Levels of circulating anti-muscarinic and anti-adrenergic antibodies and their effect on cardiac arrhythmias and dysautonomia in murine models of Chagas disease.

SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as ß 1 and ß 2 adrenergic (anti-ß 1-AR and anti-ß 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-ß 1-AR Ab are detected only in the chronic phase, while anti-ß 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60 dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-ß 1-AR, anti-ß 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.

A validated enantioselective LC-MS/MS assay for the simultaneous determination of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma: application to a clinical pharmacokinetic study.

Carvedilol is widely prescribed for the treatment of hypertension, heart failure and left ventricular dysfunction following myocardial infarction. A sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable and efficient bioanalysis of the (R)- and (S)-enantiomers of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma. Following plasma extraction using supported liquid extraction (SLE) in a 96-well plate format, extracted samples were derivatized with 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl isothiocyanate (GITC). Chromatographic separation was achieved by gradient elution on an ACQUITY UPLC HSS T3 analytical column. The impact of several potentially interfering isobaric metabolites on the quantification of the 4'-hydroxyphenyl metabolite (R)- and (S)-enantiomers was minimized by implementation of a combination of chromatographic and mass spectrometric techniques. Derivatized analytes and stable-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over concentration ranges of 0.200-100 ng/mL for (R)- and (S)-carvedilol and 0.0200-10.0 ng/mL for (R)- and (S)-4'-hydroxyphenyl carvedilol. Intra- and inter-assay precision values for replicate quality control samples were within 11.9% for all analytes during the assay validation. Mean quality control accuracy values were within ±9.4% of nominal values for all analytes. Assay recoveries were high (>76%) and internal standard normalized matrix effects were minimal. The four analytes were stable in human plasma for at least 24 h at room temperature, 89 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of the (R)- and (S)-enantiomers of both carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma in support of a human pharmacokinetic study.

Safety and pharmacokinetic studies of silodosin, a new α1A-adrenoceptor selective antagonist, in healthy Chinese male subjects.

The objectives of the study were to assess the safety and pharmacokinetics of silodosin capsules in 82 healthy male Chinese subjects. To evaluate the safety after single-dosing escalation, 40 subjects were equally divided into 4 groups (2, 4, 8, 12 mg) by a randomized, double-blind and placebo-controlled design. To assess the pharmacokinetics after single-dosing, 30 subjects were equally divided into 3 groups (4, 8, 12 mg). To assess the safety and pharmacokinetics via multiple-dosing, 12 subjects were included as a group (4 mg once daily at day 1 and day 7; 4 mg twice daily at day 2 through day 6). The safety observations showed that mild adverse events, including postural hypotension, dizziness, and headache, were observed. After single-dosing at doses of 4, 8, and 12 mg, the mean area under the concentration-time curve from 0 to 36 h (AUC(0-36)) values were 136.82±46.38, 270.17±54.66, and 474.63±108.50 µg/l·h and the mean maximal silodosin concentration in plasma (C(max)) values were 26.70±7.48, 48.47±12.35, and 94.07±22.59 µg/l, respectively. After multiple-dosing, the C(max) value at day 7 was 33.84±19.54 µg/l, and the AUC(0-24) value at day 7 was 193.19±68.96 µg/l·h. The accumulation ratio of the AUC value was 1.55 by comparing the multiple-dosing with the single-dosing. It is concluded that silodosin is safe and tolerated in healthy Chinese male subjects at the dosing levels used in this study. The mean C(max) and AUC values of silodosin increased proportionally with dose escalation, showing characteristics of linear pharmacokinetics.

Antiadrenergic and hemodynamic effects of ranolazine in conscious dogs.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 µg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 µg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 µM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 µM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and ß) effects.

Fibers with polypyrrole and polythiophene phases for isolation and determination of adrenolytic drugs from human plasma by SPME-HPLC.

