Pharmacokinetic profile and metabolite identification of bornyl caffeate and caffeic acid in rats by high performance liquid chromatography coupled with mass spectrometry.

Bornyl caffeate was initially discovered as a bioactive compound in medicinal plants. Despite the promising pharmacological activities including anti-tumor and antibacterial activities, the pharmacokinetics of the compound remain open. This work developed a high performance liquid chromatography-tandem mass spectrometric method for the determination of bornyl caffeate and caffeic acid (major metabolite and a main unit of bornyl caffeate) in vivo. Successful application of the method included identification of its metabolites and investigation on the drug pharmacokinetics. A total of 30 compounds were identified as the metabolites of bornyl caffeate in rats. We attributed these metabolites to phase I metabolic routes of reduction, oxidation, hydrolysis and phase II metabolic reactions of glucuronidation, sulfation, O-methylation and glycine. Glucuronidation, sulfation, O-methylation and reduction were the main metabolic pathways of bornyl caffeate. The method presented a linear range of 1-4000 ng mL-1. The pharmacokinetic profile of bornyl caffeate was found to be a three compartment open model, while caffeic acid fitted to a two compartment open model when it was administered alone or served as the main metabolite of bornyl caffeate. The time to peak concentration (T max) and the maximum plasma concentration (C max) of bornyl caffeate were 0.53 h and 409.33 ng mL-1. Compared with original caffeic acid, the compound displayed an increased half-life of elimination (T 1/2ß), area under the concentration time curve from 0 to t (AUC0-t ) and area under the concentration time curve from 0 to ∞ (AUC0-∞), a decreased half-life of absorption (T 1/2α) and an identical C max. Taking together, we concluded that bornyl caffeate is able to rapidly initiate therapeutic effect and last for a relatively long time in rats; metabolic pathways of O-methylation and reduction is key to interpret the mechanism and toxicity of bornyl caffeate.

Identifying the antiasthmatic target of doxofylline using immobilized ß2 -adrenoceptor based high-performance affinity chromatography and site-directed molecular docking.

As a xanthine derivative, doxofylline is believed to be dominant for fighting against asthma in practice. Unlike other xanthines, the antiasthmatic effects of doxofylline lack any definite proof of target and mediating mechanism according to previous reports. In this work, the interaction between doxofylline and ß2 -AR was investigated by high performance affinity chromatography using frontal analysis and nonlinear model. The methodology involved the immobilization of ß2 -AR on the silica gel by a random linking method, the determination of the binding parameters by frontal analysis and nonlinear chromatography and the exploration of the binding mechanism by site-directed molecular docking. The association constant for doxofylline binding to immobilized ß2 -AR was determined to be 7.70 × 10(4) M(-1) by nonlinear chromatography and 5.91 × 10(4) M(-1) by frontal analysis. Ser(169) and Ser(173) were the binding sites for the receptor-drug interaction on which hydrogen bond was believed to be the main driven force during the interaction. These results indicated that the antiasthmatic effects of doxofylline may be behind the mediating mechanism of ß2 -AR. High performance affinity chromatography based on immobilized receptor has potential to become an alternative for drug target confirmation and drug-receptor interaction analysis. Copyright © 2016 John Wiley & Sons, Ltd.

A method of chemiluminescence coupled with ultrafiltration for investigating the interaction between ibuprofen and human serum albumin.

In acidic media, ibuprofen substantially enhanced the weak chemiluminescence (CL) produced by sodium sulfite and potassium permanganate. The increased signals were linearly correlated with ibuprofen concentrations ranging from 1.2 × 10(-3) to 4.8 µM, with a detection limit of 4.8 × 10(-4) µM. Two ultrafiltration (UF) membranes were used to construct a unit for trapping 0.15 and 0.75 µM human serum albumin (HSA) and coupled online with the CL system. At low HSA concentrations, the numbers of bound molecules per binding site were calculated to be 0.9 for Sudlow site I and 6.2 for Sudlow site II. The association constants on these binding sites were 5.9 × 10(5) and 3.4 × 10(4) M(-1), respectively. Our CL-UF protocol presents a rapid and sensitive method for studies on drug-protein interaction.

One-step method for plasma determination of ibuprofen by chemiluminescence-coupled ultrafiltration and application in a pharmacokinetic study.

