Simultaneous determination of ITF 296 and two metabolites in plasma by a validated HPLC method.

A simple and sensitive HPLC validated method was developed for the simultaneous determination of ITF 296 (a new orally active nitrate) and its metabolites ITF 1124 and ITF 1577 in biological samples. Quantitation was performed by peak height ratios between the three compounds and the internal standard (ITF 1721). Detection limit of the method was 0.005 microgram/ml using 1 ml of plasma. The results indicated that the method is reproducible, accurate, precise, sensitive and specific for the measurement of the three compounds in plasma samples. Those characteristics allowed us to conclude that the method is suitable for analysing samples obtained after administration of ITF 296 to humans at therapeutic dosage and to animals for formulation studies.

Pharmacokinetic profile of ITF 296 in rats and dogs.

The pharmacokinetic profile of ITF 296 was studied after a single oral or i.v. dose administered to rats and dogs. ITF 296 and its first metabolite, ITF 1124, were measured in plasma and urine by a validated HPLC method with UV detection. Similar results were obtained in both animal species. After i.v. administration, the initial decline in plasma levels (distribution phase) was rapid, followed by a slower elimination phase. After oral administration, ITF 296 was rapidly and almost completely absorbed from the gastrointestinal tract, the maximal plasma levels being reached between 10 min and 1 h after administration in both species. Only traces of the parent compound were found in the urine of the two species, suggesting that ITF 296 was extensively metabolized. Preliminary data indicate that renal excretion is the major route of elimination of the biotransformed products. The denitrated metabolite was present in plasma from the earliest sampling time after either the i.v. or the oral route. After oral administration the drug was not significantly metabolized during passage through the liver, suggesting good bioavailability. The development of ITF 296 as a new orally active nitrate is supported by its pharmacokinetic profile in rats and dogs.

Cyclosporine pharmacokinetics in rats and interspecies comparison in dogs, rabbits, rats, and humans.

The pharmacokinetics of cyclosporine, a potent immunosuppressive agent, are described for rats given 5 mg/kg iv, measuring blood concentrations by a specific HPLC method. Cyclosporine followed first-order kinetics and blood concentrations vs. time data were adequately described by a three-compartment open model system. The mean half-life of the lambda 1 phase was 0.11 hr, that of the lambda 2 phase was 1.82 hr, and the half-life of the estimated lambda 3 phase was 23.79 hr. The mean apparent volume of distribution and Vss were 5.69 and 4.54 liters/kg, respectively. The mean blood total body clearance was 3.38 ml/min/kg. The literature was reviewed to obtain comparable data from other mammalian species, and satisfactory kinetic information was found for humans, dogs, and rabbits. Interspecies variants in physiological parameters and cyclosporine pharmacokinetics were considered and treated as a property and consequence of body size (allometry), nullifying anatomical and physiological differences between species. A relationship between pharmacokinetic time (a variable in terms of chronological time) and body weight was found. It is suggested that the metabolic capacity (based on liver weight) to eliminate cyclosporine is similar in humans, rabbits, and rats, whereas the dog showed a potentially double capacity to metabolize the drug. However, metabolic and pharmacodynamic studies are also needed to predict toxicity accurately among species.

The effect of chronic oral ketoconazole on in vivo drug metabolism in the rat.

To verify the potential in vivo effects of ketoconazole, an antifungal agent with a wide spectrum of activity, on liver function, caffeine and phenytoin were chosen as probes for hepatic oxidative drug metabolism. Chronic treatment with ketoconazole (80 mg/kg oral) in the rat did not affect both pharmacokinetic profile of probes and a few biochemical parameters related to liver functions. These results indicate that further specific hepatic drug-metabolizing monoxygenase activities responsible to ketoconazole are to be identified to explain ketoconazole-drug interactions reported in vivo.

Caffeine disposition after oral administration to pregnant rats.

Caffeine disposition was studied over 24 h in rats on the 12th day of pregnancy given 80 mg/kg of drug as a single oral dose or in four divided doses every three hours. Peak blood levels of caffeine were reached at three hours after the single dose, and at 10 h (at half the previous value) after the first of the divided doses. At the end of the experiment both caffeine and its dimethylxanthine metabolites were higher in blood, amniotic fluid and fetuses after divided doses than after the single dose. Urinary excretion over 24 h was the same for the two groups. The overall conclusions underline that caffeine per se and not its metabolites are responsible for the teratogenic effects.

