Pharmacokinetics of the R- and S-enantiomers of oxybutynin and N-desethyloxybutynin following oral and transdermal administration of the racemate in healthy volunteers.

PURPOSE: To characterize the enantiomers of oxybutynin (OXY) and N-desethyloxybutynin (DEO) following transdermal and oral administration. METHODS: OXY was administered either as a single transdermal system over a 96 h wear period or as a single 5 mg immediate-release tablet to 18 healthy male and female subjects in a randomized, open-label, two-way crossover design. Blood samples were collected for 108 h after application of the transdermal system and for 6 h after oral administration. Plasma concentrations of the R- and S-enantiomers of OXY and DEO were assayed by LC-MS/MS. Enantiomer in vitro skin flux was evaluated using human cadaver skin. RESULTS: In vitro skin flux studies demonstrated equal absorption of R and S- OXY. Plasma concentrations and pharmacokinetic parameters of the R-enantiomers of OXY and DEO were slightly lower than the S-enantiomers following transdermal OXY. The relative AUC values were S-OXY>S-DEO>R-OXY>R-DEO. The AUC ratios of DEO/ OXY were less than 1 for both the R- and S- enantiomers. Following oral dosing, plasma DEO concentrations greatly exceeded OXY resulting in relative AUC values of R-DEO>S-DEO>S-OXY>R-OXY. The mean AUC ratios of S- and R-DEO/OXY were 3.25 and 8.93, respectively. CONCLUSIONS: Stereoselective metabolism of OXY was evident following both transdermal and oral administration of OXY. The reduced pre-systemic metabolism of transdermally administered OXY compared to oral administration resulted in not only significantly lower DEO plasma concentrations, but also a different metabolite pattern. The differences between R-OXY and R-DEO following the two routes of administration support the potential for comparable clinical efficacy and reduced anticholinergic side-effects with transdermal treatment.

Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: Cells die by one of two mechanisms, necrosis or programmed cell death. Necrosis has been implicated in stroke and occurs when the cytoplasmic membrane is compromised. Programmed cell death requires protein synthesis and often involves endonucleolytic cleavage of the cellular DNA. We assessed the potential contribution of programmed cell death to ischemia-induced neuronal death. METHODS: Cycloheximide (protein synthesis inhibitor; 1 mg/kg per 24 hours) or vehicle (1 mL/kg per 24 hours) was continuously infused into the right cerebral ventricle of spontaneously hypertensive rats. Neocortical focal ischemia was produced by tandem occlusion of the right common carotid artery and the ipsilateral middle cerebral artery. After 24 hours the brain was stained with 2% 2,3,5-triphenyltetrazolium and the ischemic zone quantitated. Protein synthesis was determined by [3H]methionine incorporation into acid-precipitated protein. DNA integrity was determined in isolated DNA by gel electrophoresis and in whole cells by flow cytometry. RESULTS: Continuous cycloheximide infusion caused approximately 70% reduction in cortical protein synthesis. Cycloheximide also reduced the size of the infarction produced by focal cerebral ischemia when compared with controls (ischemic brain volume, 147.5 +/- 25.9 and 188.7 +/- 16.8 mm3 for cycloheximide and saline, respectively; P < .01), suggesting that protein synthesis may contribute to cell death. Purified DNA from the ischemic zone showed evidence of endonucleolytic degradation when fractionated by gel electrophoresis. Flow cytometric analysis demonstrated increased propidium iodide fluorescence in intact cells isolated from ischemic cortex, indicating an increased accessibility of degraded DNA to the intercalating dye. CONCLUSIONS: New protein synthesis appears to contribute to ischemic cell death in which endonucleolytic DNA degradation is apparent. These observations implicate programmed cell death in ischemic injury and may open unique therapeutic approaches for the preservation of neurons in stroke.

[3H]TA-3090, a selective benzothiazepine-type calcium channel receptor antagonist: in vitro characterization.

Binding of the new benzothiazepine calcium channel blocker, (+)-(2S,3S)-3-acetoxy-8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro-2- (4- methoxyphenyl)-1,5-benzothiazepine-4-(5H)-one maleate, [3H]TA-3090), was characterized and its specificity for rat myocardial benzothiazepine receptors described. Scatchard plots and nonlinear regression analysis of specific [3H]TA-3090 binding best fit a one-site binding model (Kd = 8.8 +/- 2.7 nM, Bmax = 132 +/- 38 fmol/mg protein). Kinetically derived affinity constants were in close agreement (Kd = 7.86 nM) with those obtained from analysis of equilibrium binding data. In comparison, under identical conditions [3H]diltiazem exhibited a Kd of 38 nM and Bmax, 106 fmol/mg protein. Specific binding was saturable, reversible and stereoselective (d-cis-TA-3090 Ki = 14 nM; 1-cis-TA-3090 Ki = 2700 nM). Competitions for [3H]TA-3090 binding were conducted with nifedipine, propranolol, prazosin, quinuclidinyl benzilate, verapamil and yohimbine. Only the calcium channel blockers nifedipine and verapamil inhibited specific [3H]TA-3090 binding. Nifedipine could maximally inhibit only 52% of specifically bound [3H]TA-3090 at 10 microM. In contrast, however, 10 microM verapamil completely inhibited specific radioligand binding (Ki = 93 +/- 28 nM) but with six times less efficacy than TA-3090. Thus, these data demonstrate that [3H]TA-3090 is a potent radioligand selective for the benzothiazepine binding site and is consistent with the hypothesis that [3H]TA-3090 interacts with a myocardial benzothiazepine receptor site.