In this study, polypyrrole (PPy) and polythiophene (PTh) SPME coatings and their ability to extract selected adrenolytic drugs with different physico-chemical properties from standard solutions and human plasma samples were evaluated. In measurements metoprolol, oxprenolol, mexiletine, propranolol, and propaphenon were investigated. The main parameters such as extraction time, desorption conditions and pH influence were examined. Inter-day precisions were in range 0.1-2.0%, 1.1-2.9%, 1.3-2.6%, 0.1-2.6% and 0.3-2.1% for metoprolol, oxprenolol, mexiletine, propranolol and propaphenon, respectively. Accuracies were less than 15%, which was evaluated by analyzing preparation samples of five replicates. The method was successfully applied to human plasma samples spiked with selected adrenolytic drugs. The method was linear in the concentration range from 1 to 10microg/ml for all of studied adrenolytic drugs using human plasma samples. The PTh-SPME coating displayed higher extraction efficiency towards the target analytes in comparison to PPy-SPME. The reproducibility of the extraction using polypyrrole and polythiophene fibers was confirmed by variation coefficients lower than 8% and 3%, respectively.

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics.

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel alpha- and beta-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C(18) (250 mm x 4.0 mm i.d., 5 microm) column at 50 degrees C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20-10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.

Determination of adrenolytic drugs by SPME-LC-MS.

Five adrenolytic drugs have been analyzed by liquid chromatography-mass spectrometry (LC-MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5-13%, however RSD values for the drugs in human plasma were 2.5-4.5%. Using LC-MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11-0.18 and 0.39-0.54 ng mL(-1), respectively, for the five drugs.

Stereoselective analysis of carvedilol in human plasma and urine using HPLC after chiral derivatization.

An enantioselective high-performance liquid chromatographic method for the analysis of carvedilol in plasma and urine was developed and validated using (-)-menthyl chloroformate (MCF) as a derivatizing reagent. Chloroform was used for extraction, and analysis was performed by HPLC on a C18 column with a fluorescence detector. The quantitation limit was 0.25 ng/ml for S(-)-carvedilol in plasma and 0.5 ng/ml for R(+)-carvedilol in plasma and for both enantiomers in urine. The method was applied to the study of enantioselectivity in the pharmacokinetics of carvedilol administered in a multiple dose regimen (25 mg/12 h) to a hypertensive elderly female patient. The data obtained demonstrated highest plasma levels for the R(+)-carvedilol (AUC(SS) (0-12) 75.64 vs 37.29 ng/ml). The enantiomeric ratio R(+)/S(-) was 2.03 for plasma and 1.49 for urine (Ae(0-12) 17.4 vs 11.7 microg).

Population pharmacokinetics of S(-)-carvedilol in healthy volunteers after administration of the immediate-release (IR) and the new controlled-release (CR) dosage forms of the racemate.

Carvedilol is a beta(1)-, beta(2)-, and alpha(1)-adrenoreceptor blocker indicated for treatment of hypertension and mild-to-severe congestive heart failure. The objective of this study was to develop and evaluate a single population model that describes S(-)-carvedilol pharmacokinetics from both the immediate-release (IR) and the new controlled-release dosage forms of the racemate. Carvedilol IR data (1270 measurements) were obtained from 2 open-label studies (50 mg/25 mg Q12 hours for 2 doses). Carvedilol CR data (2058 measurements) were obtained from an open-label, nonrandomized, dose-rising (10, 20, 40, and 80 mg), 4-period balanced crossover study. All data were simultaneously analyzed using NONMEM V. Leverage analysis and internal evaluations were conducted for the final model. A 2-compartment model with first-order absorption and elimination provided the best fit. The model included different absorption rates (KAs) for the CR and IR morning (IR(AM)) and evening (IR(PM)) doses; incorporating change-points at certain times. Estimates of KAs indicated that the absorption was slower at equivalent times and extended for CR relative to IR carvedilol. Oral clearance of S(-)-carvedilol was 149 L/h. The IR(PM) and the CR doses had bioavailability (F(rel)) of 0.80 and 0.76, respectively, relative to the IR(AM) dose. The inter-subject variability in KAs was lower for the CR dosage form than the original IR dosage form. Estimation of interoccasion variability on KAs and F(rel) for the CR dosage form improved the fit. The model performed well in simulation and leverage analysis indicated its robustness. The model will be a useful tool for future simulation studies.

