1/2. Additionally, a higher efflux level (1.71-fold of MDCK-mock) of DHC was observed in MDCK-MDR1 cells than in MDCK-mock cells. Therefore, DHC is a weak substrate for MDR1. Studies using primary neonatal rat cardiomyocytes showed that OCT1/3 inhibitors (quinidine, decynium-22, and levo-tetrahydropalmatine) prevented the accumulation of DHC, whereas OCTN2 inhibitors (mildronate and l-carnitine) did not affect its accumulation. Moreover, the coadministration of OCT1/3 inhibitors (levo-tetrahydropalmatine, THP) decreased the concentration of DHC in the mouse heart. Based on these findings, DHC may be accumulated partly by OCT1/3 transporters and excreted by MDR1 in the heart. THP could alter the distribution of DHC in the mouse heart. SIGNIFICANCE STATEMENT: We reported the cardiac transport mechanism of dehydrocorydaline, highly distributed to the heart after oral administration of Corydalis yanhusuo extract or dehydrocorydaline only. Dehydrocorydaline (an OCT1/3 and MDR1 substrate) accumulation in primary cardiomyocytes may be related to the transport activity of OCT1/3. This ability, hampered by selective inhibitors (levo-tetrahydropalmatine, an inhibitor of OCT1/3), causes a nearly 40% reduction in exposure of the heart to dehydrocorydaline. These results suggest that OCT1/3 may contribute to the uptake of dehydrocorydaline in the heart.

Rapid quantification of tenofovir in umbilical cord plasma and amniotic fluid in hepatitis B mono-infected pregnant women during labor by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Tenofovir (TFV) is a first-line antiviral agent against hepatitis B virus (HBV) and is recommended for the prevention of mother-to-infant transmission of HBV. To study the distribution of TFV in umbilical cord plasma and amniotic fluid of HBV-infected pregnant women, a rapid and sensitive method for TFV determination was developed and validated. METHODS: The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analytes were separated on an Acquity UPLC HSS T3 column under gradient elution with methanol and 0.01% ammonia solution in 10 mM ammonium acetate/water. This is the first reported method for the determination of TFV using alkaline rather than acidic mobile phases. Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed. RESULTS: Detection of TFV was achieved within 4 min. The calibration curves for TFV quantification showed excellent linearity in the range of 1-500 ng/mL. The intra- and interbatch precision and accuracy ranged from -4.35% to 6.92%. This method was successfully applied to determination of samples from 50 HBV mono-infected women undergoing tenofovir disoproxil fumarate therapy. The mean concentrations of TFV in the umbilical cord and amniotic fluid samples were 29.2 (4.6-86) and 470.9 (156-902) ng/mL, respectively, which showed a moderate positive correlation (r = 0.5299, P<0.001). CONCLUSIONS: A simple, rapid but sensitive bioanalytical method to determine TFV concentration in both umbilical cord plasma and amniotic fluid using LC/MS/MS was developed and applied to HBV-infected women during labor who were undergoing TDF therapy, which will help us understand the efficacy and safety of tenofovir during pregnancy.

SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides To Enhance the Nucleotide-Binding Oligomerization Domain-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages.

There is increasing evidence that proton-coupled oligopeptide transporters (POTs) can transport bacterially derived chemotactic peptides and therefore reside at the critical interface of innate immune responses and regulation. However, there is substantial contention regarding how these bacterial peptides access the cytosol to exert their effects and which POTs are involved in facilitating this process. Thus, the current study proposed to determine the (sub)cellular expression and functional activity of POTs in macrophages derived from mouse bone marrow and to evaluate the effect of specific POT deletion on the production of inflammatory cytokines in wild-type, Pept2 knockout and Pht1 knockout mice. We found that PEPT2 and PHT1 were highly expressed and functionally active in mouse macrophages, but PEPT1 was absent. The fluorescent imaging of muramyl dipeptide-rhodamine clearly demonstrated that PEPT2 was expressed on the plasma membrane of macrophages, whereas PHT1 was expressed on endosomal membranes. Moreover, both transporters could significantly influence the effect of bacterially derived peptide ligands on cytokine stimulation, as shown by the reduced responses in Pept2 knockout and Pht1 knockout mice as compared with wild-type animals. Taken as a whole, our results point to PEPT2 (at plasma membranes) and PHT1 (at endosomal membranes) working in concert to optimize the uptake of bacterial ligands into the cytosol of macrophages, thereby enhancing the production of proinflammatory cytokines. This new paradigm offers significant insight into potential drug development strategies along with transporter-targeted therapies for endocrine, inflammatory, and autoimmune diseases.

