Inactivation of low-Km rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro. A catalase-mediated reaction.

The inactivation of the affinity chromatography purified low-Km rat liver mitochondrial aldehyde dehydrogenase (ALDH)--free of catalase activity--by the alcohol sensitizing agent cyanamide was studied in vitro. This ALDH-purified preparation was not susceptible to cyanamide inactivation at concentrations up to 2.5 mM. On the other hand, ALDH activity appears to be irreversibly inhibited when the incubation mixture contained ALDH, catalase, NAD+ and cyanamide. Influence of catalase, NAD+ and cyanamide concentrations in the incubation mixtures on the ALDH activity were also established. The time course of the concentration of cyanamide in an incubation mixture when ALDH activity was inhibited by cyanamide in the presence of catalase and NAD+, was evaluated by HPLC. No disappearance of cyanamide was observed for a period of time up to 24 hr. This result suggests that no metabolic conversion of cyanamide to an active inhibitory form takes place, as has been suggested recently.

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry.

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC-MS-MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range -5.9 to 8.5%, expressed as the RSD and the relative error, respectively.

Simultaneous determination of paracetamol and chlorpheniramine in human plasma by liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of paracetamol and chlorpheniramine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists in the extraction of paracetamol and chlorpheniramine with diethyl ether, followed by the determination of both drugs by an LC-MS-MS method, using 2-acetamidophenol as internal standard. The intra-assay and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 0.5 microg/ml of plasma for paracetamol and 0.2 ng/ml for chlorpheniramine. The concentration working range was established between 0.5 microg/ml and 25 microg/ml for paracetamol and between 0.2 ng/ml and 50 ng/ml for chlorpheniramine. This method has been used for analyzing more than 1200 human plasma samples from a clinical study with 24 volunteers.

Pharmacokinetics of cyanamide in dog and rat.

A pharmacokinetic study of cyanamide, an inhibitor of aldehyde dehydrogenase (E.C. 1.2.1.3) has been made in the beagle dog and Sprague-Dawley rat. Cyanamide plasma levels were determined by a sensitive high performance liquid chromatographic assay, specific for cyanamide. In the dog, i.v. administration of cyanamide at 1, 2 and 4 mg kg-1, produced a dose-dependent pharmacokinetic behaviour. Statistically significant changes were observed in plasma clearance values (12.6 to 19.7 mL kg-1 min-1), half life values (39 to 61 min) and mean residence times (50 to 79 min). Peak plasma concentrations, after oral administration of 4 mg kg-1 were achieved at 30 min and oral bioavailability was about 65%. In the rat after i.v. or oral administration, cyanamide (2 mg kg-1) had a half life of 30 min, a total plasma clearance of 117 mL kg-1 min-1 and a mean residence time of 26 min. Oral bioavailability was about 69%.

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.

A pharmacokinetic-pharmacodynamic linking model for the alpha 2-adrenergic antagonism of idazoxan on clonidine-induced mydriasis in the rat.

The relationship between concentration and inhibitory effect of the alpha 2-adrenoceptor antagonist idazoxan on clonidine-induced mydriasis has been studied in the rat using pharmacokinetic-pharmacodynamic simultaneous modelling. Fifteen minutes after the anaesthesia of rats with sodium pentobarbitone (55 mg kg-1, i.p.), and 5 min after the administration of clonidine (0.3 mg kg-1, i.v.) to rats pretreated with idazoxan (3 mg kg-1, i.v., and 3 and 10 mg kg-1, orally) at different time intervals, pupil diameters were assessed. The pharmacokinetics of idazoxan were adequately described by a monoexponential equation. Using a pharmacokinetic-pharmaco-dynamic linking model, the concentration-effect relationships of idazoxan were derived, and were quantified by the inhibitory simple Emax model. At the effect compartment, the estimated apparent IC50 was 153.6 ng mL-1. Values of clearance, volume of distribution and elimination half-life were 71.2 mL kg-1 min-1, 3134 mL kg-1 and 30.5 min, respectively. These results could contribute to better characterization of the pharmacodynamic and toxicological profiles of idazoxan in experimental models in which a different pharmacokinetic behaviour of the drug is presumed.

Comparative binding of lactate dehydrogenase to mitochondrial fractions.

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.

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.

Lack of correlation between pharmacokinetic and pharmacodynamic behaviour of cyanamide in man. A preliminary report.

A pharmacokinetic and dynamic study of cyanamide, an inhibitor of aldehyde dehydrogenase (ALDH) used as an adjuvant in the aversive therapy of chronic alcoholism, has been carried out in man after oral administrations. Cyanamide plasma levels were determined by a sensitive and specific high performance liquid chromatographic assay. Blood ALDH activity were estimated after oral administration of 0.3, 1 and 1.5 mg/kg of cyanamide. One i.v. administration of 1 mg/kg was performed in order to determine the absolute bioavailability and the main pharmacokinetic parameters. Elimination half life and total plasma clearance values were 51.7 8.8 min and 14.4 2.7 mL/kg/min respectively. After oral administrations of 0.3, 1 and 1.5 mg/kg a rapid absorption rate was estimated with a Tmax values range of 10.5 to 15.5 min. The extent of absorption was not complete, oral bioavailability being 53%, 70% and 81% respectively. The presence of a first pass-effect is suggested. The inhibitory activity of cyanamide on blood ALDH reached the maximum value 4 h after its administration and decreased progressively throughout six days period. The cyanamide plasma levels time course did not correlated with the pharmacodynamic time course responses.

Inactivation mechanism of low-KM rat liver mitochondrial aldehyde dehydrogenase by cyanamide in vitro.