Caffeine distribution in acute toxic response among inbred mice.

The median lethal dose (LD50) and the median lethal concentration (LC50) of caffeine administered intravenously (i.v.) and orally (p.o.) were calculated in adult male CD2F1/Crl BR mice. Acute toxic behavioral responses to the drug were observed after administration via the two routes. Deaths followed severe tetanic convulsions: some animals had transient convulsions and survived, and others presented no acute toxicity. In a further study, the LD50 of caffeine was given i.v. and by gavage to animals randomly paired. At the death of one of the two mice the other was killed and caffeine assayed in blood and tissues of both. Different patterns of distribution were observed inter- and intra-route of administration, animals which died always having higher drug levels, although they had received the same dose. These findings suggest that besides the well-known factors affecting acute toxic response to exogenous compounds, drug distribution also has to be taken into account in a multifactorial toxicological investigation.

Pharmacokinetics of paraxanthine, one of the primary metabolites of caffeine, in the rat.

The pharmacokinetic of paraxanthine, one of the primary metabolites of caffeine, is described for the first time. Groups of adult male rats were given different doses of paraxanthine as iv bolus injections. Blood cell/plasma partition and the binding of the compound to rat plasma proteins (determined by equilibrium dialysis) were investigated. The fraction bound (15%) remained constant in the concentration range of 1-100 micrograms/ml. Partition was also constant over a wide range of doses. Up to the 10 mg/kg dose, the paraxanthine followed first order kinetics and blood concentrations vs. time data were described by a one-compartment, open model system. The mean half-life and elimination rate constant were 1 hr and 0.70 hr-1, respectively. The average apparent volume of distribution was 1.50 liters/kg and total clearance was 0.90 liter/hr/kg. After larger doses (15 and 30 mg/kg), kinetics were nonlinear. The area under the blood concentration-time curve increased, but not in proportion to the dose, and modifications of pharmacokinetic parameters were shown. These findings indicate that in the rat paraxanthine is eliminated by a saturable process with an apparent Km of about 31 micrograms/ml and an apparent Vmax of about 0.40 micrograms/ml/min. Close estimates were obtained by two different methods of calculation. Our results suggest that the pharmacokinetic profile of paraxanthine could be important to understand the kinetics and the potential toxic effects of its parent compound, caffeine, in animals and man.

Prostaglandin endoperoxide synthetase and the activation of benzo(a)pyrene to reactive metabolites in vivo in guinea pigs.

The role of prostaglandin endoperoxide synthetase in the in vivo activation of benzo(a)pyrene to reactive metabolites capable of interacting irreversibly with cellular macromolecules was studied in guinea pig liver, lung, kidney, spleen, small intestine, colon, and brain. DNA and protein covalent binding experiments were made after systemic administration of acetylsalicylic acid (200 mg/kg) followed by radiolabeled benzo(a)pyrene (4 microgram/kg). Results are compared with a control situation in which the prostaglandin endoperoxide synthetase inhibitor (acetylsalicylic acid) was not administered. No decrease in the level of DNA or protein benzo(a)pyrene-derived covalent binding was observed in any of the tissues studied.

Kinetics and metabolism of theobromine in male and female non-pregnant and pregnant rabbits.

The kinetics and metabolism of theobromine (3,7- DMX ) were investigated in male rabbits after a single oral dose and 14 days oral dosing at 1, 5, 10, 50 and 100 mg/kg/day. Female non-pregnant and pregnant rabbits were also studied after single oral doses of 1, 5 and 50 mg/kg. No significant difference was found in the pharmacokinetic profile of 3,7- DMX due to either sex, pregnancy or after chronic oral administration for 14 days. Intravenous (i.v.) administration of 3,7- DMX at 1 and 5 mg/kg resulted in calculated kinetic parameters in close agreement with oral doses. Irrespective of sex, there was a reduction in the absorption rate constant as the dose increased, coupled with a linear dose-related increase in AUC values. No qualitative difference in the metabolism of 3,7- DMX in the rabbit was observed as linked to sex, pregnancy or treatment schedule. Twenty-five percent of the administered dose of 3,7- DMX was excreted unchanged, the major metabolite being 7-methylxanthine (40%). There appeared to be a shift in the metabolic pathway at 100 mg/kg/day in the males and at 50 mg/kg/day in the females with more unchanged 3,7- DMX excreted. Only at these highest doses (100 mg/kg for males and 50 mg/kg for pregnant rabbits) was there a tendency toward accumulation.