Assay of tocainide enantiomers in plasma by solid-phase extraction and indirect chiral high-performance liquid chromatography after derivatization with (−)-menthyl chloroformate

A fast high-performance liquid chromatographic (HPLC) assay was developed for determination of tocainide enantiomers in plasma. Subsequent to solid-phase extraction of tocainide from plasma, homochiral derivatization with ()-menthyl chloroformate enabled separation of the enantiomers by a conventional reversed-phase HPLC system. The detection was performed by UV absorption at 262 nm. An enantiomeric resolution of 1.0 was obtained. Linearity of the method was investigated and found to be good in the range from 1.0 to 20.0 μg/ml tocainide enantiomer and the limit of quantitation was 1.0 μg/ml. The method was applied to a study of the distribution and elimination pharmacokinetics of tocainide enantiomers in the rabbit. No difference in distribution or elimination between the enantiomers was found nor did the enantiomers affect the disposition of one another when administered together as the racemate.     

Enantioselective assay of nisoldipine in human plasma by chiral high-performance liquid chromatography combined with gas chromatographic−mass spectrometry: applications to pharmacokinetics

Nisoldipine, a second-generation dihydropyridine calcium antagonist, is a racemate compound used in the treatment of hypertension and coronary heart disease. This study presents an enantioselective HPLC-GC–MS method for the analysis of nisoldipine in human plasma and establishes confidence limits for its application to pharmacokinetic studies. Plasma samples were basified and extracted with toluene. The enantiomers were resolved on a Chiralcel^® OD-H column using hexane–ethanol (97.5:2.5, v/v) and the (+)- and (−)-fractions were collected separately with the diode array detector switched off. For the quantification of the nisoldipine enantiomers a GC–MS with an Ultra 1 Hewlett-Packard column was used with the detector operated in the single-ion monitoring mode with electron-impact ionization (m/z 371.35 and 270.20 for nisoldipine and m/z 360.00 for the internal standard, nitrendipine). The method proved to be suitable for pharmacokinetic studies based on the low quantification limit (0.05 ng/ml for each enantiomer) and the broad linear range (0.05–50.0 ng/ml for each enantiomer). Low coefficients of variation (<15%) were demonstrated for both within-day and between-day assays. No interference from drugs associated with nisoldipine treatment was observed. The enantioselective pilot study on the kinetic disposition of nisoldipine administered in the racemic form to a hypertensive patient using a multiple dose regimen revealed the accumulation of the (+)-enantiomer with an AUC^0–24 (+)/(−) ratio of approximately 8. Both enantiomers were quantified in plasma at a time interval of 24 h. This HPLC-GC–MS method is reliable, selective and sensitive enough to be used in clinical pharmacokinetic studies on the enantioselective disposition of nisoldipine in humans.     

Stereoselectivity in central analgesic action of tocainide and its analogs

The antiarrhythmic drug tocainide (5a) and some related chiral α-amino and α-imino anilides (5b–e) were synthesized in optically active form. The antinociceptive effects of the different stereoisomers of these compounds were examined and it was found that the analgesic effect of tocainide is due only to its (?)-(R)-enantiomer. Benzyl replacement for methyl group at the stereogenic centre of tocainide causes loss of activity while both enantiomers of the αiminoxilidide 5e and of the strictly related tocainide analog 5d produce an analgesic effect without any stereoselectivity. Pharmacological tests and [³H] quinuclidinyl benzilate ([³H]QNB) binding assay, taken together, seem to show that the antinociceptive effect of (?)-(R)-tocainide, like the analgesia induced by lidocaine, procaine, and mexiletine, is due to a central presynaptic cholinergic mechanism of action. © 1993 Wiley-Liss, Inc.     

Relation between the energy state of the myocardium and the release of products of anaerobic metabolism

The relationships between cellular energy parameters and succinate, alanine and creatine release from isolated guinea pig hearts were studied during a 50 min perfusion (0.2 ml/min) with 5.5 mM glucose or 5 mM sodium acetate. Compared to glucose-perfused hearts, a more rapid ATP depletion accompanied by an increased succinate and creatine release was observed during underperfusion with acetate. The succinate and alanine accumulation in the myocardial effluent was related to a decrease in tissue ATP; the creatine release showed a close inverse correlation with the tissue phosphocreatine/creatine ratio. Hyperbolic and linear relationships were found between these indices for glucose- and acetate-perfused hearts, respectively. The logarithm of tissue ATP had negative linear correlations with the perfusate succinate/creatine ratio for the both substrates. The experimental results suggest that succinate, creatine and alanine assays in the myocardial effluent may be used for the assessment of the energy state of ischemic heart.     