Multiple Drug Transporters Contribute to the Placental Transfer of Emtricitabine.

Emtricitabine (FTC) is a first-line antiviral drug recommended for the treatment of AIDS during pregnancy. We hypothesized that transporters located in the placenta contribute to FTC transfer across the blood-placenta barrier. BeWo cells, cell models with stable or transient expression of transporter genes, primary human trophoblast cells (PHTCs), and small interfering RNAs (siRNAs) were applied to demonstrate which transporters were involved. FTC accumulation in BeWo cells was reduced markedly by inhibitors of equilibrative nucleoside transporters (ENTs), concentrative nucleoside transporters (CNTs), organic cation transporters (OCTs), and organic cation/carnitine transporter 1 (OCTN1) and increased by inhibitors of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs). ENT1, CNT1, OCTN1, MRP1/2/3, and BCRP, but not ENT2, CNT3, OCTN2, or multidrug resistance protein 1 (MDR1), were found to transport FTC. FTC accumulation in PHTCs was decreased significantly by inhibitors of ENTs and OCTN1. These results suggest that ENT1, CNT1, and OCTN1 probably contribute to FTC uptake from maternal circulation to trophoblasts and that ENT1, CNT1, and MRP1 are likely involved in FTC transport between trophoblasts and fetal blood, whereas BCRP and MRP1/2/3 facilitate FTC transport from trophoblasts to maternal circulation. Coexistence of tenofovir or efavirenz with FTC in the cell medium did not influence FTC accumulation in BeWo cells or PHTCs.

Maternal Plasma l-Carnitine Reduction During Pregnancy Is Mainly Attributed to OCTN2-Mediated Placental Uptake and Does Not Result in Maternal Hepatic Fatty Acid ß-Oxidation Decline.

l-Carnitine (l-Car) plays a crucial role in fatty acid ß-oxidation. However, the plasma l-Car concentration in women markedly declines during pregnancy, but the underlying mechanism and its consequences on maternal hepatic ß-oxidation have not yet been clarified. Our results showed that the plasma l-Car level in mice at gestation day (GD) 18 was significantly lower than that in nonpregnant mice, and the mean fetal-to-maternal plasma l-Car ratio in GD 18 mice was 3.0. Carnitine/organic cation transporter 2 (OCTN2) was highly expressed in mouse and human placenta and upregulated as gestation proceeds in human placenta, whereas expressions of carnitine transporter (CT) 1, CT2, and amino acid transporter B0,+ were extremely low. Further study revealed that renal peroxisome proliferator-activated receptor α (PPARα) and OCTN2 were downregulated and the renal l-Car level was reduced, whereas the urinary excretion of l-Car was lower in late pregnant mice than in nonpregnant mice. Meanwhile, progesterone (pregnancy-related hormone) downregulated the expression of renal OCTN2 via PPARα-mediated pathway, and inhibited the activity of OCTN2, but estradiol, corticosterone, and cortisol did not. Unexpectedly, the maternal hepatic level of l-Car and ß-hydroxybutyrate (an indicator of mitochondrial ß-oxidation), and mRNA levels of several enzymes involved in fatty acid ß-oxidation in GD 18 mice were higher than that in nonpregnant mice. In conclusion, OCTN2 mediated l-Car transfer across the placenta played a major role in maternal plasma l-Car reduction during pregnancy, which did not subsequently result in maternal hepatic fatty acid ß-oxidation decrease.

