Synonymous mutation rs2515641 affects CYP2E1 mRNA and protein expression and susceptibility to drug-induced liver injury.

Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 (CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results:CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.

CYP2E1 and miRNA-378a-3p contribute to acetaminophen- or tripterygium glycosides-induced hepatotoxicity.

Increased expression of CYP2E1 may represent the main factor contributing to oxidative stress-mediated liver damage in drug-induced liver injury (DILI). However, the regulation mechanism of CYP2E1 expression is poorly described. The present study was aimed to investigate the role of CYP2E1 in acetaminophen (APAP)- or tripterygium glycosides (TG)-induced hepatotoxicity as well as the regulation of CYP2E1 and miR-378a-3p expression by APAP or TG. Rats were randomly divided and treated with APAP, TG, chlormethiazole (CMZ), APAP + CMZ and TG + CMZ, respectively, for 4 weeks. Then, blood and liver samples were collected. Serum and hepatic biochemical parameters were measured using commercial kits. Liver histopathology was tested by H&E staining. Expression levels of CYP2E1 mRNA and miR-378a-3p were detected by qRT-PCR. CYP2E1 protein expression was determined by Western blot. Our results showed that CMZ effectively restored the hepatic histopathological changes, oxidative stress biomarkers and TNF-α levels induced by APAP or TG. CYP2E1 mRNA and/or protein expression levels were dramatically increased after chronic APAP or TG treatment, while this induction was significantly reversed by CMZ co-treatment. Of note, miR-378a-3p expression levels were significantly suppressed after APAP, TG and/or CMZ treatment. These results suggested that CYP2E1 were highly induced after chronic APAP or TG treatment, which in turn play an important role in APAP- or TG-induced hepatotoxicity. These inductions of CYP2E1 expression were probably carried out by inhibition of miR-378a-3p. Our findings might provide a new molecular basis for DILI.

Characterization of Triptolide-Induced Hepatotoxicity by Imaging and Transcriptomics in a Novel Zebrafish Model.

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.

Pharmacokinetics and metabolism of Chf197, a ligustrazine derivative, in rats.

Ligustrazine is the most abundant and bioactive ingredient in Rhizoma Chuanxiong, a Chinese medicinal herb commonly used for the treatment of cardiovascular diseases. Chf197 is one of the structurally modified ligustrazine derivatives in a purpose of overcoming the rapid metabolism and short half-life of original. The plasma and urine pharmacokinetics of Chf197 in rats were studied after intravenous or intraperitoneal injection of Chf197 with the validated RP-HPLC method. The pharmacokinetic parameters of Chf197 injected intravenously 20 mg/kg were as follows: Cmax , 1.44 ± 0.4 mg/L; Tmax , 0.08 h; t1/2 , 3.03 ± 1.67 h; AUC, 3.85 ± 3.88 h/L; Vd , 31.66 ± 11.79L/kg; and CL, 9.29 ± 4.92 l/h/kg. Dose-dependent pharmacokinetics was observed, and a significantly higher dose-normalized AUC after intravenous administration was obtained than that after intraperitoneal administration. A possible metabolite was detected at about 3.1 min, and full-scan mass spectrum was adopted to predict its possible structure.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of bergenin.

Glucuronidation plays critical role in the elimination of bergenin; however the metabolic mechanism of UDP-glucuronosyltransferases (UGTs) in the process remains to be investigated. In this study, the kinetics of bergenin glucuronidation by pooled human liver microsomes (HLMs) and 12 recombinat UGT isozymes were investigated. The glucuronidation of bergenin can be shown in HLMs with a Km value of 231.62 ± 14.08 µm and a Vmax value of 2.17 ± 0.21 nmol/min/(mg protein). Among the 12 human UGTs investigated, UGT1A1 was identified as the major isoform catalyzing the glucuronidation of bergenin [Km value of 200.37 ± 26.73 µm and Vmax value of 1.88 ± 0.26 nmol/min/(mg protein)]. The bergenin glucuronosyltransferase activities in HLMs and UGT1A1 were inhibited by phenylbutazone, estradiol and bilirubin. The results demonstrate that bergenin glucuronidation in HLMs is specifically catalyzed by UGT1A1.

In vivo metabolism study of bergenin in rats by HPLC-QTOF mass spectrometry.

Bergenin is the major component of Ardisia creanta sims and Rodgersia sambucifolia hemsl with many biological activities. Although bergenin has been used to treat human diseases in China for man years, there is no report regarding its metabolism. This is the first report to separate and identify the metabolites of bergenin in vivo. In the study, HPLC/Q-TOF-MS/MS was used to investigate the metabolites of bergenin in vivo by analyzing the rat body fluid and feces samples. Three metabolites of bergenin were finally identified by the TIC chromatograms, and the structures were also confirmed by their MS(2) spectra.

