Studies comparing in vivo:in vitro metabolism of three pharmaceutical compounds in rat, dog, monkey, and human using cryopreserved hepatocytes, microsomes, and collagen gel immobilized hepatocyte cultures.

The in vivo metabolism of three pharmaceutical compounds, EMD68843, EMD96785, and EMD128130, was compared in fresh and cryopreserved hepatocyte (CPH) suspensions and microsomes from rat, dog, monkey, and human livers and fresh human and rat hepatocyte collagen gel immobilized cultures (GICs). Half of the major in vivo metabolites was produced by phase 1 (hydroxylation, oxidation, hydrolysis, N-dealkylation) and half by phase 2 metabolism (mostly glucuronidation but also sulfation and glycine conjugation). The identity and percentage of phase 1 and 2 metabolites from each compound produced in hepatocytes compared well with that in each species in vivo. Glucuronidation was more extensive in GICs than in CPHs. In contrast, CPHs but not GICs, produced sulfate metabolites. Microsomes (supplemented with NADPH only) produced most of the phase 1 but no phase 2 metabolites. Metabolism in CPHs was the same as in fresh hepatocyte suspensions. Discrete species differences in metabolism were detected by CPHs and microsomes. Cytochrome P450 and glucuronosyl S-transferase contents of CPHs did not account for species differences in the percentage of phase 1 and 2 metabolites or the rate of disappearance of the parent compounds in these cells. These data show a good correlation between major metabolites formed in vivo and in vitro. CPHs and GICs, unlike microsomes, carried out sequential phase 1 and 2 metabolism. Each in vitro system has its own advantages, however, for short-term metabolism studies CPHs may be more useful since they are readily available, easier and quicker to prepare than GICs, and have more comprehensive enzyme systems than microsomes.

Pharmacokinetics and first clinical experiences with an antihypertensive dopamine (DA2) agonist.

The pharmacokinetic properties and first clinical experiences with the antihypertensive dopamine (DA2) agonist, carmoxirole, are summarized. In man carmoxirole was rapidly absorbed. On oral administration the maximum plasma concentration was reached after 2-3 h. The drug was metabolized, mainly to an ester-type glucuronide, and was excreted (unchanged carmoxirole plus glucuronide) largely by the kidneys. The plasma half-life of the parent compound was 5.5 h. For the dose range tested (0.5 to 1.5 mg) the pharmacokinetics were linear. The drug was rapidly distributed in animals but only very small amounts penetrated the blood-brain barrier. Carmoxirole did not affect supine blood pressure in healthy subjects, but under the conditions of the Schellong's test some orthostatic reactions occurred with high doses. In patients the blood pressure was reduced for at least 8 h after single oral doses. On repeated administration for several weeks a relevant antihypertensive effect was still measurable 12 and 24 h after dose. The most frequently reported adverse events have been headache, dizziness, tiredness, nausea, and gastric disorders. These symptoms are considered to be mainly due to blood pressure reduction, as is frequently observed at the beginning of antihypertensive therapy. In patients the incidence of orthostatic reactions is appreciably lower than in healthy subjects, and in both change of position was sufficient to relieve the symptoms.

The melanin binding of bisoprolol and its toxicological relevance.

Unexpectedly high accumulations of bisoprolol were detected in iris and ciliary body and in retina+choroid of beagles after 4 weeks of conjunctival and oral administration. This phenomenon gave reason to assume that these high concentrations in the pigmented structures of the eye might be related to melanin binding. According to literature a series of drugs, e.g. chloroquine, rifampicine, chlorpromazine, benzodiazepines, and also beta-adrenoceptor antagonists exhibit melanin-binding properties. By means of autoradiography it could be demonstrated in pigmented mice that after iv and po administration 14C-labelled bisoprolol was selectively bound to the melanin-containing parts of the eye, irrespective of the mode of administration. Since the melanin-bound radioactivity could be extracted from the eye of mice and was eliminated with a t1/2 of approx. 7 days, the melanin binding of bisoprolol is considered to be reversible. Other beta-blocking agents like timolol and befunolol used already for a long time in the therapy of glaucoma have been reported to bind specifically to the melanin of the eye, and show comparable long half-lives, similar to bisoprolol. Usually, autoradiographic studies on drug distribution are performed with albino animals. This leads to a lack of information on melanin binding and may result in misinterpretation concerning the retention of substances, especially in pigmented compartments of the eye. Therefore, in autoradiographic studies of new investigational drugs during preclinical development, one should use both pigmented and albino animals.