Clozapine-induced reduction of l-carnitine reabsorption via inhibition/down-regulation of renal carnitine/organic cation transporter 2 contributes to liver lipid metabolic disorder in mice.

Clozapine, an atypical antipsychotic drug, is widely utilized for the treatment of schizophrenia; however, clozapine-induced metabolic disorders, such as fatty liver and weight gain, warrant increased attention. Considering the crucial role of l-carnitine (L-Car) in fatty acid oxidation and carnitine/organic cation transporter (OCTN) 2 in renal reabsorption of L-Car, we aimed to study whether clozapine-induced liver lipid metabolic disorder is associated with L-Car dysregulation via inhibition/down-regulation of renal OCTN2. Our results reveal that clozapine inhibits L-Car uptake in MDCK-hOCTN2 cells with an IC50 value of 1.78 µM. Additionally, clozapine significantly reduces the uptake of L-Car in HK-2 cells, mouse primary cultured proximal tubular (mPCPT) cells and HepG2 cells. Acute (intraperitoneal injection) and 21-day successive oral administration of clozapine at 12.5, 25, and 50 mg/kg to mice resulted in 2-3-fold greater renal excretion of L-Car than in the vehicle group, and the concentration of L-Car in plasma and liver was significantly decreased. Concomitantly, mRNA and protein levels of mOctn2 in the kidney were markedly down regulated. Additionally, 28-day oral administration of clozapine induced increased triglyceride (TG) and total cholesterol (TCHO) levels in mouse livers, while L-Car (40 mg/kg - 1 g/kg) attenuated clozapine-induced liver TG and TCHO increase in a dose-dependent manner. These results indicate that clozapine-induced reduction of L-Car reabsorption via inhibition/down-regulation of renal OCTN2 contributes to liver lipid metabolic disorder. L-Car supplementation is probably an effective strategy to attenuate clozapine-induced abnormal lipid metabolism.

Jatrorrhizine reduces 5-HT and NE uptake via inhibition of uptake-2 transporters and produces antidepressant-like action in mice.

1. Jatrorrhizine is an active ingredient found in various traditional Chinese medicinal plants. Based on our previous finding that jatrorrhizine was a potent inhibitor of OCT2 and OCT3, the aim of the present study was to explore whether jatrorrhizine has an antidepressant-like action action via inhibition of uptake-2 transporters. 2. In vitro uptake tests showed that jatrorrhizine strongly inhibited PMAT-mediated MPP+ uptake with an IC50 value of 1.05 µM and reduced 5-HT and NE uptake mediated by hOCT2, hOCT3 and hPMAT with IC50 values of 0.1-1 µM (for OCT2 and OCT3) and 1-10 µM (for PMAT). 3. In mouse synaptosomes, jatrorrhizine suppressed 5-HT and NE uptake in a concentration dependently manner, where the role of uptake-2 inhibition is significant. 4. The antidepressant-like action of jatrorrhizine was evaluated by mouse tail suspension test (TST). The TST showed that one week of jatrorrhizine (5, 10 and 20 mg/kg, i.p.) or venlafaxine (20 mg/kg, i.g.) can significantly reduce the duration of immobility when compared with vehicle control group. 5. The concentration of jatrorrhizine shows a dose-dependent increase in brain tissues. 6. Our study suggested that jatrorrhizine might be used as an antidepressant agent via inhibition of uptake-2 transporters.

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells.

The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d3-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d3-l-histidine and low affinity for GlySar, with Km values of 16.3 ± 1.9 µM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.

Co-administration of nuciferine reduces the concentration of metformin in liver via differential inhibition of hepatic drug transporter OCT1 and MATE1.