A simple one-step method is established for plasma determination of ibuprofen and its pharmacokinetic study. The method involves simple sample pre-treatment by dilution, rapid separation by ultrafiltration (UF) and online sensitive detection by chemiluminescence (CL) based on significant intensity enhancement of ibuprofen on the weak CL of potassium permanganate and sodium sulphite in an acidic system. The calibration curve for ibuprofen is linear in the range 0.1-50.0 µg/mL in rat plasma. Average recoveries of ibuprofen at 0.80, 12.0 and 40.0 µg/mL amounted to 98.0 ± 4.2%, 101.2 ± 3.6% and 99.3 ± 5.4%, respectively. Standard deviations of intra- and inter-day measurement precision and accuracy are within ±10.0%. The detection limit for ibuprofen is 10.0 µg/L in plasma samples. Pharmacokinetic study of ibuprofen by the validated method shows that the mean plasma drug concentration-time course confirms to a classical two-compartment open model with first-order absorption. The proposed method will be an alternative for pre-clinical pharmacokinetic study of ibuprofen and other non-steroidal anti-inflammatory drugs.

Evaluation of the effects of cytochrome P450 nonsynonymous single-nucleotide polymorphisms on tanshinol borneol ester metabolism and inhibition potential.

Nonsynonymous single-nucleotide polymorphisms (nsSNPs) in cytochrome P450 (P450) genes may affect drug metabolism and drug-drug interactions (DDIs), potentially leading to adverse drug reactions. Functional characterization of the nsSNPs in P450 genes is important to help us understand the impact of genetic factors on P450-mediated drug metabolism and DDIs. To evaluate the effects of P450 nsSNPs on the metabolism and inhibition potential of a candidate drug, tanshinol borneol ester (DBZ), we obtained and experimentally validated eight yeast-expressed human P450 isoforms and their nsSNP variants and tested DBZ using these recombinant P450 enzymes. The results suggested that CYP2C8 is the major enzyme responsible for DBZ metabolism. In addition, compared with prototypic CYP2C8, the allelic variant, CYP2C8.3, produced a 54% decrease in the intrinsic clearance of DBZ. The inhibitory potency of DBZ toward CYP3A4 was greater than that toward other P450 isoforms, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Moreover, the inhibitory potency toward three CYP3A4 allelic variants, CYP3A4.2, CYP3A4.12, and CYP3A4.16, was reduced 2- to 10-fold relative to prototype CYP3A4. These results provide useful information for understanding the influence of P450 genetic polymorphisms on DBZ metabolism and may help to design future clinical trials of DBZ. Our results suggest applications for in vitro P450 assays both for basic research in pharmacogenomics and for drug development.

Metabolism of tanshinol borneol ester in rat and human liver microsomes.

Tanshinol borneol ester (DBZ) is an experimental drug that exhibits efficacious anti-ischemic activity in rats. Although the specific metabolic properties of DBZ are still unknown, previous studies in rats have strongly suggested that DBZ is extensively metabolized after administration and thus probably acts as a prodrug. Because the enzymes involved in drug metabolism differ between humans and rats in isoform composition, expression, and catalytic activity, the pharmacokinetics of the same drug in the two species may also differ. Establishing the differences between DBZ metabolism in human and rat liver microsomes can help to predict DBZ pharmacokinetics in humans and aid in the assessment of its potential efficacy, toxicity, and mechanism of action. In this work, the microsomal stabilities and metabolic kinetics of DBZ in rat and human liver microsomes were compared, and the DBZ metabolites generated in human liver microsomes (HLMs) were identified. The results suggested that DBZ is more stable in HLMs than in rat liver microsomes (RLMs). The intrinsic clearance of DBZ in HLMs was 10- to 17-fold lower than that in RLMs, which indicates lower DBZ clearance in humans. Metabolite analysis suggested that DBZ is hydroxylated by liver microsomal enzymes, resulting in the production of five metabolites. Although the kinetics of metabolite formation in HLMs and RLMs were different, the same metabolites were generated, indicating that the same metabolic pathway is present in both species. The results obtained from this work suggest the potential for DBZ to act as a prodrug with anti-ischemic activity in humans.

Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues beta2-adrenoceptor using an affinity chromatographic selection method.

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to beta(2)-adrenoceptor (beta(2)-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a beta(2)-AR affinity column. The retention compounds eluted from the beta(2)-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the beta(2)-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized beta(2)-AR. Their association constants were (2.28+/-0.11)x10(4)/M and (3.00+/-0.10)x10(4)/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized beta(2)-AR, and the corresponding association constants were (2.20+/-0.09)x10(-4)/M and (6.78+/-0.001)x10(5)/M.

Isopropyl 3-(3,4-dihydroxy-phen-yl)-2-hydroxy-propanoate.

The title compound, C(12)H(16)O(5), is a derivative of ß-(3,4-dihydroxy-phen-yl)-α-hydr-oxy acid. The crystal packing is stabilized by inter-molecular O-H⋯O hydrogen bonds.

2-Acetamido-3-(4-hydr-oxy-3-methoxy-phen-yl)acrylic acid.

In the title compound, C(12)H(13)NO(5), the azlactone of vanillin, the acrylic acid side chain has a trans extended conformation. There are inter-molecular N-H⋯O and O-H⋯O hydrogen bonds in the crystal structure.