Stereoselective disposition of S- and R-licarbazepine in mice

The stereoselective disposition of S-licarbazepine (S-Lic) and R-licarbazepine (R-Lic) was investigated in plasma, brain, liver, and kidney tissues after their individual administration (350 mg/kg) to mice by oral gavage. Plasma, brain, liver, and kidney concentrations of licarbazepine enantiomers and their metabolites were determined over the time by a validated chiral HPLC-UV method. The mean concentration data, attained at each time point, were analyzed using a non-compartmental model. S-Lic and R-Lic were rapidly absorbed from gastrointestinal tract of mouse and immediately distributed to tissues supplied with high blood flow rates. Both licarbazepine enantiomers were metabolized to a small extent, each parent compound being mainly responsible for the systemic and tissue drug exposure. The stereoselectivity in the metabolism and distribution of S- and R-Lic was easily identified. An additional metabolite was detected following R-Lic administration and S-Lic showed a particular predisposition for hepatic and renal accumulation. Stereoselective processes were also identified at the blood-brain barrier, with the brain exposure to S-Lic almost twice that of R-Lic. Another finding, reported here for the first time, was the ability of the mouse to perform the chiral inversion of S- and R-Lic, albeit to a small extent.     

Study of stereoselective pharmacokinetics of anisodamine enantiomers in rabbits by capillary electrophoresis

The purpose of this study was to determine the pharmacokinetics of anisodamine enantiomers in plasma after oral and intravenous administration of racemic anisodamine in rabbits. A capillary electrophoresis method for the simultaneous separation of two pairs of enantiomers in plasma has been firstly developed and validated. Using a 75 mM phosphate buffer containing 25 mM carboxymethylated-gamma-cyclodextrin at pH 2.5, good resolution was achieved on a 45-cm uncoated fused-silica capillary at the voltage of 20 kV and 25 degrees C. The pharmacokinetics of individual anisodamine enantiomers were characterized using the CE assay, the sole method of enantiomeric separation for anisodamine. Pharmacokinetic analysis of results indicated that anisodamine enantiomers showed non-stereoselective disposition or stereoselective disposition in different rabbits. For the rabbits with non-stereoselective disposition, similar pharmacokinetic characteristics were observed between (6S, 2'S)- and (6R, 2'R)-, or (6S, 2'R)- and (6R, 2'S)-anisodamine. For the rabbits with stereoselective disposition, (6S, 2'S)- and (6R, 2'S)-anisodamine were below the established LOD, while the two remaining enantiomers also had similar pharmacokinetic profiles. Further investigations remain necessary to find out the underlying mechanism about the stereoselective disposition of (6S, 2'S)- and (6R, 2'S)-anisodamine.     

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies

Pregabalin (Lyrica) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a gamma-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.     

Measurement of the activation time of oxidative phosphorylation in isolated mouse hearts

The purpose of this study was to develop a technique for determination of the dynamic regulation of oxidative myocardial metabolism in the mouse. The response time of myocardial oxygen consumption (MVO(2)) to a step in heart rate was determined in Langendorff-perfused mouse hearts. We examined the effect of glucose-only perfusate and glucose combined with 1, 3, or 6 mM pyruvate. Left ventricular systolic pressure (LVSP) decreased, yet the rate-pressure product (RPP) and MVO(2) increased with upward steps in heart rate. Pyruvate increased LVSP, RPP, and MVO(2) at the lower concentrations; however, when 6 mM pyruvate was added, LVSP and RPP became depressed while MVO(2) remained elevated. The mean response time of oxygen consumption to a step in heart rate from 270 to 350 beats/min was 9.8 s (n = 7) in the glucose-only perfused hearts. Perfusion with glucose plus 6 mM pyruvate decreased the response time to 5.3 s. These results are similar to those found in the rabbit heart and lay the groundwork for further examination of the dynamic regulation of oxidative myocardial metabolism in genetically altered mice. We concluded that the activation time of oxidative phosphorylation in the mouse is similar to that in larger species, despite the high mitochondrial content and natural heart rate of the mouse.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Is the damaging action of catecholamines on the myocardium realized via hyperstimulation of the beta receptors?

Experiments on an isolated rat heart were made to compare the damaging action on the myocardium of catecholamines (noradrenaline, adrenaline and isoproterenol) differing in the affinity for beta-receptors. The damage to myocardial cells was evaluated from the release into the perfusate of intracellular enzymes (creatine phosphokinase and lactate dehydrogenase) and the number of contracture damaged myocytes. Noradrenaline exerted the most powerful damaging action on the myocardium at a concentration of 10(-6) M. Perfusion of the heart with isoproterenol at concentrations of 10(-6) M and 10(-5) M did not lead to the affection of cardiomyocytes. It was isoproterenol concentration exceeding noradrenaline concentration 100 times that produced an increase in the rate of the release of the enzymes to the perfusate and a rise of the number of contractures in the myocardium, with the above increase being less than that provoked by adrenaline and noradrenaline (10(-6) M). It is concluded that the mechanism of the cardiotoxic effect of catecholamines cannot be reduced only to their effect on myocardial beta-receptors.     