Bisoprolol--comparative toxicokinetic study after oral and conjunctival administration in beagles.

Beagles were treated with bisoprolol, a beta 1-selective adrenoceptor antagonist, for 30 days with the following daily doses: oral: 30 mg/kg; conjunctival: 0.5% solution (approx. 0.04 mg/kg) and 5% solution (approx. 0.4 mg/kg). Drug concentrations were determined in plasma and various eye tissues on days 1, 16, and 30, and on day 59, i.e. on day 29 of the follow-up period. Bisoprolol concentrations in plasma and most eye tissues were considerably higher after oral than after conjunctival treatment. The highest tissue concentrations were observed in the iris (+ciliary body) and retina (+choroid) with tissue/plasma concentration ratios between 100 and 150 after oral and 1000 to 3000 after conjunctival instillation (5% solution). In plasma no accumulation of the drug was observed which is in accordance with its plasma half-life of 4 to 5 h. In contrast to this, concentrations in the iris and retina increased from day 1 to day 16 and 30 by 3 to 8 times and the half-life of bisoprolol in these tissues was estimated to be between 3 to 5 days.

Basic pharmacokinetics of bisoprolol, a new highly beta 1-selective adrenoceptor antagonist.

The basic pharmacokinetics of bisoprolol were investigated in three independent studies involving 23 healthy volunteers. After administering 20 mg of 14C-bisoprolol orally, mean elimination half-lives of 11 hours for the unchanged drug and 12 hours for total radioactivity were observed. The enteral absorption of bisoprolol was nearly complete. Fifty percent of the dose was eliminated renally as unchanged bisoprolol and the other 50% metabolically, with subsequent renal excretion of the metabolites. Less than 2% of the dose was recovered from the feces. Intraindividual comparison of the pharmacokinetic data measured after oral and intravenous administration of 10 mg bisoprolol to 12 subjects yielded an absolute bioavailability of 90%. Total and renal clearance were calculated as 15.6 L/hr and 9.6 L/hr, respectively. The volume of distribution was 226 L. Concomitant food intake did not influence the bioavailability of bisoprolol.

Determination of the new beta-blocker bisoprolol and of metoprolol, atenolol and propranolol in plasma and urine by high-performance liquid chromatography.

High-performance liquid chromatographic methods using fluorimetric detection have been developed for the determination in plasma and urine of bisoprolol, atenolol, metoprolol and propranolol. Bisoprolol, metoprolol and propranolol were extracted at alkaline pH with dichloromethane, atenolol with n-butanol-dichloromethane (25:75). After evaporation of the organic solvent the compounds were chromatographed on silica gel Si-60 columns (normal phase) using aqueous ammonium phosphate buffer (pH 4) containing 3-7% acetonitrile as eluent (method 1). Alternatively, the compounds were acetylated prior to chromatography on reversed-phase columns (RP-8), using acetonitrile-water mixtures as eluents (method 2). The detection limit was 1-2 ng/ml in plasma and 10 ng/ml in urine for bisoprolol and metoprolol with either method. For atenolol the detection limit was 5 ng/ml in plasma or 50 ng/ml in urine (method 1), for propranolol 1 ng/ml in plasma (method 2).

Pharmacodynamic profile of bisoprolol, a new beta 1-selective adrenoceptor antagonist.

