Enantioselective LC/ESI-MS/MS analysis and pharmacokinetic and tissue distribution study of (2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol in rats.

A sensitive and stereospecific liquid chromatography-tandem mass spectrometry method for the quantitative determination of TWo8 enantiomers ((2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol) was developed and validated in rat serum and some tissues. Racemic TWo8 is a new chemical entity, and it has been shown to possess pharmacological activity in vivo. The assay involved the diastereomeric derivatization of racemic TWo8 with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate. The TWo8 diastereoisomers quantification was performed on a triple quadrupole mass spectrometer employing an electrospray ionization technique. The precursor to the product ion transition for TWo8 derivatives and for the internal standard (carbamazepine) was m/z 776.4 → 387.2 and 237.4 → 194.4, respectively. The assay was validated with a linear range of 10-2000 ng/ml of racemic TWo8. The inter-day precisions for (-)-(S)-TWo8 and (+)-(R)-TWo8 were 2.1% to 14.9% and 1.3% to 14.8%, respectively. The inter-day accuracy for (-)-(S)-TWo8 and (+)-(R)-TWo8 was within 86% to 114% and 91% to 114%, respectively. A pilot pharmacokinetic study of this new ß-adrenolytic compound has shown that (-)-(S)-TWo8 is eliminated faster than its antipode. The terminal half-lives of (-)-(S)-TWo8 and (+)-(R)-TWo8 were 3.2 and 3.9 h, respectively. The compound distribution into different organs, evaluated in tissue homogenate samples following TWo8 intravenous administration, showed an enantioselective penetration of TWo8 enantiomers in the liver (p < 0.03), in the kidney (p < 0.001), and in the lungs (p < 0.05). The developed method using liquid chromatography-tandem mass spectrometry method with electrospray ionization could be employed for quantitative determination of compounds with similar structure.

Stereospecific effects of benzo[d]isothiazolyloxypropanolamine derivatives at beta-adrenoceptors: synthesis, chiral resolution, and biological activity in vitro.

We performed the asymmetric synthesis of four enantiopure benzo[d] isothiazo-3-or 5-yloxypropanolamine derivatives, previously described as competitive antagonists at beta-adrenoceptors. The chemical characterization of each enantiomer was accomplished by (1)H NMR and HPLC/DAD/CD. The direct chromatographic separation of the enantiomers via chiral HPLC was investigated. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). The enantiomers obtained had enantiomeric purities suitable for biological assays. Tested in isolated rat cardiac and intestinal tissues to evaluate their effects at beta(1)- and beta(3)-adrenoceptors, the (S)-enantiomers revealed a higher degree of antagonism than (R)-enantiomers at both subtypes, even though their activity was greater at the cardiac beta(1)-subtype. The potent and cardiospecific antagonistic effect exerted by the compounds tested suggests that the benzisothiazole moiety could be an interesting scaffold for discovering new chiral beta-blocking drugs.

Development of retention prediction models for adrenoreceptor agonists and antagonists on a polyvinyl alcohol-bonded stationary phase in hydrophilic interaction chromatography.

Retention prediction models based on multiple linear regression (MLR) and artificial neural network (ANN) for adrenoreceptor agonists and antagonists chromatographed on a polyvinyl alcohol-bonded stationary phase under hydrophilic interaction chromatography were described. The models showed the combined effects of solute structure and mobile phase composition on the retention behavior of the analytes. Using stepwise MLR, the retentions of the studied compounds were satisfactorily described by a five-predictor model; the predictors being the %ACN, the logarithm of the partition coefficient (log D), the number of hydrogen bond donors (HBD), the desolvation energy for octanol (FOct), and the total absolute atomic charge (TAAC). The inclusion of the solute-related descriptors suggested that hydrophilic interactions such as hydrogen bonding and also ionic interactions are possible mechanisms by which analytes are retained on the studied system. ANN prediction models were also derived using the predictors derived from MLR as inputs and log k as outputs. The best network architectures were found to be 5-3-1 for the datasets at pH 3.0 and 4.0, and 5-4-1 for the dataset at pH 5.0. The optimized ANNs showed better predictive properties than the MLR models for both training and test sets under all pH conditions.

