Effect of food and polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on pharmacokinetics of abiraterone and its metabolites in Chinese volunteers.

AIMS: Abiraterone acetate, a prodrug of abiraterone (ABI), provides an efficient therapeutic option for metastatic castration-resistant prostate cancer patients. ABI undergoes extensive metabolism in vivo and is transformed into active metabolites Δ4 -abiraterone and 3-keto-5α-abiraterone as well as inactive metabolites abiraterone sulfate and abiraterone N-oxide sulfate. We aimed to examine the effect of polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on the pharmacokinetics of ABI and its metabolites. METHODS: In this study, 81 healthy Chinese subjects were enrolled and divided into 2 groups for fasted (n = 45) and fed (n = 36) studies. Plasma samples were collected after administering a 250 mg abiraterone acetate tablet followed by liquid chromatography-tandem mass spectrometry analysis. Genotyping was performed on a MassARRAY system. The association between SLCO2B1, CYP3A4, UGT1A4 genotype and pharmacokinetic parameters of ABI and its metabolites was assessed. RESULTS: Food effect study demonstrated high fat meal remarkedly increased systemic exposure of ABI and its metabolites. The geometric mean ratio and 90% confidence interval of area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration (AUC0-t ) and maximum plasma concentration (Cmax ) of ABI in fed state vs. fasted state were 351.64% (286.86%-431.04%) and 478.45% (390.01%-586.94%), respectively, while the corresponding results were ranging from 145.11% to 269.42% and 150.10% to 478.45% for AUC0-t and Cmax of ABI metabolites in fed state vs. fasted state, respectively. The SLCO2B1 rs1077858 had a significant influence on AUC0-t and Cmax , while 7 other SLCO2B1 variants prolonged half-life of ABI under both fasted and fed conditions. As for ABI metabolites, the systemic exposure of Δ4 -abiraterone, abiraterone sulfate and abiraterone N-oxide sulfate as well as the elimination of 3-keto-5α-abiraterone were significantly affected by SLCO2B1 polymorphisms. Polymorphisms in CYP3A4 and UGT1A4 did not significantly affect pharmacokinetics of ABI and its metabolites. CONCLUSION: Polymorphisms in SLCO2B1 were significantly related to the pharmacokinetic variability of ABI and its metabolites under both fasted and fed conditions.

Au3+ -Functionalized UiO-67 Metal-Organic Framework Nanoparticles: O2•- and •OH Generating Nanozymes and Their Antibacterial Functions.

The synthesis and characterization of Au3+ -modified UiO-67 metal-organic framework nanoparticles, Au3+ -NMOFs, are described. The Au3+ -NMOFs reveal dual oxidase-like and peroxidase-like activities and act as an active catalyst for the catalyzed generation of O2•- under aerobic conditions or •OH in the presence of H2 O2 . The two reactive oxygen species (ROS) agents O2•- and •OH are cooperatively formed by Au3+ -NMOFs under aerobic conditions, and in the presence of H2 O2. The Au3+ -NMOFs are applied as an effective catalyst for the generation ROS agents for antibacterial and wound healing applications. Effective antibacterial cell death and inhibition of cell proliferation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacterial colonies are demonstrated in the presence of the Au3+ -NMOFs. In addition, in vivo experiments demonstrate effective wound healing of mice wounds infected by S. aureus, treated by the Au3+ -NMOFs.

mPEG2k-PCLx Polymeric Micelles Influence Pharmacokinetics and Hypoglycemic Efficacy of Metformin through Inhibition of Organic Cation Transporters in Rats.