The pharmacodynamic profile of bisoprolol, a new beta 1-selective adrenoceptor antagonist, was investigated in four independent studies including 36 healthy male volunteers. Using the model of exercise-induced tachycardia (ET) the beta-adrenoceptor blocking properties of bisoprolol (2.5-40 mg) were examined in comparison to metoprolol (50 and 100 mg), propranolol (40 and 80 mg) and atenolol (50 and 100 mg). The maximal reduction of ET was achieved between 1 and 4 h following single oral administration. The dose-response relationship using individual maximal reduction of ET showed, on a molar basis, that bisoprolol is about 5, 7 and 10 times more effective than propranolol, atenolol and metoprolol, respectively. In the model of insulin-induced hypoglycaemia bisoprolol behaved as a beta 1-selective adrenoceptor antagonist. There was a good correlation (r = 0.94) between the log bisoprolol concentration and the reduction in exercise-induced tachycardia. Bisoprolol is a potent new cardioselective beta-adrenoceptor antagonist with a competitive action at beta 1-adrenoceptors.

Pharmacokinetics and metabolism of bisoprolol-14C in three animal species and in humans.

The pharmacokinetic properties of bisoprolol-14C were studied in Wistar rats, beagle dogs, and Cynomolgus monkeys. Bisoprolol is well absorbed in these species; independent of the route of administration (i.v. or p.o.), 70-90% of the 14C-dose was recovered in urine. Faecal excretion was approximately 20% in rats and less than 10% in dogs and monkeys. Rats excreted approximately 10% of the dose in bile after i.v. as well as after oral administration. The plasma half-life of the unchanged drug was approximately 1 h in rats, 3 h in monkeys, and 5 h in dogs. The bioavailability was 40-50% in monkeys, approximately 80% in dogs, and 10% in rats. Studies in rats have shown that the drug is rapidly taken up by the tissues. After i.v. administration, high levels of radioactivity were found in lung, kidneys, liver, adrenals, spleen, pancreas, and salivary glands. After oral administration, the highest concentration occurred in the liver and kidneys. With the exception of plasma and liver, unchanged bisoprolol was the major radioactive constituent in all tissues studied. Both the blood-brain and placental barriers were penetrated, but only to a small degree. No accumulation of radioactivity in tissues was observed after repeated dosing (1 mg/kg/day). The metabolism of bisoprolol was studied in the same three animal species and in humans. The major metabolites are the products of O-dealkylation and subsequent oxidation to the corresponding carboxylic acids. The amount of bisoprolol excreted unchanged in the urine is 50-60% of the dose in humans, 30-40% in dogs, and approximately 10% in rats and monkeys.

Concentration kinetics of propranolol, bisoprolol, and atenolol in humans assessed with chemical detection and a subtype-selective beta-adrenoceptor assay.

After oral administration of single doses of 240 mg of (+/-)propranolol (prop), 200 mg of (+/-)-atenolol (aten), and 100 mg of (+/-)-bisoprolol (biso) to six healthy male volunteers, the plasma concentration time profile was investigated. To measure total plasma concentrations of the parent racemic mixture of drug administered, a HPLC-assay of drug concentrations was used. To detect active metabolites and stereoselective pharmacokinetics of the racemates, plasma concentrations were also monitored by means of a subtype-selective receptor assay, using a beta 1-adrenoceptor preparation from rat salivary glands. It is shown that relevant amounts of active metabolites do not become apparent for either of the three drugs investigated. Furthermore, for neither of them can significant stereoselective elimination characteristics be seen. Monophasic elimination characteristics with t1/2 of 4.8 +/- 0.42 (prop), 6.87 +/- 0.46 (aten), and 9.19 +/- 0.38 h (biso) become apparent. The maximum concentrations observed after administration of the doses mentioned previously were 220 +/- 71 (prop), 904 +/- 104 (aten), and 445 +/- 32 (biso) (ng/ml plasma). One can conclude from comparison with the results from receptor-binding studies that the 100 mg dose of biso is five- to seven-fold more potent than the 200 mg dose of aten, with respect to antagonism versus beta 1-adrenoceptor-mediated effects.

Sucralfate: pharmacokinetics, metabolism and selective binding to experimental gastric and duodenal ulcers in animals.

