Network Pharmacology and Bioactive Equivalence Assessment Integrated Strategy Driven Q-markers Discovery for Da-Cheng-Qi Decoction to Attenuate Intestinal Obstruction.

BACKGROUND: Intestinal obstruction (IO) is a kind of acute abdomen with high morbidity and mortality. Patients suffer from poor quality of life and tremendous financial pressure. Da-Cheng-Qi decoction (DCQD), a classical purgation prescription, has clinically been proven to be an effective treatment for IO. PURPOSE: Network pharmacology integrated with bioactive equivalence assessment was used to discover the quality marker (Q-marker) of DCQD against IO. METHODS: As there is hardly any targets recorded in database, thus the collection of IO targets was conducted by searching those of alternative diseases which have similar pathological symptoms with IO. In order to improve the reliability of the obtained targets, IO metabolomics data was introduced. Active compounds combination (ACC) was focused as potential Q-markers via component-target network analysis and function query from the identified components corresponding to the common targets. Bioequivalence between ACC and DCQD was assessed from the aspects of intestine motility (somatostatin secretion), inflammation (IL-6 secretion) and injury (wound healing assay) in vitro and was further validated in ileus rat model. PPI network analysis of core targets followed by gene pedigree classification and experimental validation confirmed the potential intervention pathway. RESULTS: A combination of 11 ingredients, including emodin, physcion, aloe-emodin, rhein, chrysophanol, gallic acid, magnolol, honokiol, naringenin, tangeretin, and nobiletin was finally confirmed bioequivalence with DQCD to some extent and could serve as Q-markers for DCQD to attenuate IO. PI3K/AKT was verified as a possible affected pathway that DCQD exerted the effectiveness against IO. CONCLUSION: For the disease with few recorded targets, searching those of alternative diseases which have similar pathological symptoms could be a feasible and effective approach. The proposed network pharmacology integrated bioactive equivalence evaluation paradigm is efficient to discover Q-marker of herbal formulae.

Biomarker Discovery for Cytochrome P450 1A2 Activity Assessment in Rats, Based on Metabolomics.

Cytochrome P450 1A2 (CYP1A2) is one of the major CYP450 enzymes (CYPs) in the liver, and participates in the biotransformation of various xenobiotics and endogenous signaling molecules. The expression and activity of CYP1A2 show large individual differences, due to genetic and environmental factors. In order to discover non-invasive serum biomarkers associated with hepatic CYP1A2, mass spectrometry-based, untargeted metabolomics were first conducted, in order to dissect the metabolic differences in the serum and liver between control rats and ß-naphthoflavone (an inducer of CYP1A2)-treated rats. Real-time reverse transcription polymerase chain reaction and pharmacokinetic analysis of phenacetin and paracetamol were performed, in order to determine the changes of mRNA levels and activity of CYP1A2 in these two groups, respectively. Branched-chain amino acids phenylalanine and tyrosine were ultimately focalized, as they were detected in both the serum and liver with the same trends. These findings were further confirmed by absolute quantification via a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics approach. Furthermore, the ratio of phenylalanine to tyrosine concentration was also found to be highly correlated with CYP1A2 activity and gene expression. This study demonstrates that metabolomics can be a potentially useful tool for biomarker discovery associated with CYPs. Our findings contribute to explaining interindividual variations in CYP1A2-mediated drug metabolism.

Determination of cetirizine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study.

A rapid, sensitive and selective HPLC-MS/ MS method was developed and validated for the quantification of cetirizine dihydrochloride (CAS 83881-51-0) in human plasma using mosapride citrate as internal standard (IS, CAS 112885-42-4). Following liquid-liquid extraction, the analytes were separated using a mobile phase consisting of methanol and aqueous ammonium acetate solution (10 mM) (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 398 --> 201 for cetirizine and m/z 422 --> 198 for mosapride. The analysis time for each run was 8.0 min. The assay exhibited a linear dynamic range of 0.5-500 ng/ml for cetirizine dihydrochloride in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/ml with a relative standard deviation of less than 15% (all the concentration data in this study related to the salt (cetirizine dihydrochloride)). Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC-MS/MS method has been successfully applied to a bioequivalence study in 20 healthy male Chinese volunteers.

Determination of dexmedetomidine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a pharmacokinetic study.

A rapid, sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-ESI-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of dexmedetomidine (DMED) in human plasma. Dexmedetomidine and the internal standard (ondansetron) were extracted in a single step with diethyl-ether from 1.0 mL of alkalinized plasma. The mobile phase was a mixture of acetonitrile and 0.5% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min(-1). The detection was performed on a triple quadrupole tandem mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions m/z 201.0-->95.1 for DMED and m/z 294.1-->170.1 for the IS. The assay exhibited a linear dynamic range of 5-5000 pg mL(-1) with the correlation coefficient above 0.9995. The lower limit of quantification (LLOQ) was 5 pg mL(-1) with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated HPLC-MS/MS method has been successfully applied to study the pharmacokinetics of three level doses of DMED in Chinese healthy volunteers.

Bioequivalence assessment of two formulations of spironolactone in Chinese healthy male volunteers.

The bioavailability of a new spironolactone ((7alpha,17alpha)-7-(acetylthio)-17-hydroxy-3-oxopregn-4-ene-21-carboxylic acid gamma-lactone, CAS 52-01-7) formulation (test) was compared with a commercially available original formulation (reference) of the drug in 20 Chinese healthy male volunteers, aged between 21 and 27. The trial was designed as an open, randomized, single blind two-sequence, two-period crossover study. Under fasting conditions, each subject received a single oral dose of 100 mg spironolactone as a test or reference formulation with a 7-day washout period between the two formulations. The plasma concentrations of spironolactone and its active metabolite canrenone (CAS 976-71-6) were analyzed by a sensitive liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method. The pharmacokinetic parameters included AUC(0.t), AUC(0-infinity), C(max), t1/2, and T(max). Values of AUC(0-t) demonstrate nearly identical bioavailability of spironolactone from the examined formulations. The AUC(0.12) of spironolactone was 148.35 +/- 39.5 and 144.39 +/- 53.02 ng x h/ml for the test and reference formulation, respectively. The AUC(0-60) of the metabolite canrenone was 1873.36 +/- 318.10 and 1911.28 +/- 355.60 ng h/ml for test and reference formulation, respectively. The maximum plasma concentration (C(max)) of spironolactone was 48.34 +/- 21.16 ng/ml for the test and 47.40 +/- 23.40 ng/ml for the reference product and the C(max) of the metabolite was 122.90 +/- 27.70 and 123.35 +/- 27.29 ng/ml for the test and reference product, respectively. No statistical differences were observed for C(max) and the area under the plasma concentration-time curve for both spironolactone and its active metabolite canrenone. 90% confidence limits calculated for C(max) and AUC from zero to infinity (AUC(0-infinity)) of spironolactone and its metabolite were included in the bioequivalence range (80%-125% for AUC). This study shows that the test formulation is bioequivalent to the reference formulation for spironolactone and its main active metabolite canrenone.

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma.

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.