Increasing evidence has shown that nanocarriers have effects on several efflux drug transporters. To date, little is known about whether influx transporters are also modulated. Herein, we investigated the impact of amphiphilic polymer micelles on the uptake function of organic cation transporters (OCTs) and the influence on the pharmacokinetics and pharmacodynamics of metformin, a well-characterized substrate of OCTs. Five types of polymeric micelles (mPEG2k-PCL2k, mPEG2k-PCL3.5k, mPEG2k-PCL5k, mPEG2k-PCL7.5k, and mPEG2k-PCL10k) were prepared to evaluate the inhibition of hOCT1-3-overexpressing Madin-Darby canine kidney cells. The mPEG2k-PCLx micelles played an inhibitory role above the critical micelle concentration. The inhibitory potency could be ranked as mPEG2k-PCL2k > mPEG2k-PCL3.5k > mPEG2k-PCL5k > mPEG2k-PCL7.5k > mPEG2k-PCL10k, which negatively declined with the increase of molecular weight of the hydrophobic segment. The inhibitory effects of polymeric micelles on the hOCT1 isoform were the most pronounced, with the lowest IC50 values ranging from 0.106 to 0.280 mg/mL. The mPEG2k-PCL2k micelles distinctly increased the plasma concentration of metformin and significantly decreased Vss by 35.6% (p < 0.05) after seven consecutive treatments in rats, which was interrelated with the restrained metformin distribution in the liver and kidney. The uptake inhibition of micelles on hepatic and renal rOcts also diminished the glucose-lowering effect of metformin and fasting insulin levels in the oral glucose tolerance test. Consistent with the inhibitory effects, the mRNA and protein levels of rOct1 and rOct2 were decreased in the liver, kidney, and small intestine. The present study demonstrated that mPEG2k-PCLx micelles could inhibit the transport function of OCTs, indicating a potential risk of drug-drug interactions during concomitant medication of nanomedicine with organic cationic drugs.

Simultaneous LC-MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self-microemulsifying drug-delivery systems.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid-liquid extraction using methyl tert-butyl ether-dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB-C18 column (100 × 2.1 mm, 3 µm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide-d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra- and inter-day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel-loaded self-microemulsifying drug-delivery systems.

Accurate determination of a novel vasodilatory ß-blocker TJ0711 using LC-MS/MS: Resolution of an isobaric metabolite interference in dog plasma.

A rapid, robust and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for bioanalysis of TJ0711, a novel vasodilatory ß-blocker in dog plasma. This assay is able to chromatographically separate TJ0711 from its isobaric metabolite as well as glucuronide conjugates. Chromatographic separation was achieved on a Welch Ultimate-XB C18 column (2.1 × 100 mm, 3 µm). The analyte and internal standard (propranolol) were extracted from plasma by liquid-liquid extraction using ethyl acetate. The mass spectrometric detection was carried out in positive ion multiple reaction monitoring mode. Good linearity was obtained over the concentration range of 0.5-500 ng/mL (r > 0.99) for TJ0711. Moreover, the method had good accuracy (RE ranging from -2.70 to -0.32%) and precision (RSD < 7.55%). TJ0711 was stable in dog plasma for at least 6 h at ambient temperature, for at least 30 days at -20°C and after three freeze-thaw cycles. This method was successfully applied to a preclinical pharmacokinetic study and the results demonstrated linear pharmacokinetics of TJ0711 over a dose range from 0.03 to 0.3 mg/kg. No significant gender differences were observed in TJ0711 plasma pharmacokinetic parameters.

Enantioselective determination of 1-[4-(2-methoxyethyl)phenoxy]-3-[2-(2-methoxyphenoxy)ethylamino]-2-propanol hydrochloride, a novel antihypertensive agent, in rat plasma and tissues by liquid chromatography-tandem mass spectrometry.