Studies on Ca2+ channel antagonists. 5-[(3,4-Dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2- isopropylpentyl isothiocyanate, a chemoaffinity ligand derived from verapamil.

Reduction of 1 (verapamil) afforded amine 2, which was converted with thiophosgene to isothiocyanate 3, a chemoaffinity ligand for Ca2+ channels. Compound 3 showed concentration-dependent negative inotropic effects in rat right myocardial ventricular strips, EC50 = (4.56 +/- 3.40) X 10(-6) M (mean +/- SD), being slightly less potent than 4 (gallopamil), EC50 = (1.95 +/- 1.22) X 10(-6) M. It displaced [3H]gallopamil in rat myocardial membranes, IC50 = (3.42 +/- 2.51) X 10(-7) M, approximately equipotent with 1. It showed irreversible antagonism of [3H]gallopamil binding when preincubated at 10(-5) M; only 25% of [3H]gallopamil binding vs. control was observed. This agent may be a useful chemoaffinity ligand to aid in characterization of Ca2+ channels.

The interaction of phenylalkylamine calcium channel blockers with the 1,4-dihydropyridine binding site.

The interaction of verapamil and other phenylalkylamine calcium channel blockers with the 1,4-dihydropyridine receptor was examined. Studies characterizing the interaction and relationship between calcium channel blocking potency and binding affinity were performed in rat myocardium. The 1,4-dihydropyridines, nifedipine and nitrendipine, interacted competitively. The apparent Kd and Bmax of nitrendipine were 270 +/- 140 pM and 390 +/- 76 fmol/mg protein, respectively. In contrast, the interaction of the phenylalkylamines with the 1,4-dihydropyridine receptor was not competitive. At a 3H-nitrendipine concentration of 0.12 nM, verapamil displaced only 60% of specifically bound radioactivity and progressively less as the concentration of 3H-nitrendipine increased. Kinetic data indicated that the interaction of both D600 and verapamil with the 1,4-dihydropyridine receptor was not cooperative. At infinite dilution the dissociation rate constant (k-1) was not altered in the presence of 10(-5) M D600. We examined the hypothesis that 3H-nitrendipine interacts at several sites with similar affinities and that the phenylalkylamines interact at one of these sites. Although D600 could not further displace 3H-nitrendipine in the presence of a maximally displacing concentration of nifedipine (10(-6) M), nifedipine could further displace 3H-nitrendipine in the presence of a maximally displacing concentration of D600 (10(-5) M). These results are consistent with competitive interactions at multiple sites but do not explain the diminished ability of the phenylalkylamines to displace progressively less radioactivity at increasing 3H-nitrendipine concentrations. No relationship between binding affinity and pharmacologic potency of the phenylalkylamines was found suggesting that the interaction of the phenylalkylamines with the 1,4-dihydropyridine receptor is not responsible for their calcium channel blocking effects.

Extraction, separation, and detections of 14C-diazepam and 14C-metabolites from brain tissue of mature and old rats.

A rapid method for simultaneous determination of brain concentrations of diazepam and each of its three major metabolites in brain tissue by a reverse isotope dilution procedure is presented. Radiolabeled diazepam and metabolites were extracted from brain tissue of mature and senescent rats with ethyl ether. After the ether was evaporated the benzodiazepines were separated from the residue by passing the water soluble portion through C-18 bonded-phase extraction columns. High pressure liquid chromatography (HPLC) was used to separate the benzodiazepines from each other. Reverse isotope dilution analysis was used to quantify diazepam and its metabolites. The percent recovery of diazepam and its metabolites from the brain of mature or senescent rats did not vary significantly.

Aging: effects on beta-carboline binding in hippocampal subfields.

The CA1 and CA4/area dentata subfields of the hippocampus in young, mature and old rats were examined for age-related change in propyl beta-carboline-3-carboxylate (PrCC) binding. 3H-PrCC bound with high affinity (Kd = 0.82 nM) to one binding site when ethyl beta-carboline-3-carboxylate was used to delineate specific binding. An age dependent change in the maximum number of 3H-PrCC binding sites in the CA1 and CA4/area dentate subfields of the hippocampus were assessed by using a saturating (6 nM) concentration of 3H-PrCC. Although 3H-PrCC specific binding at a saturating concentration (6 nM) was significantly less in both the CA1 and CA4/area dentata subfields of the senescent rat, the magnitude of the decrease was greater in the CA1 region. In addition, the affinity of the 3H-PrCC binding site in both subfields probably did not vary significantly with age. Therefore, the CA1 and CA4/area dentate of the rat hippocampus may not only lose BZ1 receptors or the BZ1 receptor conformation with age, but the severity of receptor or receptor conformation loss varies with the subfield.

Characterization of the respiratory activity of (D-Ala2)methionine-enkephalinamide.

The effects of central administration of (D-Ala2)methionine-enkephalinamide (EA) on respiration were studied in the unanesthetized rat. EA induced a dose-dependent depression of respiratory rate and tidal volume which was reversed by the subcutaneous administration of naloxone HCl (10 mg/kg). Increasing atmospheric CO2 concentration stimulated EA-induced respiratory depression and the peptide produced a parallel shift to the right of both the respiratory rate -- PaCO2 and tidal volume -- PaCO2 curves. Following chronic administration of EA tolerance to the respiratory depression was observed and chronic exposure to morphine produced partial tolerance to the respiratory depression induced by EA. It was concluded that EA desensitizes the central chemoreceptors to CO2 and the action of EA on respiration parallels that of morphine.