Whole-genome methylation profiling of peripheral blood mononuclear cell for acute exacerbations of chronic obstructive pulmonary disease treated with corticosteroid.

OBJECTIVE: Although association studies in the general population may be relevant for determining susceptibility to chronic obstructive pulmonary disease (COPD), they may be less applicable for pharmacogenetics research in participants who have already acquired the disease. PATIENTS AND METHODS: A genome-wide methylation profiling (generated by HumanMethylation450 BeadChips study was performed on peripheral blood mononuclear cells of 24 patients with AECOPD (acute exacerbation COPD), with good and poor responsiveness to standard corticosteroid treatment. Pyrosequencing was used to replicate the selected CpG sites in 50 patients with AECOPD with standard corticosteroid treatment. RESULTS: The results showed the patients with AECOPD with good and poor response to standard corticosteroid treatment have a distinct DNA methylation pattern. A total of 23 CpG loci located in 19 known gene regions, including gene-body and promoter, appeared to be significantly differentially methylated. Replication by pyrosequencing revealed that one CpG site in PSMD8 showed the same trend of differential methylation and reached to statistical significance as the microarray result. CONCLUSION: Our preliminary findings provide evidence for molecular heterogeneity in patients with AECOPD, which may contribute to significant differences in their response to COPD treatment.

Quantification of the major metabolites of bromhexine in human plasma using RRLC-MS/MS and its application to pharmacokinetics.

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8microm particle size reversed-phase C(18) column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5mM ammonium acetate at a flow rate of 0.7mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1-70.9%, 60.5-68.4%, 57.0-63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9-96.7%, 89.6-94.8%, 90.4-91.4%, and 103%, respectively. After an oral administration of 8.0mg bromhexine to five healthy male subjects, AUC(0-24h) values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5+/-31.9, 34.0+/-14.5, and 15.8+/-6.89ngh/mL, respectively; while C(max) values were 24.6+/-5.16, 3.11+/-1.13, and 5.36+/-2.55ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t(1/2) which gave 3.6+/-0.5, 8.4+/-2.7, and 6.4+/-2.5h, respectively.

Molecularly imprinted solid-phase extraction for the selective determination of bromhexine in human serum and urine with high performance liquid chromatography.

In this work, a novel method is described for the determination of bromhexine in biological fluids using molecularly imprinted solid-phase extraction as the sample cleanup technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and bromhexine as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of bromhexine from human serum and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for molecularly imprinted solid-phase extraction (MISPE) consisted of conditioning 1 mL methanol and 1 mL of deionized water at neutral pH, loading of 5 mL of the water sample (25 microg L(-1)) at pH 6.0, washing using 2 mL acetonitrile/acetone (1/4, v/v) and elution with 3x 1 mL methanol/acetic acid (10/1, v/v). The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of bromhexine. Results from the HPLC analyses showed that the calibration curve of bromhexine using MIP from human serum and urine is linear in the ranges of 0.5-100 and 1.5-100 microg L(-1) with good precisions (3.3% and 2.8% for 5.0 microg L(-1)), respectively. The recoveries for serum and urine samples were higher than 92%.

Bioequivalence study of bromhexine by liquid chromatography-electrospray ionization-mass spectrometry after oral administration of bromhexine hydrochloride tablets.

A simple, sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry method was developed for the quantitative determination of bromhexine in human plasma. Sample preparation involved simple liquid-liquid extraction. Simvastatin was used as internal standard. The separation of the analyte, internal standard and possible endogenous compounds were accomplished on a Shim-pack ODS column (150 mm x 4.6 mm i.d., 5 microm) with methanol-water (98:2, v/v) as mobile phase. Detection was performed in positive selected ion monitoring (SIM) mode at m/z 264.1 (for bromhexine) and m/z 441.7 (for IS). The method was validated over the range of 0.5-60 ng/mL and the results were acceptable. The method could offer the advantages of shorter run time (5.5 min) and lower LLOQ (0.5 ng/mL) with a decreased plasma volume requirement (250 microL) and it had been successfully applied to a bioequivalence study in healthy Chinese volunteers after single oral administration of 16 mg bromhexine.

[Determination of bromhexine in plasma by gas chromatography-electron capture detection and pharmacokinetic studies].

This paper reports a gas chromatography-electron capture detection(GC-ECD) method for the study of pharmacokinetics of bromhexine in healthy human body. A2 m x 3 mm i.d. silanized glass column packed with 5% SE-30 was used. The carrier gas was nitrogen. The internal standard was 5-chloro-2-amino-diphenyl ketone. After NaH2PO4-Na2HPO4 buffer (pH 6.0) being added, the plasma was extracted with n-hexane-dichloromethane (9:1, V/V). A good linearity was obtained from 1.0 microgram/L to 50.0 micrograms/L of bromhexine in human plasma, r = 0.9994. The detection limit of bromhexine in plasma was 0.5 microgram/L. The average recovery was 97.5%. The pharmacokinetics of bromhexine was determined by this method after a single oral dose of 8 mg bromhexine capsule given to each of 8 volunteers. The results showed that the plasma concentration-time courses conformed to one compartment model. The established GC-ECD method is a good method for the determination of bromhexine in human plasma. The method is rapid, simple, precise and sensitive.