Nuciferine (NF), one of the main and effective components in Nelumbo nucifera Gaertn. leaf extracts, is a promising drug candidate for the treatment of obesity-related diseases, while metformin is a first line therapeutic drug for type 2 diabetes mellitus. Since nuciferine and metformin are likely to be co-administered, the aim of the present study was to evaluate whether co-administration of nuciferine would influence the liver (target tissue) distribution and the anti-diabetic effect of metformin by inhibiting hepatic organic cation transporter 1 (OCT1) and multidrug and toxin extrusion 1 (MATE1). The data demonstrated that nuciferine significantly reduced metformin accumulation in MDCK cells stably expressing human OCT1 (MDCK-hOCT1) or hMATE1 (MDCK-hMATE1), and primary cultured mouse hepatocytes. Furthermore, the presence of nuciferine in the basal compartment caused a concentration-dependent reduction of intracellular metformin accumulation in MDCK-hOCT1/hMATE1 cell monolayers. Compared with the metformin treatment-alone group, co-administration of nuciferine (40 mg/kg) markedly reduced the metformin concentration in mouse livers at 30 and 60 min after a single oral dose of metformin (200 mg/kg), and subsequently impaired the glucose-lowering effect of metformin (200 mg/kg), but the glucose-lowering effect became no different at 90 and 120 min. Therefore, nuciferine influenced the liver concentration and glucose-lowering effect of metformin only for a period of time after dose, administration of nuciferine and metformin with an interval might prevent the drug-drug interaction mediated by OCT1 and MATE1.

Development and Validation of a HPLC-ESI-MS/MS Method for Simultaneous Quantification of Fourteen Alkaloids in Mouse Plasma after Oral Administration of the Extract of Corydalis yanhusuo Tuber: Application to Pharmacokinetic Study.

The tuber of Corydalis yanhusuo is a famous traditional Chinese medicine and found to have potent pharmacological effects, such as antinociceptive, antitumor, antibacterial, anti-inflammatory, and anti-depressive activities. Although there are several methods to be developed for the analysis and detection of the bioactive ingredients' alkaloids, so far, only few prominent alkaloids could be quantified, and in vitro and in vivo changes of comprehensive alkaloids after oral administration are still little known. In this study, we first developed a simple and sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to quantify the comprehensive alkaloids of extracts of C. yanhusuo in mouse plasma, using nitidine chloride as an internal standard. As results, at least fourteen alkaloids, including an aporphine (oxoglaucine), a protopine (protopine), five tertiary alkaloids (corydaline, tetrahydroberberine, tetrahydropalmatine, tetrahydrocolumbamine, and tetrahydrocoptisine) and seven quaternary alkaloids (columbamine, palmatine, berberine, epiberberine, coptisine, jatrorrhizine, and dehydrocorydaline) could be well quantified simultaneously in mouse plasma. The lower limits of quantification were greater than, or equal to, 0.67 ng/mL, and the average matrix effects ranged from 96.4% to 114.3%. The mean extraction recoveries of quality control samples were over 71.40%, and the precision and accuracy were within the acceptable limits. All the analytes were shown to be stable under different storage conditions. Then the established method was successfully applied to investigate the pharmacokinetics of these alkaloids after oral administration of the extract of Corydalis yanhusuo in mice. To the best of our knowledge, this is the first document to report the comprehensive and simultaneous analyses of alkaloids of C. yanhusuo in mouse plasma. It was efficient and useful for comprehensive pharmacokinetic and metabolomic analyses of these complex alkaloids after drug administration.

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants.

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 µM) > mouse (15.6 µM) ≈ rat (11.9 µM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans.

Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir.

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na+ free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.

Histone H3 Lysine 4 Trimethylation, Lysine 27 Trimethylation, and Lysine 27 Acetylation Contribute to the Transcriptional Repression of Solute Carrier Family 47 Member 2 in Renal Cell Carcinoma.