Enantioselective biodistribution studies of 1-[4-(2-methoxyethyl)phenoxy]-3-[2-(2-methoxyphenoxy)ethylamino]-2-propanol hydrochloride (TJ0711), a novel antihypertensive agent, require the accurate and precise quantification of each TJ0711 enantiomer in biological fluids and tissues. Here we report a simple and sensitive liquid chromatography with tandem mass spectrometry method for simultaneous determination of (R)-TJ0711 and (S)-TJ0711 in rat plasma and tissue samples using protein precipitation. The influence of column type, temperature, mobile phase composition, and flow rate on the retention and enantioselectivity was evaluated. The separation of the TJ0711 enantiomers was ultimately achieved on a SUMICHIRAL OA-2500 column in 15 min using isocratic elution with ethanol/hexane (40:60) at a flow rate of 0.8 mL/min. Good linearities of spiked analyte concentration from 5 to 2000 ng/mL were achieved and the correlation coefficients (R) were greater than 0.99. The intra- and inter-day accuracy and precision for both analytes were <15% at all concentration levels, and the extraction recoveries were consistent among the five quality control concentrations. This assay was successfully applied to quantify plasma and tissue concentrations of TJ0711 enantiomers in a preclinical study.

[Quantification of amlodipine in human plasma by LC-MS/MS method: elimination of matrix effects by improving chromatographic conditions].

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of amlodipine in human plasma. The influence of alkalizer, extraction solvent and the chromatographic conditions on the matrix effects was investigated. The stable isotope-labeled amlodipine (amlodipine-d(4)) was used as an internal standard. Sample preparation involved simple liquid-liquid extraction procedure using methyl tertiary butyl ether. Chromatographic separation was achieved on a Welch Ultimate XB-C18 (100 mm × 2.1 mm, 3 µm) column with acetonitrile/2 mmol·L(-1) ammonium formate (p H 3.0） under gradient condition at a flow rate of 0.6 m L·min(-1). Detection was performed using electrospray ionization (ESI) in positive ion multiple reaction monitoring (MRM) mode. The linear range of the analyte was 0.1-20.0 µg·L(-1), with the lower limit of quantitation (LLOQ) of 0.1 µg·L(-1). The matrix factor for low, medium, high concentration quality control samples and internal standard was (93.9 ± 1.8)%, (95.8 ± 4.9)%, (93.9 ± 1.5)% and (97.9 ± 5.3)%, respectively. The method showed excellent specificity, linearity, intra-day and inter-day accuracy and precision, extraction recovery and stability, according to the CFDA guidance for bioanalytical method validation. The matrix effect was significantly improved through optimizing the chromatographic conditions. This economical, simple, robust, sensitive and specific method is entirely able to meet the requirement of the determination of amlodipine in human plasma samples obtained from bioequivalence studies.

PBPK modeling to unravel nonlinear pharmacokinetics of verapamil to estimate the fractional clearance for verapamil N-demethylation in the recirculating rat liver preparation.

We applied physiologically based pharmacokinetic (PBPK) modeling to study the dose-dependent metabolism and excretion of verapamil and its preformed metabolite, norverapamil, to unravel the kinetics of norverapamil formation via N-demethylation. Various initial verapamil (1, 50, and 100 µM) and preformed norverapamil (1.5 and 5 µM) concentrations, perfused at 12 ml/min, were investigated in the perfused rat liver preparation. Perfusate and bile were collected over 90 minutes, and livers were harvested at the end of perfusion for high-performance liquid chromatography analysis. After correction for the adsorption of 10%-25% dose verapamil and norverapamil onto Tygon tubing and binding to albumin and red blood cell, fitting of verapamil and formed and preformed norverapamil data with ADAPT5 revealed nonlinearity for protein binding, N-demethylation (V(max,met1)(VER --> NOR) = 96.6 ± 33.4 nmol/min; K(m,met1)(VER --> NOR) = 10.4 ± 4.1 µM), formation of other metabolites (V(max,met2(VER -->others) 288 ± 51 nmol/min; K(m.met2)(VER -->others )= 14.1 ± 4.9 µM), as well as biliary excretion (V(max,sec)(VER)= 0.911 ± 0.505 nmol/min; K(m,sec)(VER) = 4.75 ± 2.29 µM). The hepatic clearance of verapamil (CL(L)(VER) decreased with the dose (8.16-10.2 ml/min), with values remaining high relative to perfusate blood flow rate among the doses. The hepatic clearance of preformed norverapamil (11 ml/min) remained unchanged for the concentrations studied and approximated perfusate blood flow rate, suggesting a high norverapamil extraction ratio. The fractional formation of norverapamil and biliary excretion of verapamil based on fitted constants were 31.1% and 0.64% of CL(L)(VER), respectively. Enantiomeric disposition and auto-inhibition of verapamil failed to perturb these estimaties according to PBPK modeling, due to the low values of the Michaelis-Menten constant, Km, and inhibition parameter, kI.