Determination of bromhexine and ambroxol in pharmaceutical dosage forms, urine and blood serum.

Data presented in this paper show that bromhexine and its pharmacologically active metabolite can easily be determined by capillary zone electrophoresis. The composition of the running buffer had a significant effect on the reproducibility of the migration time for which a carrier solution containing 30 mM phosphate buffer (pH 3.0), 5 M urea and 10% (v/v) acetonitrile was used. The method was validated with respect to its response linearity and reproducibility. The method is suitable for the determination of bromhexine and ambroxol in several samples such as pharmaceuticals, urine and serum. Photodiode-array detection permitted the rapid identification of both drugs in the sample analyzed.

Flow-injection extraction-spectrophotometric determination of bromhexine with orange IV.

An automatic flow-injection photometric method for the determination of bromhexine is proposed. The drug was determined by formation of an ion-pair with orange IV, extraction into 1,2-dichloroethane and measurement of the absorbance at 412 nm of the organic phase. A linear calibration graph was obtained at concentrations of 5 x 10(-6)-1.6 x 10(-4) M of bromhexine. Up to 40 samples h-1 can be processed with an RSD of 0.32-0.88%. The method was applied to the determination of bromhexine in blood serum and a pharmaceutical preparation.

Characterization of bromhexine and ambroxol in equine urine: effect of furosemide on identification and confirmation.

The purpose of this study was two-fold: (1) to develop a simple and sensitive screening procedure for identifying and confirming bromhexine and ambroxol and, (2) to determine the effect of furosemide on the detection of bromhexine, ambroxol, or their metabolites in urine. Female horses (450-550 kg) treated with bromhexine or ambroxol (1 g, p.o.) were used. Urine samples were collected up to 48 h post-drug administration and analysed. Blind samples were used in evaluating the sensitivity of these methods and reproducibility of the results. Bromhexine and ambroxol were extensively metabolized in the horse. These agents and their respective metabolites were identified and confirmed using thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Hydroxy-bromhexine and desmethyl-bromhexine were major metabolites found to be unique to bromhexine-treated horses. These metabolites selectively absent from ambroxol-treated horse urine provide a chemical means to distinguish bromhexine from ambroxol administration in horses. These specific metabolites were similarly identified and confirmed in "blind" horse urine samples. The concomitant presence of furosemide (300 mg, i.v.) with bromhexine or ambroxol did not mask the presence of these agents or alter their metabolite profile. By application of the methods described in this study, bromhexine and ambroxol metabolites in horse urine can be easily identified and confirmed.

Determination of bromhexine in plasma by reversed-phase liquid chromatography. Interference of lipoproteins on extraction.

Extraction of the hydrophobic tertiary amine bromhexine from plasma using cyclohexane-heptafluorobutanol (99.5:0.5, v/v) was studied at different pH values. The extraction yield from buffer solutions was quantitative at pH greater than 4.1, but from plasma the extraction yield decreased with increasing pH. Furthermore, at pH 8.4 the extraction yield varied greatly (56-99%) in different human plasma. The addition of lipoproteins to phosphate buffer, at pH 8.1, decreased the extraction yields considerably. Quantitative extraction from plasma was obtained by using a very long extraction time at pH 8.4 or by decreasing the pH to 5.4. The chromatographic system consisted of a reversed-phase column (Nucleosil C18, 5 microns) with an acidic mobile phase (methanol-phosphate buffer, pH 2) containing an aliphatic tertiary amine. UV detection at 308 or 254 nm was used. The limit of quantitation was 5 ng/ml using 3.00 ml of plasma and detection at 254 nm. The intra-assay precision for bromhexine was better than 3.6% at 5 ng/ml.

Lack of detectable N-nitroso-N-methyl-N-cyclohexylamine in humans after administration of bromhexine.

The possible formation of N-nitroso-N-methyl-N-cyclohexylamine (NMCA) from the drug bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammonium hydrochloride) and nitrite was investigated in humans using three different approaches: 1. analysis on metabolites of NMCA in human urine; 2. analysis on NMCA in human gastric juice; 3. in vitro incubation of human gastric juice with therapeutic bromhexine doses. Diet given to volunteers was varied during these investigations with respect to nitrate content. Experiments with a maximum load of 200 mg nitrate to stimulate nitrite formation were performed. Results of in vivo experiments did not indicate any formation of NMCA. In one out of 39 ex-vivo/in-vitro experiments (with a load of 100 mg nitrate in drinking water) 0.5 ng NMCA/ml gastric juice could be detected which is near the detection limit. Finally, this study showed that bromhexine is not secreted by saliva. This allows to conclude that nitrite and bromhexine do not reach the stomach simultaneously over a longer period of time. In consequence, medication with bromhexine is not regarded to represent a risk due to nitrosamine formation.