Does a stable isotopically labeled internal standard always correct analyte response? A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma.

A stable isotopically labeled (SIL) analogue is believed to be the most appropriate internal standard in a quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. It is assumed that a SIL internal standard always compensates for variability in chemical derivatization, sample extraction and LC/MS/MS analysis due to its nearly identical chemical and physical properties to the unlabeled analyte. Hence, the analyte to internal standard peak area ratio should be constant despite any variations in sample processing or analysis. However, in our laboratories, a deuterium labeled internal standard of carvedilol demonstrated an unexpected behavior-the analyte to internal standard peak area ratio changed with two specific lots of commercially supplied human plasma. Several experiments, including dilution of the extract with LC mobile phase and post-column infusion of the carvedilol solution followed by the injection of extracted blank plasma, have indicated that a high level of matrix suppression affected the ionization of the carvedilol-S enantiomer and its deuterated internal standard differently in these two lots of plasma. For the first time, it was clearly demonstrated that a slight difference in retention time between the analyte and the SIL internal standard, caused by deuterium isotope effect, has resulted in a different degree of ion suppression between these two analogues. This difference was significant enough to change the analyte to internal standard peak area ratio and affect the accuracy of the method.

Application of capillary electrophoresis with field-amplified sample injection for the detection of new adrenoreceptor antagonist enantiomers in plasma in the low ng/mL concentration range.

Throughout the separation of chiral basic drugs by capillary electrophoresis (CE) with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral selector, the sensitivity of detection has been improved by using field-amplified sample injection (FASI). In the present work, this on-line stacking method has been used to detect low ng/mL levels of cationic enantiomers of a new adrenoreceptor antagonist in plasma. A systematic study of the parameters affecting on-line concentration of these enantiomers (nature of the preinjection plug, composition of sample solvent, injection times of water and sample plugs) has been performed enabling the detection sensitivity of antagonist enantiomers to be improved by 180 times compared with usual hydrodynamic injection. The quantification of each adrenoreceptor antagonist enantiomer in plasma samples was then performed in the 2-100 ng/mL (or 8-400 nM) concentration range after a solid-phase extraction step. Using this FASI-CE-UV procedure, the limit of quantification (LOQ) for each enantiomer was in the low ng/mL concentration range (3 ng/mL or 10 nM).

Multiple dose pharmacokinetics of fiduxosin under fasting conditions in healthy elderly male subjects.

Selective alpha1a-adrenoceptor antagonists are effective agents for treatment of benign prostatic hyperplasia, a disorder occurring in middle-aged and elderly males. The objective of this study was to determine the pharmacokinetics of fiduxosin, a novel alpha1a-adrenoceptor antagonist, following multiple dose administration. This was carried out in a Phase I, randomized, double-blind, placebo-controlled, parallel group, multiple oral dose study of fiduxosin. Single once-daily oral doses of 30, 60, 90 or 120 mg of fiduxosin or placebo were administered to healthy elderly male subjects (n = 48; 8 active and 4 placebo per dosing group) for 14 consecutive days. Fiduxosin plasma concentration-versus-time profiles for days 1, 7 and 14 were used to assess fiduxosin pharmacokinetics. Steady state was achieved by day 7. At steady-state mean Tmax (time to maximum plasma concentration), CL/F (apparent oral clearance) and Vbeta/F (apparent volume of distribution) ranges were 1.8-7.8 h, 27.3-47.2 L h(-1) and 846-1399 L, respectively. Tmax and VbetaF were independent of dose. Cmax (maximum plasma concentration), Cmin (minimum plasma concentration) and AUC24 (area under plasma concentration vs time curve from 0 to 24 h) for days 7 and 14 were linearly proportional with dose overthe 30-120 mg/day dose range and were unchanged from day 7 to day 14. It was concluded that fiduxosin multiple-dose pharmacokinetics were dose-independent and time-invariant over the 30-120 mg/day dose range under fasting conditions.