Influence of dose and route of administration on the enantioselective pharmacokinetics of TJ0711 hydrochloride in rats.

The enantioselective pharmacokinetics of TJ0711 hydrochloride were studied in rats given different doses of rac-TJ0711 hydrochloride via intravenous and oral routes. R- and S-TJ0711 hydrochloride were both rapidly absorbed, and the average AUC0-∞ of R-TJ0711 hydrochloride was greater than that of S-TJ0711 hydrochloride after intragastric administration, with an R/S AUC ratio 1.11 and 1.35 for 30 and 50 mg/kg dose group, respectively. In contrast, the average AUC0-∞ of R-TJ0711 hydrochloride was smaller than that of S-TJ0711 hydrochloride after intravenous injection, with an R/S AUC ratio 0.57 and 0.73 for 10 and 20 mg/kg dose group, respectively. R-TJ0711 hydrochloride plasma half-lives were shorter than those of S-TJ0711 hydrochloride for all groups. AUC0-4h and Cmax between the two enantiomers were significantly different after oral administration of 50 mg/kg dose of the racemate, while no significant differences between the two enantiomers were found for all the pharmacokinetic parameters of the 30 mg/kg dose group. Significant differences between the two enantiomers were detected for nearly all the pharmacokinetic parameters after intravenous administration, except for the VZ of 20 mg/kg dose group. This study suggests that dose and route of administration will influence the enantioselectivity in the pharmacokinetics of TJ0711 hydrochloride in rats.

Study of the in vitro metabolism of TJ0711 using ultra high performance liquid chromatography with quadrupole time-of-flight and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry.

TJ0711 (1-[4-(2-methoxyethyl)phenoxy]-3-[2-(2-methoxyphenoxy)ethylamino]-2-propanol) is a novel ß-adrenoreceptor blocker with vasodilating activity. The aim of this study was to investigate the in vitro metabolic properties of TJ0711 from both qualitative and quantitative aspects using mouse, rat, dog, and human liver microsomes as well as rat hepatocytes. Two modern liquid chromatography with tandem mass spectrometry systems, ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry, were utilized for the analysis. To better characterize the metabolic pathways of TJ0711, two major metabolites were incubated under the same conditions as that for TJ0711. TJ0711 was extensively metabolized in vitro, and a total of 34 metabolites, including 19 phase I and 15 phase II metabolites, were identified. Similar metabolite profiles were observed among species, and demethylation, hydroxylation, carboxylic acid formation, and glucuronidation were proposed as the major metabolic routes. Significant interspecies differences were observed in the metabolic stability studies of TJ0711. Furthermore, gender differences were significant in mice, rats, and dogs, but were negligible in humans. The valuable information provided in this work will be useful in planning and interpreting further pharmacokinetic, in vivo metabolism and toxicological studies of this novel ß-blocker.

Nitric oxide releasing d-α-tocopheryl polyethylene glycol succinate for enhancing antitumor activity of doxorubicin.