Intratumoral pharmacokinetics of oligonucleotides in a tissue-isolated tumor perfusion system

The intratumoral pharmacokinetics of model oligonucleotides were studied in Walker 256 tissue-isolated tumor preparations using an in situ single-pass vascular perfusion technique. A 20-mer phosphodiester (PO) oligonucleotide, its fully phosphorothioated (PS) oligonucleotide counterpart, and an 18-mer phosphorothioated oligonucleotide containing four 2'-O-methylribonucleosides at both the 3'-end and 5'-end (PS-OMe) were used. These oligonucleotides were administered to the tumor in two ways, by constant arterial infusion and by direct intratumoral injection. In the case of constant arterial infusion, the experiments were carried out using perfusate with or without 4.7% bovine serum albumin (BSA). The protein binding of PO, PS, and PS-OMe to BSA was 46%, 87%, and 94%, respectively. No marked difference was observed between the degree of accumulation of the three types of oligonucleotides in the tumor when BSA was present in the perfusate. PS and PS-OMe showed higher degrees of accumulation in tumors compared with PO when no BSA was present. These results indicate that free (i.e., protein unbound) PS-OMe and PS have superior tumor accumulation characteristics. In the intratumoral injection experiments, PS-OMe was retained longer in tumor tissue compared with PS, suggesting that it might be useful for direct local injection into solid tumors. Thus, the present study provides useful information about the basic disposition characteristics of oligonucleotides in solid tumors.     

Bioanalytical chiral chromatographic technique and docking studies for enantioselective separation of meclizine hydrochloride: Application to pharmacokinetic study in rabbits

Enantiomeric resolution and molecular docking studies of meclizine hydrochloride on polysaccharide-based chiral stationary phase comprising cellulose tris(4-methylbenzoate) chiral selector (150 × 4.6 mm, 3.0 μm) were presented. The mobile phase used was acetonitrile:10mM ammonium bicarbonate (95:05, v/v). The developed technique was used to perform the enantioselective assay of meclizine hydrochloride in its marketed formulation. The elution order of meclizine hydrochloride enantiomers was determined by docking studies. Target compound was extracted from rabbit plasma using protein precipitation technique, followed by development of bioanalytical chiral separation method using the same matrix. Application of the method to determine pharmacokinetic parameters of meclizine hydrochloride enantiomers was performed using Phoenix WinNonlin 8.1 software. The results demonstrated stereoselective disposition of meclizine hydrochloride enantiomers in rabbits.     

Naloxone reduces release of creatine kinase in the isolated ischemic rat heart

The effects of naloxone, propranolol, or both on the release of creatine kinase (CK) from the isolated ischemic rat heart were studied. Naloxone at concentrations of 1.1 and 3.6 mmole liter-1 in the perfusate at a rate of 1-2 ml min-1 reduced the release of CK from the isolated ischemic rat heart during myocardial ischemia in a dose-dependent manner. Propranolol at a concentration of 7 mumole liter-1 in the perfusate also reduced the release of CK. Addition of naloxone (1.1 mmole liter-1) to propranolol further reduced the release of CK. The effect of the joint administration of the two drugs seemed to be additive.     

The effects of pantothenic acid,cysteine and dithiothreitol in intact,reperfused pig hearts

The objective of this study was to augment myocardial tissue levels of amphiphiles using a treatment protocol of pantothenic acid, cysteine and dithiothreitol (DTT) in 24hr fasted pigs and to test their influence on mechanical recovery in reperfusion. Eighteen pig hearts were extracorporeally perfused aerobically, subjected to regionally reversible ischemia in the left anterior descending perfusion system and reperfused. Nine hearts served as a placebo group; nine hearts were treated. All hearts received trace-labeled palmitate to measure fatty acid oxidation and were perfused with an infusion of 20% Intralipid to augment perfusate levels of fatty acids. Fasting alone in the presence of carbon substrates in the coronary perfusate was not sufficient to de-inhibit pantothenic acid kinase such that CoA synthesis was not enhanced. Tissue contents of triacylglycerols and phospholipids in reperfused myocardium were no different than in aerobic heart muscle but free CoA and free and total carnitine were reduced, suggesting a leakage of cytosolic contents across injured sarcolemma. Treatment significantly impaired mechanical recovery during reflow, presumable due to the noxious properties of DTT whose reported effects in heart muscle are wide ranging, difficult to predict in intact hearts and may be harmful.     

