Nitrosation of tryptophan residue(s) in serum albumin and model dipeptides. Biochemical characterization and bioactivity.

Nitrosation of bovine serum albumin with acidified NaNO2 was compared to that of carboxymethyl-bovine serum albumin in which the thiol group is covalently blocked. Differential ultraviolet-visible (UV-Vis) spectroscopy and a modified Saville assay indicated that a non-cysteine residue(s) in carboxymethyl-bovine serum albumin was nitrosated. The nitrosated carboxymethyl-bovine serum albumin exhibited similar vasorelaxation activity as that observed with nitrosated bovine serum albumin. Identification of the nitrosated non-cysteine residue(s) was studied using 16 model dipeptides, each of which contained a glycyl residue and a variable residue. Using photolysis-chemiluminescence analysis, modified Saville assay, differential UV-Vis spectroscopy, and bioassays, L-glycyl-L-tryptophan (Gly-Trp) was found to be the only dipeptide that underwent significant nitrosation under these conditions. Liquid chromatography-UV-Vis spectroscopy-mass spectrometry showed that the NO group was attached to the indole nitrogen of tryptophan. Nitrosated Gly-Trp exhibited dose-dependent vasorelaxation and platelet inhibiting activity with apparent EC50 values of 1.1 +/- 0. 3 and 3.5 +/- 0.9 microM, respectively. Because N-nitroso-Gly-Trp does not release NO radical via spontaneous homolytic N-NO bond fission nor freely diffuse through cellular membranes, the ability of this compound to induce NO.-like biological effects suggests the existence of a (membrane-associated) transnitrosation system that facilitates delivery of -NO to its specific biologic target(s).

Peroxynitrite-induced apoptosis in HL-60 cells.

Peroxynitrite (ONOO-), an anion and a potent oxidant, generated by the interaction of nitric oxide (NO) and superoxide is able to induce apoptosis in HL-60 human leukemia cells in a time- and concentration-dependent manner. Characteristic morphology of apoptosis can be observed 3 h after HL-60 cells are exposed to 10 microM ONOO-. Treatment of HL-60 cells with increasing concentrations of ONOO- from 1 to 100 microM confirms the concentration dependence of apoptosis as evidenced by: 1) degradation of nuclear DNA of these cells into integer multiples of approximately 200 base pairs; 2) colorimetric DNA fragmentation assay; and 3) evidence of condensation of chromatin and nuclear fragmentation shown by propidium iodide staining. Under the same conditions, peroxynitrite causes apoptosis in another transformed cell line, U-937 cells, but is ineffective at inducing apoptosis in normal endothelial cells derived from human umbilical cord and normal human peripheral blood mononuclear cells. This direct evidence of peroxynitrite inducing apoptosis implicated a new function of this potent oxidant.

Multiple dose pharmacokinetic study of cicletanine in healthy volunteers.

Cicletanine hydrochloride, a furopyridine derivative, is a new type of antihypertensive drug. A pharmacokinetic study was performed in 8 non-patient subjects who were given 50 mg oral daily doses for 7 days. Cicletanine plasma levels were measured by HPLC. An open bicompartmental model was fitted to the experimental data using an extended non-linear regression method. Additionally plasma levels obtained after the first administration were used to determine pharmacokinetic parameters, which were considered as representative of a single dose administration. The results showed no significant differences between parameters estimated after the first dose and repeated dosing. Mean half-life values were 7.3 and 7.9 hours respectively. The mean peak and trough concentration values in the last interval studied were 1730 and 44 ng/mL respectively. The accumulation index was negligible (1.15). The similarity in the values obtained after the first and repeated administration suggests that cicletanine displays a linear pharmacokinetic behaviour at a dose of 50 mg.

Oral idazoxan bioavailability in rat. Relevance of intestinal and hepatic first-pass effect.