Nitric oxide (NO) has attracted much attention for its antitumor activity and synergistic effects when codelivered with anticancer agents. However, due to its chemical instability and short half-life, delivering gaseous NO directly to tumors is still challenging. Herein, we synthesized a NO releasing polymer, nitrate functionalized d-α-tocopheryl polyethylene glycol succinate (TNO3). TNO3 was able to self-assemble into stable micelles in physiological conditions, accumulate in tumors, and release ∼90% of NO content in cancer cells for 96 h. It further exhibited significant cancer cell cytotoxicity and apoptosis compared with nitroglycerine (GTN). Notably, TNO3 could also serve as an enhancer for the common chemotherapeutic drug doxorubicin (DOX). Codelivering TNO3 with DOX to hepatocarcinoma HepG2 cancer cells strengthened the cellular uptake of DOX and enabled the synergistic effect between NO and DOX to induce higher cytotoxicity (∼6.25-fold lower IC50). Moreover, for DOX-based chemotherapy in tumor-bearing mice, coadministration with TNO3 significantly extended the blood circulation time of DOX (14.7-fold t1/2, 6.5-fold mean residence time (MRT), and 13.7-fold area under curve (AUC)) and enhanced its tumor accumulation and penetration, thus resulting in better antitumor efficacy. In summary, this new NO donor, TNO3, may provide a simple but effective strategy to enhance the therapeutic efficacy of chemotherapeutic drugs.

Establishment and Evaluation of a Mouse Model of Experimental Ulcerative Colitis Induced by the Gavage Administration of Dextran Sulfate Sodium.

Given the critical role of dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mouse models in the appraisal of associated therapeutic drugs, the optimization of the administration method and dosages is of paramount importance. Therefore, UC was induced in mice through the gavage administration of a DSS solution instead of free drinking water. The effects of varying daily dosages (2, 4, 6, and 8 g/kg) and frequencies (once or twice) of administration on the body weight and survival rate of the model mice were evaluated. Concurrently, the inflammatory indicators and tissue sections of the model mice were thoroughly evaluated. The results revealed that when the daily dosage reached 8 g/kg, the dosage exhibited a high level of toxicity, resulting in a high mortality rate among the mice. The DSS administration of 6 g/kg*2 not only elicited conspicuous symptoms, significant weight loss, substantial shortening of the colon, and significant changes in various inflammatory indicators, such as myeloperoxidase (MPO), nitric oxide (NO), reactive oxygen species (ROS), and glutathione (GSH), but it also maintained a high survival rate in the UC mice. The findings from this experiment lay a solid experimental foundation for future research on drugs intended for the treatment of UC.

A topical thermosensitive hydrogel system with cyclosporine A PEG-PCL micelles alleviates ulcerative colitis induced by TNBS in mice.

Ulcerative colitis (UC) is an idiopathic, chronic, relapsing disease. In most cases, only the distal colon is affected, and the colonic stasis or fast colonic transit through the inflamed colon usually results in reduced exposure of the distal inflamed colon. Although the immunosuppressant cyclosporine A (CsA) has been used in patients with severe colitis who do not respond to corticosteroids, the clinical application of CsA remains limited due to the systemic toxicities and insufficient accumulation at the site of action for the intravenous and oral routes. In this study, we loaded CsA into the amphipathic poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) micelles and then embedded them in hydrogels consisting of chitosan, poloxamer 188, and poloxamer 407 to construct a thermosensitive and mucoadhesive hydrogel drug delivery system (PLCP). The PLCP presented a high drug-loading capacity and showed a stable and rapid gelation rate after rectal administration into the body. Compared to CsA-loaded micelles and Sandimmun (Neoral®), the developed thermosensitive gel exhibited prolonged retention on the inflamed colon, as seen from in vitro adhesion and in vivo distribution experiments. It also fast mitigated colitis symptoms in TNBS-treated mice by regulating the expression levels of proinflammatory cytokines (TNF-α, IL-1ß, COX-2, and iNOS2), anti-inflammatory cytokines (IL-10, Nrf2, NQO1, and HO-1), and other relevant biochemical factors. Our results suggested that CsA-loaded micelle thermal hydrogel system could be a promising strategy by enhancing the retention in the diseased colon and promoting the relief and recovery of UC.

Molecular mechanisms of hepatoprotective effect of tectorigenin against ANIT-induced cholestatic liver injury: Role of FXR and Nrf2 pathways.