Retention prediction of adrenoreceptor agonists and antagonists on unmodified silica phase in hydrophilic interaction chromatography.

The development of retention prediction models for adrenoreceptor agonists and antagonists chromatographed on an unmodified silica stationary phase under the hydrophilic interaction chromatographic (HILIC) mode at three pH conditions (3.0, 4.0 and 5.0) is described. The models were derived using multiple linear regression (MLR) and an artificial neural network (ANN) using the logarithm of the retention factor (log k) as the dependent variable. In addition to the effects of the solute-related variables (molecular descriptors), the percentage of acetonitrile (%ACN) was also used as a predictor to gauge the influence of the mobile phase on the retention behavior of the analytes. Using stepwise MLR, the retention behavior of the studied compounds at pH 3.0 were satisfactorily described by a four-predictor model; the predictors being the %ACN, the logarithm of the partition coefficient (log D), the number of hydrogen bond acceptors (HBA), and the magnitude of the dipole moment (DipolMag). In addition to these four predictors, the total absolute atomic charge (TAAC) was found to be a significant predictor of retention at pH 4.0 and 5.0. Among the five descriptors, %ACN had the strongest effect on the retention, as indicated by its higher standardized coefficient than those obtained for the other four predictors. The inclusion of these four predictors which are related to the molecular properties of the compounds (log D, HBA, DipolMag, and TAAC) suggested that hydrophilic interactions, hydrogen bonding and ionic interactions are possible mechanisms by which analytes are retained on the studied system. The reliability and predictive ability of the derived MLR equations were tested using cross-validation and a test set which was not used when fitting the model. The models derived from MLR produced adequate fits, as proven by the high R2 values obtained for all calibration and training sets (0.9497 and above), and their good predictive power, as indicated by the high cross-validated q2 (0.9465 and above) and high R2 (0.9305 and above) values obtained for the test sets. ANN prediction models were also derived using the predictors derived from MLR as inputs and log k as output. A comparison of the models derived from both ANN and MLR revealed that the trained ANNs showed better predictive abilities than the MLR models, as indicated by their higher R2 values and their lower root mean square error of predictions (RMSEP) for both training and test sets under all pH conditions. The derived models can be used as references and they provide a useful tool for method development and the optimization of chromatographic conditions for the separation of adrenoreceptor agonists and antagonists.

Lysozyme: a mediator of myocardial depression and adrenergic dysfunction in septic shock in dogs.

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the beta- agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation-contraction coupling in sepsis.

The effects of epiphyseal peptides on the release of immunoglobulins in Peyer's patches in rats in vitro.

The effects of epiphyseal peptides (1 microg/ml) on the release of immunoglobulins into the incubation medium by isolated Peyer's patches from non-immunized mice and mice immunized orally against ovalbumin were studied during 40-min incubations. The possibility that epiphyseal peptides act on adrenoreceptors of cells in secondary lymphoid organs in the small intestine was assessed using alpha- and beta-adrenoreceptor blockers, i.e., phentolamine HCl (0.02 mg/ml) and anaprilin (0.06 mg/ml) respectively. Basal levels of secretory activity in control Peyer's patches from immunized rats were 2.4 times (p < 0.01) greater than for the lymphoid organs of non-immunized animals. The effects of epiphyseal peptides on the secretory activity of antibody-forming cells depended on the functional state of Peyer's patches. Application of epiphyseal peptides led to a 35% increase (p < 0.05) in the release of immunoglobulins from Peyer's patches in non-immunized rats and a 25% decrease (p < 0.05) in the release of antibody into the incubation medium from the lymphoid organs of immunized animals. These data lead to the suggestion that the activatory effect of epiphyseal peptides on antibody-forming cells in Peyer's patches from non-immunized animals is associated with alpha-adrenoceptors, while their inhibitory action on immunoglobulin release by the small intestine lymphoid organs from immunized animals is not mediated via adrenoceptors.