Neuropeptide Y (NPY) reduces myocardial perfusion and inhibits the force of contraction of the isolated perfused rabbit heart

Isolated rabbit hearts, perfused under constant pressure (Langendorff technique) were used to study the effect of neuropeptide Y (NPY) on heart rate, force of heart contraction and rate of myocardial perfusion. No significant net change in heart rate was noted. A dose-dependent negative inotropic effect was consistently demonstrated which was characterised by slow onset and was often preceded by a transient positive inotropic response. Addition of small doses of NPY resulted in a prompt reduction in flow of the perfusate through the coronary vasculature. Since NPY is present locally in cardiac nerves, these effects may have physiological importance.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.     

Interaction of polystyrene microspheres with liver cells: roles of membrane receptors and serum proteins

Our previous studies have demonstrated that serum would play an important role in the hepatic disposition of polystyrene microspheres (MS) and that complement C3 should be involved as the serum opsonin. In this study, we tried to identify the entity of other serum opsonins and dysopsonin for the hepatic uptake of MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion studies using a recirculation procedure in rats. Pretreatment of the liver by trypsin significantly suppressed the serum-dependent hepatic uptake of both MSs, suggesting that some protein components on the cell surface should be necessary for the serum-dependent phagocytosis of MSs. Pretreatment of the serum by the anti-fibronectin antibody resulted in a significant reduction in the hepatic disposition of MS-500 (49% of control), suggesting that fibronectin should also work as the opsonin for the hepatic uptake of MS-500. The hepatic disposition of both MSs in the presence of serum was inhibited by the addition of N-acetylgalactosamine into the perfusate, suggesting the possible involvement of lectin in the serum-dependent hepatic uptake of MSs. Furthermore, a more intensive hepatic disposition of MSs was observed in the presence of plasma compared with that in the presence of serum in the perfusate, suggesting the possible involvement of blood coagulation factors, such as fibrinogen, as the opsonin in the hepatic disposition of MSs.     

Enantioselective kinetic disposition of albendazole sulfoxide in patients with neurocysticercosis

The enantioselectivity of the kinetic disposition of albendazole sulfoxide (ASOX) was investigated in 18 patients with neurocysticercosis treated with a multiple dose regimen of albendazole for 8 days (5 mg/kg every 8 h). Serial blood samples were collected on the eighth day of treatment during the last dose interval, with prorogation up to 12 h. Albendazole sulfone (ASON) and enantiomers of ASOX were analyzed in plasma samples by HPLC using a Chiralpak AD column and detection by fluorescence. The pharmacokinetic parameters showing statistically significant differences between the (+) ASOX and (-) ASOX enantiomers are presented as respective means (95% CI) as follows: maximum plasma concentration, Cmax = 301.6 (179.7-423.5) vs 54.9 (21.9-87.9) ng.ml-1; elimination half-life, t1/2 = 5.2 (4.1-6.3) vs 3.3 (2.8-3.8) h, area under the plasma concentration-time curve, AUCss0-8 = 1719.2 (978.6-2459.8) vs 261.4 (102.9-419.8) ng.h.ml-1 and apparent clearance, Cl/fm = 5.8 (3.8-7.8) vs 54.0 (35.2-72.7) l.h-1.kg-1. The mean value of 9.2 (7.6-10.9) for the AUC0-8(+)-ASOX/AUC0-8(-)-ASOX ratio demonstrated plasma accumulation of the (+) enantiomer. Sulfone formation capacity, expressed by the AUCss0-8 ratio ASON/ASOX + ASON, was 8.0 (7.0-8.9). The present data indicate enantioselectivity in the kinetic disposition of ASOX in patients with neurocysticercosis.     

The role of adenosine in the isolatedRana ridibunda heart

Summary In an attempt to study the metabolic role of adenosine in the amphibian heart, we perfusedRana ridibunda hearts under conditions of decreased oxygen supply or increased oxygen demand and measured the rate of adenosine appearance as well as the concentrations of adenine nucleotides. Anoxia was associated with a significant increase in the myocardial and perfusate concentration of adenosine and its degradation products, inosine and hypoxanthine, while changes were also observed in the concentrations of adenine nucleotides and creatine phosphate. Furthermore, adenosine production inRana ridibunda hearts was enhanced under conditions of increased cardiac work induced by perfusion pressure elevation. Adenosine production was inversely proportional to the energy charge value calculated from the tissue content of adenine nucleotides under conditions of anoxia and increased heart work. The results are in accordance with the proposed role of adenosine as a physiological metabolic vasodilator in the coronary circulation of the mammalian heart and support the hypothesis that adenosine can be involved in regulating blood vessel resistance inRana ridibunda heart under conditions of low myocardial oxygen tension. Thus it appears that adenosine could act as a vasodilatory substance inRana ridibunda heart.     

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).