Cholestatic liver injury is caused by toxic action or allergic reaction, resulting in abnormality of bile formation and excretion. Few effective therapies have become available for the treatment of cholestasis. Herein, we found that tectorigenin (TG), a natural isoflavone, showed definite protective effects on alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury, significantly reversing the abnormality of plasma alanine/aspartate aminotransferase, total/direct bilirubin and alkaline phosphatase, as well as hepatic reactive oxygen species, catalase and superoxide dismutase. Importantly, the targeted metabolomic determination found that BA homeostasis could be well maintained in TG-treated cholestatic mice, especially the levels of glycocholic acid, tauromuricholic acid, taurocholic acid, taurolithocholic acid, tauroursodeoxycholic acid and taurodeoxycholic acid. Overall, primary/secondary and amidated/unamidated bile acid (BA) levels were significantly altered upon ANIT stimulation but could be restored by TG intervention to certain extents. In addition, TG boosted the expression of farnesoid x receptor (FXR), which in turn upregulated multidrug resistance protein 2 (MRP2) and bile salt export pump (BSEP) to accelerate the excretion of BA. Meanwhile, TG enhanced the expression of Nrf2 and its upstream genes PI3K/Akt and downstream target genes HO-1, NQO1, GCLC and GCLM to strengthen the antioxidant capacity. Taken together, TG plays a vital role in maintaining BA homeostasis and ameliorating cholestatic liver injury through regulating FXR-mediated BA efflux and Nrf2-mediated antioxidative pathways.

Enteric Polymer-Based Amorphous Solid Dispersions Enhance Oral Absorption of the Weakly Basic Drug Nintedanib via Stabilization of Supersaturation.

The pH−induced crystallization of weakly basic drugs in the small intestine limits oral bioavailability. In this study, we investigated the solubilization and inhibitory effects on nintedanib in the presence of enteric polymers (HPMCAS LG, HPMCAS MG, Eudragit L100 55, and Eudragit L100). These polymers provided maintenance of supersaturation by increasing the solubility of nintedanib in PBS 6.8 in a concentration-dependent manner, and the improved ranking was as follows: Eudragit L100 > Eudragit L100 55 > HPMCAS MG > HPMCAS LG. After being formulated into amorphous solid dispersions (ASDs) by a solvent evaporation method, the drug exhibited an amorphous state. The pH shift dissolution results of polymer-ASDs demonstrated that four polymers could effectively maintain the drug supersaturation even at the lowest ratio of nintedanib and polymer (1:1, w/w). Eudragit L100−ASD could provide both acid resistance and the favorable mitigation of crystallization in GIF. In comparison to the coarse drug, the relative bioavailability of Eudragit L100−ASD was 245% after oral administration in rats, and Tmax was markedly delayed from 2.8 ± 0.4 h to 5.3 ± 2.7 h. Our findings indicate that enteric ASDs are an effective strategy to increase the intestinal absorption of nintedanib by improving physiologically generated supersaturation and subsequent crystallization.

Simultaneous determination of abiraterone and its five metabolites in human plasma by LC-MS/MS: Application to pharmacokinetic study in healthy Chinese subjects.

In this study, a rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify abiraterone (ABI), a widely used anti-metastatic castration-resistant prostate cancer drug, and its metabolites comprising Δ4-abiraterone (D4A), 3-keto-5α-abiraterone (5αA), abiraterone N-oxide (A-NO), abiraterone sulfate (A-Sul) and abiraterone N-oxide sulfate (A-NO-Sul) in human plasma. The analytes were extracted by protein precipitation with acetonitrile and ideal chromatographic separation was achieved on ACE-C18 column (2.1 × 50 mm, 5 µm) using a gradient elution. Triple Quad™ 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source was used and the multiple reaction mode (MRM) was performed. In terms of method validation, good linearity was observed in preassigned validated concentration range for each analyte of interest. Both intra- and inter-batch accuracy was within the range of 87.6-113.8% for all analytes, while intra- and inter-batch precision was below 14.0%. Additionally, both low matrix effects and high recovery were obtained. All analytes remained stable in human plasma at room temperature for 4 h, on wet ice for 8 h, at - 80 °C for 42 d, over three freeze-thaw cycles and under auto-sampler temperature (4 °C) for 48 h post sample preparation. Subsequently, the validated LC-MS/MS method was applied for pharmacokinetic study in healthy Chinese volunteers following an oral dose of 250 mg abiraterone acetate tablet under fasted conditions. Our study for the first time reported the pharmacokinetic parameters of the ABI metabolites in Chinese subjects.