Influence of a fluidifying agent (bromhexine) on the penetration of antibiotics into respiratory secretions.

The authors report the results of a study designed to evaluate the possible increase of penetration into bronchial secretions of antibiotics when combined with a fluidifying agent: bromhexine. The study was carried out in a double-blind experiment: erythromycin in 22 patients (group I) or amoxycillin in 26 patients (group II) were administered orally; in both groups several patients were given a placebo, instead of bromhexine. Antibiotics were administered at the usual dosage: 0.5 g X 2 for erythromycin (3 days); 1 g X 2 for amoxycillin (7 days); with the latter, two different doses of bromhexine were administered simultaneously: 48 mg/day and 96 mg/day in ten and eight patients respectively. Samples of bronchial secretions were collected by means of fibreoptic bronchoscopy at the second hour for erythromycin and for amoxycillin; simultaneous serum samples were also collected. The results of the study showed in both groups a significant increase of the ratios between bronchial levels and simultaneous serum concentrations when combined with bromhexine; in patients receiving amoxycillin with 96 mg of bromhexine the percentage penetration was noticeably higher (7.5%) than in those treated with 48 mg bromhexine (4.3%). These results confirm the efficacy of bromhexine as a drug able to disrupt mucopolysaccharides of bronchial secretions and, as a result, to increase the bronchial penetration of antimicrobial drugs as evaluated on the basis of percentage penetration ratio.

Bioavailability of bromhexine tablets and preliminary pharmacokinetics in humans.

The absorption of bromhexine from Bromhexin tablets 8 mg, DAK has been compared with that from Bisolvon tablets 8 mg, Boehringer Ingelheim, in a two-way complete crossover study. Four tablets of each of the two bromhexine products, corresponding to a single dose of 32 mg of bromhexine hydrochloride (77.6 mumol), was administered to each of the 10 volunteers. The plasma concentration was followed over the 4-hour period following each administration. By means of Pratt's test for paired data no statistically significant difference (p greater than 0.10) between the two products was found with respect to maximum plasma concentration (89 and 84 nmol.1(-1), respectively), the times for their occurrence (1.3 and 1.0 h, respectively), and the area under the plasma concentration-time curves (140 and 132 nmol.1(-1).h, respectively). It is concluded that Bromhexin, DAK and Bisolvon are bioequivalent. Provisional pharmacokinetic data for bromhexine, after oral administration, in man were obtained. The first-pass effect and the biological half-life were estimated by combining plasma and 30 h urine data from four of the volunteers. The first-pass effect was estimated to be c. 75 per cent, the biological half-life to be c. 6 h, and c. 0.1 per cent of the dose was found as unmetabolized bromhexine in the urine. The data indicate that the pharmacokinetics of bromhexine may be described as a two-compartment open model.

Assay of bromhexine in human plasma by capillary gas--liquid chromatography with nitrogen-selective detection and selected ion monitoring.

A specific, sensitive method for the determination of bromhexine in human plasma is described. It comprises a selective extraction procedure and a specific determination with capillary gas--liquid chromatography and nitrogen-selective flame ionization detection. The detection limit of the assay is about 0.5 ng/ml. The specificity of the assay was checked by gas chromatography--mass spectrometry. The method is applied to the pharmacokinetics of bromhexine in humans.

Electron-capture GLC determination of bromhexine in human plasma.

A specific and sensitive GLC analysis of bromhexine in human plasma is described. After addition of the internal standard and an aqueous triethanolamine solution, bromhexine is extracted at alkaline pH into n-hexane, transferred to an acidic aqueous solution, and back-extracted into n-hexane after alkalinization. Both compounds are derivatized with trifluoroacetic anhydride and quantified by GLC using , 63Ni-electron-capture detector. The method has a sensitivity of approximately 1.0 ng/ml of plasma. Linearity of plasma working curves was good. The extraction recovery from spiked plasma was 90.1 +/- 5.68% (SD). The within-run and within-day precisions (CV) were 6.0% (4.3 ng/ml, n = 8) and 8.6% (11.0 ng/ml, n = 13), respectively. The procedure was applied successfully to measurement of the plasma concentration-time profile in a human volunteer after oral drug administration.

Determination of bromhexine as its trifluoroacetyl derivative by gas-liquid chromatography with electron-capture detection.

A specific gas-liquid chromatographic method with electron-capture detection is described for the determination of bromhexine at the nanogram level. Structurally analogous internal standards were synthesized and their suitability was investigated, based on their chromatographic properties on different stationary liquid phases. The electron-capture activity of the compounds is increased by trifluoroacetylation. Reaction conditions for this derivatization were studied. Calibration graphs in the range 0-57 ng (in a total of 100 microliters of reaction mixture) showed good linearity.