In recent years, finding effective biomarkers for identifying early stage cancer and predicating prognosis is crucial for renal cell carcinoma (RCC) diagnosis and treatment. In this study, a dramatic decrease of the solute carrier family 47 member 2 (SLC47A2) mRNA in RCC comparing with the paired adjacent nontumor tissues from patients at low Tumor Node Metastasis stage was observed. Thus, patients with SLC47A2 transcriptional repression are susceptible to RCC. Little is known about the regulation mechanism of SLC47A2 We found that it was a bivalent gene that was enriched with both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). Loss of mixed lineage leukemia 1 binding at the gene promoter caused decreased H3K4me3 enrichment and H3K4me3/H3K27me3 ratio, and subsequently repressed the expression of SLC47A2 These two epigenetic markers modulated the expression of SLC47A2 simultaneously, suggesting the regulation pattern for bivalent genes. Histone H3 lysine 27 acetylation also contributed to the expression of SLC47A2 An E2F1-histone deacetylase 10 complex catalyzed deacetylation of H3K27, then prevented the enrichment of H3K4me3, and finally reduced SLC47A2 expression. Consequently, the combined effect of all these factors determined SLC47A2 transcriptional repression in RCC tissues.

The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants.

Uridine diphosphate-glucuronosyltransferase (UGT) 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGT1A1 and UGT1A9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7*1 (wild type), UGT2B7*71S (A71S, 211G>T), UGT2B7*2 (H268Y, 802C>T), UGT2B7*5 (D398N, 1192G>A), and double-recombinant allozymes formed by the dimerization of UGT1A9*1 (wild type), UGT1A9*2 (C3Y, 8G>A), UGT1A9*3 (M33T, 98T>C), UGT1A9*5 (D256N, 766G>A), UGT1A1 (wild type) with its splice variant UGT1A1b were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7*1, UGT2B7*2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7*5 allozymes perform opposite effects. The double allozymes of UGT1A9*2 or UGT1A9*5 with UGT2B7 tend to produce M6G. Interestingly, the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGT1A9*2-UGT2B7*1 double enzyme has the lowest M3G:M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGT1A1 and UGT1A9 and their allelic variants can regioselectively affect the generation of two metabolites of morphine via altering the CLint ratios of M3G to M6G. These results may predict the effectiveness of morphine antinociception in individualized opioid treatment.

Utilizing single- and double-transfected cell models expressing human organic anion transporter 1 and human cytochrome P450 1A2 to investigate the interactions with ingredients of herbal medicines.

1. Cell models expressing human drug transporters and enzymes are useful tools to understand the process of drug disposition in vitro. However, no study on transfected cells stably co-expressing human organic anion transporter 1 (hOAT1) and/or human cytochrome P450 1A2 (hCYP1A2) is available. In this study, cell models stably expressing hOAT1 and/or hCYP1A2 were established, and were used to investigate the interactions of ingredients of herbal medicines (IHMs) with hOAT1 and/or hCYP1A2. 2. The MDCK cells were stable transfected with recombinant plasmids expressing hOAT1 and/or hCYP1A2. Cellular uptake assay and CYP450 activity assay showed that the transfected cells were available. A marked high expression of hOAT1 and hCYP1A2 mRNA was also validated by quantitative RT-PCR. Totally 6 IHMs which significantly inhibited the activity of hOAT1 were screened out by employing hOAT1 expressing cells. The contribution of hOAT1 and hCYP1A2 to the toxicity of aristolochic acid I (AAI) was further determined. Compared to mock cells, all transfected cells showed a decrease in viability after being treated with AAI. 3. A method to establish transfected cell expressing drug metabolism enzymes and/or transporters was provided in our study. Three IHMs (dihydrotanshinone I, cryptotanshinone, and tanshinone I) were confirmed as novel inhibitors of hOAT1. Furthermore, a synergistic effect of hOAT1 and hCYP1A2 on AAI-induced toxicity was also observed in this investigation.