Hepatotoxicity of the Major Anthraquinones Derived From Polygoni Multiflori Radix Based on Bile Acid Homeostasis.

Polygoni Multiflori Radix (PMR), the dried root of Polygonum Multiflorum Thunb., has been widely used as traditional Chinese medicines in clinical practice for centuries. However, the frequently reported hepatotoxic adverse effects hindered its safe use in clinical practice. This study aims to explore the hepatotoxic effect of PMR extract and the major PMR derived anthraquinones including emodin, chrysophanol, and physcion in mice and the underlying mechanisms based on bile acid homeostasis. After consecutively treating the ICR mice with PMR extract or individual anthraquinones for 14 or 28 days, the liver function was evaluated by measuring serum enzymes levels and liver histological examination. The compositions of bile acids (BAs) in the bile, liver, and plasma were measured by LC-MS/MS, followed by Principal Component Analysis (PCA) and Partial Least Squares Discriminate Analysis (PLS-DA). Additionally, gene and protein expressions of BA efflux transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), were examined to investigate the underlying mechanisms. After 14-day administration, mild inflammatory cell infiltration in the liver was observed in the physcion- and PMR-treated groups, while it was found in all the treated groups after 28-day treatment. Physcion and PMR extract induced hepatic BA accumulation after 14-day treatment, but such accumulation was attenuated after 28-day treatment. Based on the PLS-DA results, physcion- and PMR-treated groups were partially overlapping and both groups showed a clear separation with the control group in the mouse liver. The expression of Bsep and Mrp2 in the physcion- and PMR-treated mouse liver was decreased after 14-day treatment, while the downregulation was abrogated after 28-day treatment. Our study, for the first time, demonstrated that both PMR extract and tested anthraquinones could alter the disposition of either the total or individual BAs in the mouse bile, liver, and plasma via regulating the BA efflux transporters and induce liver injury, which provide a theoretical basis for the quality control and safe use of PMR in practice.

The important role of glycerophospholipid metabolism in the protective effects of polyphenol-enriched Tartary buckwheat extract against alcoholic liver disease.

Alcoholic liver disease (ALD) is a mounting public health problem with significant medical, economic and social burdens. Tartary buckwheat (F. tataricum (L.) Gaertn, bitter buckwheat) is a kind of healthy and nutritious food, which has been demonstrated to protect against ALD, but the underlying mechanism has not been fully studied. Herein, we aimed to elucidate the beneficial effects of Tartary buckwheat extract (mainly composed of polyphenols including rutin, quercetin, kaempferol and kaempferol-3-O-rutinoside) in terms of lipid metabolism with the aid of lipidomic analysis. In our study, we employed C57BL/6J mice and a Lieber-DeCarli alcohol liquid diet to construct an ALD model and found that Tartary buckwheat extract was able to prevent ALD-induced histopathological lesions, liver injury and abnormal plasma lipid levels. These beneficial effects might be attributed to the regulation of energy metabolism-related genes (SIRT1, LKB1 and AMPK), lipid synthesis-related genes (ACC, SREBP1c and HMGR) and lipid oxidation-related genes (PPARα, CPT1 and CPT2). In addition, lipidomic profiling and KEGG pathway analysis showed that glycerophospholipid metabolism contributed the most to elucidating the regulatory mechanism of Tartary buckwheat extract. In specific, chronic ethanol intake reduced the level of phosphatidylcholines (PC) and increased the level of phosphatidylethanolamines (PE) in the liver, resulting in a decrease in the PC/PE ratio, which could be all significantly restored by Tartary buckwheat extract intervention, indicating that the Tartary buckwheat extract might regulate PC/PE homeostasis to exert its lipid-lowering effect. Overall, we demonstrated that Tartary buckwheat extract could prevent ALD by modulating hepatic glycerophospholipid metabolism, providing the theoretical basis for its further exploitation as a medical plant or nutritional food.