The inactivation of low-KM rat liver mitochondrial aldehyde dehydrogenase (ALDH) by the alcohol-sensitizing agent cyanamide (H2NCN) has been studied in vitro. The effect of the concentrations of NAD+ at different concentrations of catalase on the inactivation of ALDH by cyanamide (20 and 200 microM) in vitro point to an ALDH-NAD(+)-catalase complex prior to the binding to cyanamide to form the holoenzyme-inhibitor complex. Cyanamide itself could be responsible for the inactivation of ALDH. The possibility that both irreversibly inactivated ALDH and cyanamide remain free at the end of the inactivation process is discussed. The effects of pH and ionic strength on the inactivation process are also described. The pseudo-first order rate constants for inactivation of low-KM ALDH depends on both effects, suggesting that electrostatic forces are involved in the process and that a group with pK approximately 6.8, presumably a histidine residue, at the active site of ALDH could be involved. A representative equation for the inactivation process of low-KM ALDH by cyanamide in vitro has been fitted to experimental kinetic data, involving both catalase and inhibitor concentrations.

Modulation of lactate dehydrogenase activity by enzyme-protein interaction.

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.

Determination of cicletanine enantiomers in plasma by high-performance capillary electrophoresis.

A sensitive and selective high-performance capillary electrophoresis procedure was developed for the determination of S(+) and R(-) enantiomers of cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using gamma-cyclodextrins in the run buffers and ultraviolet detection. The method was linear from 10 to 500 ng/ml and the limit of detection was 10 ng/ml for each enantiomer in plasma samples. The within-run precision of the method, expressed as relative standard deviation, was 10.4 and 9.6% at 25 ng/ml for S(+) and R(-) cicletanine, and 4.2 and 4.6% at 500 ng/ml, respectively. This method has been used to follow the time course of the concentrations of the cicletanine enantiomers in human plasma after a single therapeutic dose of cicletanine given by mouth.

Determination of cyanamide in plasma by high-performance liquid chromatography.

A sensitive, selective and rapid high-performance liquid chromatographic procedure was developed for the determination of cyanamide in plasma. The procedure involved extraction with ethyl acetate, derivatization with 5-(dimethylamino)naphthalene-1-sulphonyl chloride and posterior analysis by high-performance liquid chromatography on a mu Bondapak C18 column with fluorimetric detection. Linearity ranged from 5 to 500 ng/ml and the lower limit of sensitivity of the assay was 4 ng/ml of cyanamide in plasma. The precision of the method was 3.0-8.9%, expressed as relative standard deviation over the linear range. This method has been used to elucidate the time course of the cyanamide concentration in the plasma of humans, following oral administration of cyanamide at therapeutic doses.

Pharmacokinetic study of cicletanine in healthy volunteers.

A pharmacokinetic study of cicletanine, a new class of antihypertensive drugs was performed in ten healthy volunteers after administration of 50 mg single oral dose. Cicletanine plasma and urine concentrations were measured using the HPLC technique. The main pharmacokinetic parameters were estimated applying a non-compartmental approach. The half-life ranged between 4.76 to 17.76 h; the mean oral and renal clearance were 7.3 +/- 2.5 l/h and 0.026 +/- 0.012 l/h, respectively. The urinary excretion of the product ranged between 37.5 and 64.6% of the administered dose, the amount of unchanged drug being negligible (0.4 +/- 0.3%). Both metabolic conjugation pathways glucuronidation and sulphation occurred in a similar extent 23.7 +/- 6.7% and 23.0 +/- 7% respectively. The overall pharmacokinetic parameters obtained in this study agree with those previously reported after the administration of higher doses (75-300 mg) and is in favour of a linear pharmacokinetic behaviour of cicletanine over the dose range of 50 mg to 300 mg.

Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.

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).

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.

Lack of hepatotoxicity after long-term administration of cyanamide in rats: a histological and biochemical study.

An experimental long-term hepatotoxicity study of cyanamide in Sprague-Dawley and Wistar rats has been carried out. After six months oral administration of cyanamide (2.7 and 25 mg/kg/day) no significant histological changes have been observed in the liver. Similarly, a one year intraperitoneal administration of 8 and 16 mg/kg/day has not induced any hepatic change. Specifically, no inclusion bodies in any cyanamide treated rat have been detected. Moreover, hepatic biochemical parameters have shown no significant impairment of hepatic function at doses used in human therapy (0.5-1 mg/kg).

Pharmacokinetics and oral bioavailability of carbimide in man.

A pharmacokinetic study of carbimide, an inhibitor of aldehyde dehydrogenase, used as an adjuvant in the aversive therapy of chronic alcoholism, has been carried out in male human volunteers for intravenous and oral administration. Carbimide plasma concentrations were determined by a sensitive and specific high performance liquid chromatographic method. The intravenous doses administered were 0.1, 0.3, 0.6, and 1 mg kg-1 and linear pharmacokinetics were observed for this dose range. Elimination half-life and total plasma clearance values ranged from 42 to 52 min and from 14.4 to 20.5 ml kg-1 min-1, respectively. After oral administration of 1 and 1.5 mg kg-1 of carbimide, elimination half-life values were 75 and 61 min, respectively, being higher than the corresponding value obtained after 0.3 mg kg-1 doses, i.e. 39 min. In all cases, rapid absorption was indicated by tmax values ranging from 10.5 to 15.5 min. Absorption was not complete, the oral bioavailability being 53 per cent and 70 per cent for the 0.3 and 1 mg kg-1 carbimide dose, respectively. The data indicate that there is a first-pass effect for carbimide.