A metabonomics study of epilepsy in patients using gas chromatography coupled with mass spectrometry.

Epilepsy is a cryptogenic neurological disorder characterized by recurrent seizures which may be precipitated by a variety of endogenous or exogenous factors, and usually occurs many months or years after a precipitating injury. Timely diagnosis and treatment at the early stage of epilepsy are very important for patients to prevent serious lesions and improve the quality of their life. In this study, the metabonomics approach based on the GC-MS technique, multivariate statistical analysis and the metabolism network analysis were applied to investigate metabolic changes in epileptic patients. The outcome of this study suggested that ten endogenous metabolites and five metabolism pathways were mainly involved and showed marked perturbations in epileptic patients. It not only enhances the understanding of the pathology of epilepsy, but also provides an experimental foundation for the therapeutic strategy of epilepsy. Furthermore, this work demonstrates the powerful predictive potential of the metabolic network analysis to neurological disease.

Determination of the active metabolite of moguisteine in human plasma and urine by LC-ESI-MS method and its application in pharmacokinetic study.

In this study, a sensitive and reproducible electro-spray ionization liquid chromatography-mass spectrometry (LC-ESI-MS) method was established to determine the concentration of M1, the main active metabolite of moguisteine in human plasma and urine. The analysis was performed on a Diamonsil® C18(2) column (150 mm × 4.6 mm, 5 µm) with the mobile phase consisting of 0.1% formic acid-acetonitrile (57:43, v/v, pH=3.0) at a flow rate of 0.8 mL min⁻¹. The pseudo-molecular ions [M+H]+ (m/z 312.2 for M1 and 446.3 for glipizide) were selected as the target ions for quantification in the selected ion monitoring (SIM) mode. Plasma samples were analyzed after being processed by acidification with formic acid and protein precipitation with acetonitrile. Urine samples were appropriately diluted with blank urine for analysis. Calibration curve was ranged from 0.02 to 8 µg mL⁻¹. The extraction recovery in plasma was over 90%. Both the inter- and intra-day precision values were less than 7.5%, and the accuracy was in the range from -6.0% to 6.0%. This is the first reported LC-ESI-MS method for analyzing M1 in human plasma and urine. The method was successfully applied to the pharmacokinetic study after oral administration of single-dose and multiple-dose of moguisteine tablets in healthy Chinese subjects.

Simultaneous determination of lercanidipine, benazepril and benazeprilat in plasma by LC-MS/MS and its application to a toxicokinetics study.

We aim to develop a rapid, simple, sensitive and specific LC-MS/MS method for the simultaneous quantification of lercanidipine, benazepril and benazeprilat in plasma. It is performed on the Agilent 6410 LC-MS/MS under the multiple-reaction monitoring (MRM) mode with electrospray ionization. Gliclazide was used as the internal standard (IS). Analytes and IS were extracted from plasma by solid-phase extraction. The reconstituted samples were chromatographed on a Diamond C18(150 mm × 4.6 mm, 5 µm) column. The mobile phase was composed of 0.1% acetic acid-acetonitrile (50:50, v/v), with gradient flow rates: 0.6 mL/min (0-4.55 min); 4.55-4.65 min, 1 mL/min; 1 mL/min (4.65-9.5 min); 9.5-9.6 min, 0.6 mL/min; 0.6 mL/min (9.6-10 min). Method validation demonstrated that the method was of satisfactory specificity, sensitivity, precision and accuracy in linear ranges of 1-2000 ng/mL for lercanidipine, 1-2000 ng/mL for benazepril and 1-1600 ng/mL for benazeprilat, respectively. The precision (RSD%) was better than 15, and the lower limit of quantitation was identifiable and reproducible at 1 ng/mL for the three analytes. The plasma samples were stable after being stored for more than 60 days and after two freeze-thaw cycles (-20 to -25 °C). It is demonstrated that this method was successfully applied to samples from a toxicokinetics study of a compound of lercanidipine and benazepril in beagle dogs.

Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study.

A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C(18) reversed-phase column, eluted with mobile phase of acetonitrile-ammonium acetate (5 m m; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers.

Separation and identification of norcantharidin metabolites in vivo by GC-MS method.

Norcantharidin (NCTD), the demethylated analogue of cantharidin, inhibits the proliferation of a variety of human tumor cell lines, and appears to cause the least nephrotoxic and inflammatory side effects. Although NCTD has been used to treat human cancers in China for years, there is no report regarding its metabolism up to now. This is the first report to separate and identify the main metabolites of NCTD in vivo by GC-MS using TMS derivatives. Two hydrolyzed products and five phase I or phase II metabolites were found in rat by the chromatogram comparisons of the blank with incurred biological samples. Multiple stages of fragmentation patterns were used to confirm the metabolites characterizations. The established GC-MS method can also be applied to identifying unknown metabolites of the drugs containing hydroxyl or carbonyl groups in molecular structure.