In Situ intestinal perfusion of irinotecan: application to P-gp mediated drug interaction and introduction of an improved HPLC assay.

PURPOSE: To determine experimentally the intestinal permeability of the anticancer prodrug irinotecan, and to quantify the amount of its cytotoxic metabolite SN-38 that is intestinally excreted (exsorped) as a predictor of intestinal toxicity, and to assess the effect of p-glycoprotein (p-gp) inhibitors (verapamil as a model) on the permeability and toxicity of irinotecan. METHODS: Single pass intestinal perfusion of rat's whole length small intestines is applied to assess the permeability of the parent drug and quantify the intestinally excreted metabolite. The perfusion solution contained 30µg/ml of irinotecan (control group) without or with verapamil (verapamil group). A simple reversed phase HPLC method with UV detection is developed and validated for simultaneous determination of irinotecan and SN-38 using camptothecin as an internal standard. RESULTS: HPLC-UV method found to be simple, specific, accurate, and precise. Effective permeability coefficient of irinotecan found to be 4.9±1.7 10-3 mm/min and was doubled in verapamil group (P=0.007). Average cumulative amount of SN-38 exsorped found to be 29 ng/cm over 2 hours perfusion time which was decreased to 15 ng/cm in verapamil group (P=0.016). CONCLUSIONS: in situ intestinal perfusion method was successfully applied to quantify the permeability of irinotecan and the exsorption of SN-38 in the same experiment, in a manner that robustly reflects real in vivo situation. P-gp inhibition using verapamil found to significantly enhance the intestinal permeability of irinotecan and potentially decrease the intestinal toxicity due to SN-38 exposure.

The influence of co-solvents on the stability and bioavailability of rapamycin formulated in self-microemulsifying drug delivery systems.

OBJECTIVE: This work aims to investigate the influence of various types and different contents of co-solvent on the stability and bioavailability of rapamycin formulated in self-microemulsifying drug delivery systems (SMEDDS). METHODS: A series of SMEDDS of rapamycin were prepared with different co-solvents [including PEG 400/ethanol (F1), glycerol/ethanol (F2), propylene glycol (F3), glycerol formal (F4), transcutol P (F5)]. Drug stability in aqueous media at different pH values and in vitro dispersion of SMEDDS were investigated prior to bioavailability assessment. The storage stability of rapamycin in formulations was also evaluated. RESULTS AND DISCUSSION: The AUC values of rapamycin following oral administration of F1, F3-F5 to rats were significantly higher than those of Rapamune and F0 (SMEDDS without co-solvent). Interestingly, a tendency toward increased bioavailability was seen in F1-F5, which presented the better drug stability in pH 1.2 aqueous media. However, a further increase of the content of co-solvent did not effectively improve the oral bioavailability of rapamycin. Compared with F0, F1-F5 presented significant improvement of drug storage stability. More specifically, the more--OH per unit mass co-solvent had, the better stability rapamycin presented in formulation. CONCLUSIONS: The data obtained in present study highlight the importance of co-solvents on the stability and bioavailability of rapamycin formulated in SMEDDS. Besides solubilizing drug and increasing the dispersion rate, co-solvent could markedly affect the stability of rapamycin whether in different aqueous media or during storage and contribute to the improved oral bioavailability; it can also appropriately decrease the content of surfactant without compromising the absorption of drug.