High performance liquid chromatography coupled with radioactivity detection: a powerful tool for determining drug metabolite profiles in biological fluids.

High performance liquid chromatography coupled with continuous radioactivity detection represents an advancement in drug metabolism research. Using radioactive substances labelled in biologically stable positions, all metabolites can be specifically detected by radioactivity measurement. Thus no clean-up of biological fluids is required prior to HPLC. This can prevent artefact formation from unstable metabolites, reduces recovery problems and facilitates quantitation. Separation of highly polar and unpolar metabolites is possible in a single chromatographic run using gradient elution and reversed phase materials. This technique is also well-suited for preparative isolation and purification of metabolites for subsequent structure elucidation. Various metabolite profiles of drugs labelled with carbon-14 or tritium are shown. Metabolites of the following drugs are presented: norfenefrine, etozolin, thymoxamine, naloxone, and levobunolol. We review the general methodology and report our experience with this technique. In principle, this technique may be useful for all biological systems in which tracer techniques are applied.

Metabolism of thymoxamine. III. Structure elucidation of the metabolites and interspecies comparison.

The structures of six metabolites were elucidated using rat urine after intragastric administration of 14C-thymoxamine by means of enzyme incubations, mass spectrometry and synthesis of metabolites: desacetylthymoxamine, N-demethyl-desacetylthymoxamine, the corresponding sulfates and glucuronides. The nature of the conjugates was confirmed by biosynthesis, i.e., co-administration of unlabelled thymoxamine and 35S-sulfate or 14C-glucose. The system high performance liquid chromatography-radioactivity detection was used for interspecies comparison. All biotransformation pathways are seen in rat and man. In dog and cat demethylation is a very minor reaction. Glucuronidation is not observed in the cat.

GLC method for the quantification of metabolites of thymoxamine in human plasma.

A sensitive gas-chromatographic method for quantification of the pharmacologically active metabolites I-IV of thymoxamine in plasma is described. 4-(Hydroxythymyl)-(2'methylbutylaminoethyl)ether, a compound similar to metabolite I, is used as an internal standard. Metabolites I and II the internal standard are extracted with cyclohexane from alkalinized plasma followed by back-extraction into 0.1 N hydrochloric acid. After evaporating the hydrochloric acid solution, the sample is silylated with BSTFA and analyzed by gas-chromatography on a CRS 101/Carbowax 4000 column using a thermoionic detector. For subsequent determination of metabolites III and IV, the extracted plasma is hydrolyzed under conditions in which the phenol sulfates but not the glucuronide conjugates undergo cleavage. The resulting phenols (metabolite I and II) are analyzed as described above. The sensitivity threshold for all 4 compounds is approximately 5 ng/ml plasma based on a 2 ml plasma sample.

Metabolism of thymoxamine. I. Studies with 14C-thymoxamine in rats.

Thymoxamine is rapidly and completely absorbed in rats. It is a prodrug which does not enter the systemic circulation in its unchanged form. After either oral or intravenous administration it undergoes rapid and intense metabolism involving four biotransformation reactions: Enzymatic hydrolysis to the corresponding phenol (metabolite I), Monodemethylation to metabolite II, Sulfate conjugation of I and II (metabolites III and IV) and Conjugation of I and II with glucuronic acid (metabolites V and VI). With these 6 metabolites identified approximately 95% of the radioactivity can be accounted for in plasma, urine and bile. Whereas the systemic availability of I and II is low, III and IV show high bioavailability. Metabolites I to IV are pharmacologically active, while III and IV are less potent than I and II. The radioactivity distribution in tissues is different after oral and intravenous administration consistent with the higher portion of unconjugated metabolites in the body after administration by parenteral route. Although 60% of the labelled compounds is eliminated via bile, the radioactive compounds are almost completely excreted in the urine after both routes of administration. This demonstrates complete reabsorption of the biliary metabolites. Secondary peaks of radioactivity in plasma and organs at 4 hours are explained by the participation of the metabolites in the enterohepatic circulation.

Pharmacokinetics and metabolism of gabapentin in rat, dog and man.

This paper describes the pharmacokinetic studies of 1-(aminomethyl)-cyclohexane acetic acid (gabapentin, Gö 3450, CI-945) conducted with the 14C-labelled substance following intravenous and intragastric administration to rats and dogs and oral administration to humans. Gabapentin is well absorbed in rats, dogs and in humans, with maximum blood levels, reached within 1-3 h after peroral administration. Following i.v. administration to rats, similar blood and brain levels of gabapentin are observed after a short distribution phase, whereby concentrations in cerebrum and cerebellum are comparable. The highest concentrations are found in the pancreas and kidneys and the lowest values in adipose tissue. No binding of gabapentin to human plasma proteins or human serum albumin is observed. The distribution coefficient (octanol/buffer pH 7.4) is 7.5 X 10(-2). In man, no biotransformation of gabapentin is observed. In rats, biotransformation is only minor. In dogs, however, a remarkable formation of N-methyl-gabapentin is found. Elimination half-lives range between 2-3 h in rats, 3-4 h in dogs, and 5-6 h in man. Gabapentin is nearly exclusively eliminated via the kidneys. Renal elimination was up to 99.8% in rats and approx. 80% in man following oral administration. The blood level-time course after i.v. administration to rats can well be described by a three-compartment open model. Experiments in rats and dogs demonstrate that pharmacokinetics are not sex-dependent and are not changed after multiple dosage. Pharmacokinetics are shown to be linear in the range tested of 4 to 500 mg/kg i.v. in rats.