Determinations of mifepristone and its metabolites and their pharmacokinetics in healthy female Chinese subjects.

The aim of this study is to establish an HPLC method for simultaneous determinations of mifepristone and its metabolites, mono-demethylated mifepristone, di-demethylated mifepristone and C-hydroxylated mifepristone in plasma and to evaluate the pharmacokinetic characteristics of mifepristone tablet. Twenty healthy female Chinese subjects were recruited and a series of blood samples were collected before and after 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 8.0, 12.0, 24.0, 48.0, 72.0 and 96.0 hours administration by a single oral dose of 75 mg mifepristone tablet. Mifepristone and its three metabolites were extracted from plasma using ethyl acetate and determined by high performance liquid chromatography. The main pharmacokinetic parameters of mifepristone and its metabolites, including Cmax, tmax, MRT, t(1/2), V, CL, AUC(0-96 h) and AUC(0-infinity), were calculated by Drug and Statistical Software Version 2.0. The simple, accurate and stable method allows the sensitive determinations of mifepristone and its metabolites in human plasma up to 4 days after oral administration of 75 mg mifepristone tablet and the clinical applications of their pharmacokinetic studies.

Metabolism and disposition of tribendimidine and its metabolites in healthy Chinese volunteers.

BACKGROUND: Tribendimidine is a new anthelmintic agent synthesized by Chinese scientists. It is a broad spectrum agent with high activity against parasites. However, its disposition and metabolism remain unknown. OBJECTIVE: To investigate the metabolism, disposition, and metabolites of tribendimidine in healthy human volunteers. METHODS: Twelve healthy Chinese volunteers were chosen after clinical assessment of health status and laboratory tests. They received single oral doses of tribendimidine 400 mg enteric-coated tablets. Blood and urine samples were collected at scheduled timepoints. Samples were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometric (LC-MS) and high performance liquid chromatography (HPLC) methods, respectively. RESULTS: Tribendimidine was rapidly and completely broken down to p-(1-dimethylamino ethylimino) aniline (dADT) and terephthalaldehyde (TPAL). Furthermore, dADT was partially transformed to acetylated dADT, and TPAL completely transformed to terephalic acid (TPAC). The main pharmacokinetic parameters (± SD) of dADT were as follows: elimination half life (t(½)) 4.74 ± 1.80 h; elimination rate constant (K(e)) 0.16 ± 0.06 h-1; apparent volume of distribution (Vd/F) 12.23 ± 8.69 L * kg(-1); apparent total clearance of the drug from plasma (CL/F) 1.63 ± 0.58 L * h(-1) * kg(-1); area under the plasma concentration-time curve (AUC) from time 0 to time 24 hours (AUC(24)) 4.29 ± 1.88 µg * mL(-1) * h; AUC from time zero to infinity (AUC(infinity)) 4.45 ± 1.81 µg * mL(-1) * h; maximum plasma drug concentration (C(max)) 0.64 ± 0.27 µg * mL(-1); and time to C(max) (t(max)) 4.20 ± 0.71 h. A total of 35.28% dADT and 28.50% TPAC were excreted through the urine within 24 hours after tribendimidine administration. CONCLUSION: These results reveal the disposition, metabolism, and main metabolites of tribendimidine in healthy Chinese volunteers.

Bioequivalence of nifedipine softgel and capsule in healthy Chinese volunteers by liquid chromatography-mass spectrometry.

The study aimed to compare and evaluate the bioequivalence of Calcigard-10 softgel and Adalat 10 capsule in healthy Chinese volunteers in a randomized, two-way cross over study design with a washout period of 7 days. A sensitive and reproducible electro-spray ionization liquid chromatography-mass spectrometry (ESI-LCMS) assay was developed and validated to determine nifedipine in human plasma using nitrendipine as internal standard. Nifedipine and nitrendipine were extracted from plasma using liquid-liquid extraction with methylene chloride as extraction solvent. The separation was performed by a Diamonsil ODS column (150 x 4.6 mm, 5 microm). The mobile phase was consisted of acetonitrile-5 mM ammonium acetate (52:48, v/v), delivered at flow rate of 1 mL/min. The 90% confidence intervals for the ratio values of logarithmic transformed Cmax and AUC were calculated to evaluate the bioequivalence of two preparations. The values of Cmax (92.3-112.7%), AUC0-t (84.5-95.1%) and AUC0-inf (84.4-95.5%) are within the interval criterion of 70-143% for Cmax and 80-125% for AUC. The Calcigard-10 softgel and Adalat 10 capsule are bioequivalent.