The alpha 2-antagonist idazoxan (2-(2-(1,4-benzodioxanyl)-2-imidazoline) was administered iv, hepatoportally, and orally to Sprague-Dawley rats at 1, 3, and 10 mg/kg. Idazoxan plasma levels were determined by a HPLC method. A noncompartmental treatment of data was used to estimate the main pharmacokinetic parameters. After iv administration, idazoxan exhibited a linear kinetic profile. Half-life and mean residence time values ranged from 24.4 to 27.9 and from 34.2 to 40.5 min, respectively. Total plasma clearance values and volume of distribution at steady state values ranged from 0.057 to 0.078 (liters/kg)/min and 1.95 to 3.18 liters/kg, respectively. After the oral administration of idazoxan, time to peak values ranged from 5 to 10 min. When the oral 10 mg/kg dose was compared with both 1 and 3 mg/kg doses, significant statistical differences were observed in AUC levels and in dose-normalized peak concentration values (p less than 0.05, t test). Bioavailability values obtained after the oral administration of idazoxan ranged from 12.6 to 31.5%. The bioavailability range observed after the hepatoportal administration exceeded largely and significantly the range denoted after the oral route and displayed a saturable character already noted at the 3 mg/kg dose (p less than 0.01, t test).

Pharmacokinetic profile of idazoxan in the beagle dog.

The alpha 2-antagonist idazoxan (2- (2- (1,4-benzodioxanyl))-2-imidazoline) has been given intravenously and orally to five beagle dogs at 1, 3 and 10 mg kg-1 doses. Idazoxan plasma levels were determined by a HPLC method. After intravenous administration, a linear kinetic behaviour was obtained. Half-life and mean residence time values ranged 105.2-117.1 and 138.1-154.0 min, respectively. Total plasma clearance values and volume of distribution at steady state values ranged from 25.6-32.1 (mL kg-1) min-1 and 3.60-4.36 L kg-1, respectively. After oral administration, time to peak values averaged around 1 h. Dose normalized peak concentration values ranged 161-182 ng mL-1. Bioavailability values ranged 60-88%. Low idazoxan bioavailability has been described in other animal species and attributed to a first-pass effect.

Theophylline pharmacokinetics following single and repeated administration of slow-release capsules.

The aim of this study was to evaluate the pharmacokinetic profile after single and multidose oral administration of a new slow-release theophylline formulation and the bioavailability at steady-state during two dosing intervals (5th and 8th day) in 6 healthy subjects. A dose of 6 mg/Kg (capsules) was given for the single and at fixed 12 h intervals during 10 days for the multidose schedule. Theophylline kinetics were best described by a one-compartment open model. After single dose the elimination half-life was 7.22 +/- 2.36 h, the Vd area/F was 0.50 +/- 0.07 l/kg and the total clearance/F was 0.86 +/- 0.24 ml/Kg/min, which was similar to results reported in other studies. Steady-state plasma levels were predictable from the kinetic data and were reached between the 4th and 6th dose, falling within the therapeutic range throughout the dosing interval. The percentage of fluctuation remained constant during both intervals, around 33 and 35% respectively. Bioavailability parameter values for the two intervals showed no differences either in extent or in rate. A circadian rythm was confirmed with the mean morning trough values significantly greater than the corresponding mean evening values. From these results it may be concluded that the formulation studied produces few fluctuations of theophylline levels during the 12 h interval between both administrations, thus permitting a good therapeutic cover in chronic therapy.

Pharmacokinetics and bioavailability of methocarbamol in rats.

In a pharmacokinetic study, 15, 30, 60, and 150 mg kg-1 intravenous and oral doses of methocarbamol were administered to rats. Differences observed in plasma clearance values, i.e. 0.0203, 0.0156, 0.0123, and 0.0085 1 kg-1 min-1 for 15, 30, 60, and 150 mg kg-1, respectively, suggested a dose-dependent pharmacokinetic behaviour of the drug. Elimination according to a biocompartmental open model and Michaelis-Menten kinetics fits the plasma level data. Estimated Km and Vmax values were 38.49 +/- 3.71 mg l-1 and 1.24 +/- 0.06 mg l-1 min-1, respectively. After oral administration of 15, 30, and 60 mg kg-1 the peak plasma levels were reached earlier. The tmax values were 6, 6, and 10 min, respectively. After 150 mg kg-1 oral doses, peak plasma levels were reached later (tmax = 150 min). Estimated bioavailability ranged between 77 and 112 per cent.