Ralitoline: a reevaluation of anticonvulsant profile and determination of "active" plasma concentrations in comparison with prototype antiepileptic drugs in mice.

Ralitoline (RLT) is a new thiazolidinone derivative with potent anticonvulsant activity in different seizure models. During Phase I studies, RLT was well tolerated in human volunteers and showed linear pharmacokinetics in the dose range tested (up to 150 mg). Since RLT will soon be entering clinical Phase II studies, we were interested in obtaining predictive data for effective plasma concentrations in patients. For this purpose, the anticonvulsant potency of RLT was determined in four seizure models in mice, and plasma levels were measured at time of peak drug effect. The four models were the threshold for maximal (tonic extension) electroshock seizures (MES), the threshold for clonic seizures determined by i.v. infusion of pentylenetetrazol (PTZ), the traditional MES test with supramaximal (50 mA) stimulation, and generalized clonic seizures induced by s.c. administration of PTZ. Furthermore, median minimal "neurotoxic" doses (TD50s) were determined by the rotorod and chimney test for calculation of protective indices. All data obtained for RLT were compared with data obtained with standard antiepileptic drugs: phenobarbital, phenytoin, valproate, and diazepam. The onset of anticonvulsant action after i.p. injection of RLT was very rapid, and the peak drug effect was already obtained after 2 min. In the MES models, RLT was the most potent compound. "Active" plasma levels ranged from approximately 300 ng/ml in the MES threshold test to approximately 1,300 ng/ml in the MES test. RLT was also capable of increasing the PTZ threshold, whereas, possibly because of its short duration of action in mice, it was not very active in the s.c. PTZ seizure test.(ABSTRACT TRUNCATED AT 250 WORDS)

[Metabolism of Etozolin in rat, dog and man]. / Metabolismus von Etozolin bei Ratte, Hund und Mensch.

Investigations on the metabolism of the new diuretic ethyl (Z)-(3-methyl-4-oxo-5-piperidino-thiazolidin-2-ylidene) acetate (Gö 687, etozolin, Elkapin) were carried out with urine of rat, dog and man as well as rat bile after enteral administration of the 14C-labelled substance. Seven metabolites were isolated with either the aid of high-pressure liquid chromatography (HPLC) or extraction and thin-layer chromatography. Mass spectroscopy was applied to determine the structures of the metabolites, partly by use of authentic reference substances. Because of the instability of most of the metabolites, some of them showing strong polarity, the described investigations on the metabolic profiles and the enrichment and purification of some metabolites could only be carried out with the HPLC-radioactivity detector system, which requires no clean-up for the samples. The metabolisation process of etozolin is qualitatively equal in rat, dog and man; it is characterized by 3 steps: 1. enzymatic cleavage of the ester group, which leads to the also diuretically active main metabolite (metabolite I) in the plasma of all 3 species; 2. glucuronidation of the resulting metabolite I, leading to metabolites II and III, which are diastereoisomeric esters of the two enantiomeric forms of metabolite I with beta-D-glucuronic acid. 50--60% of the urinary radioactivity can be described with these two metabolisation steps in all 3 species; 3. Oxidation of the piperidine moiety to metabolites IV--VII.

Disposition of gabapentin (neurontin) in mice, rats, dogs, and monkeys.

Gabapentin, an analog of gamma-aminobutyric acid, exhibits anticonvulsant properties in both animal models and humans. Gabapentin pharmacokinetics was studied in laboratory animals using HPLC and radiometry. Oral bioavailability was 40% in monkeys administered 25 mg/kg, 79% in mice and rats receiving 50 mg/kg, and 80% in dogs administered 50 mg/kg. Binding to plasma proteins was < 3%. Maximum blood or plasma concentrations generally occurred within 2 hr of an oral dose. In rats and monkeys, increases in maximum plasma concentrations and/or areas under the curve were less than dose-proportional following oral administration, most likely because of saturable absorption. However, intravenous pharmacokinetics in rats were linear over the dosage range of 4-500 mg/kg. Mean intravenous elimination half-life was 1.7 hr in rats, 2.9 hr (14C only) in dogs, and 3.0 hr in monkeys. In rats and dogs, repeated administration did not alter gabapentin or 14C pharmacokinetics. Additionally, gabapentin did not induce hepatic cytochrome P450 monooxygenases in rats. There were no age- (rats only) or gender-associated changes in pharmacokinetic parameters. [14C]Gabapentin was extensively distributed to tissues. In the dog, gabapentin was metabolized to N-methylgabapentin (approximately 34% of dose); whereas metabolism in mouse, rat, and monkey was minimal (< 5%). The principal route of excretion was via urine. In summary, as an antiepileptic drug, gabapentin exhibited desirable pharmacokinetic properties, such as linear elimination kinetics, not highly bound to plasma proteins, not extensively metabolized, and not an inducer of hepatic cytochrome P450.