Determination and pharmacokinetic study of hydrocodone in human plasma by liquid chromatography coupled with tandem mass spectrometry.

A rapid, highly sensitive and specific high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS-MS) quantitation method was developed and validated for the determination of hydrocodone in human plasma. Sample was extracted from 0.5mL heparinized plasma by a simple liquid-liquid extraction method and analyzed on a C18 column with a mobile phase of acetonitrile-water (78:22,v/v,0.1% acetic acid). Detection was carried out by positive elevtrospray ionization (ESI) in multiple reactions monitoring (MRM) mode of 300.3-->199.2 (m/z) for hydrocodone and 341.2-->107.2 (m/z) for canrenone (I.S.), respectively. A good linearity was obtained from 0.5 to 60 ng x mL(-1) and the lower limit of quantification (LLOQ) was 0.1 ng x mL(-1). Compared to an existing method, the extraction method, internal standard and chromatographic conditions were modified and the cost of a large amount of samples determination was decreased obviously. The method was successfully applied to the pharmacokinetic and bioequivalence studies in healthy Chinese volunteers.

Determination of bergenin in human plasma after oral administration by HPLC-MS/MS method and its pharmacokinetic study.

A highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and applied to the determination of bergenin concentration in human plasma. Bergenin and the internal standard (IS) thiamphenicol in plasma were extracted with ethyl acetate, separated on a C(18 )reversed-phase column, eluted with mobile phase of acetonitrile-water, ionized by negative ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor --> product ions of m/z 327.1 --> 192 for bergenin and 354 --> 185.1 for the IS, respectively. The linear range of the calibration curve for bergenin was 0.25-60 ng mL(-1), with the lowest limit of quantification of 0.25 ng mL(-1), and the intra/inter-day relative standard deviation (RSD) was less than 10%. The method is suitable for the determination of low bergenin concentration in human plasma after therapeutic oral doses, and has been first and successfully used for its pharmacokinetic studies in healthy Chinese volunteers.

Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass.

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers.

Determination and pharmacokinetic study of norcantharidin in human serum by HPLC-MS/MS method.

A sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry method was developed and applied to the determination of norcantharidin concentration in human serum. Norcantharidin (NCTD) and cyclophosphamide (IS) in serum were extracted with acetone, separated on a C18 reversed-phase column, gradiently eluted with a mobile phase of acetonitrile-water containing 2 mm ammonium acetate and 0.1% formic acid (pH 3), ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring mode using precursor-->product ions of m/z 169.3-->123.1 for NCTD and 261.2-->140.2 for IS, respectively. The linear range of the calibration curve for NCTD was 2.5-50 ng/mL, with a lowest limit of quantification of 2.5 ng/mL, and the intra/inter-day RSD was less than 10%. The method was suitable for determination of low NCTD concentration in human serum after therapeutic oral doses, and has been successfully used for pharmacokinetic studies in healthy Chinese volunteers.

[Determination of curcumol in plasma by HPLC-MS/MS method and its pharmacokinetics in Beagle dogs].

To establish a high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry quantitative detection method for the determination of curcumol, the main ingredient of zedoary turmeric oil fat emulsion, and investigate its pharmacokinetics in Beagle dogs, nine healthy Beagle dogs were divided into three groups, and blood samples were collected at scheduled time points after intravenous injection of 7.5, 10 and 12.5 mg x kg(-1) zedoary turmeric oil fat emulsion. The concentrations of curcumol were determined and pharmacokinetics was calculated. A good linearity was obtained from 0.25 to 100 ng x mL(-1) in plasma. The relative recoveries were from 91.33% to 103.17%, and the absolute recoveries were from 31.61% to 37.20%. The intra-day and inter-day variances (RSD) were < 15%. The main pharmacokinetic parameters of curcumol after intravenous injection of 7.5, 10 and 12.5 mg x kg(-1) zedoary turmeric oil fat emulsion were as follows, T1/2 : (2.0 +/- 0.4), (1.7 +/- 0.2) and (2.3 +/- 0.8) h, AUC(0-infinity): (15.1 +/- 2.7), (18.3 +/- 2.0) and (29.5 +/- 4.0) ng x mL(-1) x h; MRT: (0.9 +/- 0.1), (0.8 +/- 0.2) and (0.8 +/- 0.1) h, CL: (21.9 +/- 4.0), (24.9 +/- 6.0) and (18.4 +/- 1.2) L x h(-1) x kg; Vd : (65.4 +/- 26.5), (62.0 +/- 13.4) and (61.2 +/- 19.8) L x kg(-1), respectively. The developed method was rapid, highly sensitive and specific and could be used in curcumol pharmacokinetic studies in vivo. A three-compartment model was best fit to the plasma concentration--time curves obtained in Beagle dogs and the plasma AUC was increased proportionally with doses.