Pharmacokinetics, metabolism, and binding capability of a basic aluminium salt of sucrose octasulphate (sucralfate, Ulcogant) to ulcer lesions were investigated in Wistar rat, Beagle dog, Rhesus, and Cynomolgus monkey with 14C-labelled material. After i.v. injection in rats the 14C-radioactivity has a very short serum half-life of 1 h and is excreted almost completely by the kidneys. After oral administration only a very small portion of the dose is detectable in serum which is eliminated rapidly from the intravascular space. From the renal excretion data it is concluded that sucralfate is absorbed from the gastrointestinal tract of rat, Beagle dog, and Rhesus monkey only from 1% to 5% of the dose. The radioactive constituents in plasma, urine, and feces of rat and Cynomolgus monkey were analysed by HPLC. Both after i.v. and p.o. administration only unchanged substance could be detected. A possible binding of 14C-sucralfate to the ulcerated mucosa of stomach and duodenum was investigated in rats by histoautoradiography. The ulcers were induced by acetic acid. A selective accumulation of 14C-sucralfate in the area of the stomach ulcer could be demonstrated as long as 8 h after single p.o. administration. In the duodenum the autoradiographs showed a specific binding of 14C-sucralfate to the ulcerated mucosa at 1, 2, and 4 h after administration. Neither pretreatment with an H2-receptor antagonist nor simultaneous administration of an antacid influenced the binding of 14C-sucralfate. These results suggest that the mode of action of sucralfate is the formation of a protective layer which stops harmful agents like gastric juice and bile from further damaging the ulcerous lesions, thus ensuring an uninterrupted healing process. The very small extent of absorption and the rapid excretion from the organism minimize the risk of a systemic burden.

[Quantitative determination of drugs by means of gaschromatography. Further development of detectors (author's transl)]. / Quantitative Bestimmung von Arzneimitteln mit Hilfe der Gaschromatographie. Weiterentwicklung der Detektoren.

Gaschromatography is an excellent and well established method for the separation of volatile compounds. Up to now the flame ionization detector was used almost exclusively for the quantitative determination of organic compounds separated by gaschromatography. This detector is an unspecific detector and responds to all organic compounds containing at least one C-H bond. Due to that this detector can be used for the determination of almost any drug, however, the chromatographic column must have a very high resolution, especially when small amounts of drugs are to be determined in the presence of large amounts of interfering compounds. As a consequence "substrate specific" detectors were developed, among them the electron capture detector, the nitrogen-phosphor-sensitive detector and the mass spectrometer are the most important ones. The function of these detectors and their advantages for the quantitative determination of drugs are discussed in this paper.

[Metabolism of pramiverine (author's transl)]. / Metabolismus von Pramiverin

The metabolite patterns of 4,4-diphenyl-N-isopropyl-cyclohexylamine-hydrochloride (pramiverine, Sistalgin) in urine, feces, bile and serum of Wistar rats, beagle dogs, rhesus monkey and man were analyzed with radio thin-layer chromatographic techniques. The structures of six pramiverine metabolites were elucidated. Pramiverine is eliminated unchanged in only minute amounts via the renal, the biliary and the fecal route. Consequently an almost quantitative absorption and metabolism takes place. Metabolite patterns in serum and urine differ considerably indicating that some of the metabolites are excreted preferentially by the kidney while others are reabsorbed to various degrees. The identified metabolites are products of dealkylation, deamination and hydroxylation reactions.

Metabolism of dihydroaminopterin and the influence of aminopterin and related derivatives on the metabolism of (-)-5-formyl-tetrahydrofolic acid in Pediococcus cerevisiae.

[3H]Dihydroaminopterin, but not [3H]methotrexate or [3H]aminopterin, was rapidly taken up by resting cell of Pediococcus cerevisiae and inhibited the uptake of (-)-5-formyl-tetrahydrofolic acid to more than 90% at a concentration of 25 muM. On the other hand 5-formyl-tetrahydrofolic acid inhibited the uptake of dihydroaminopterin to a similar extent. Dihydroaminopterin was metabolized by Pediococcus cerevisiae, but not by Lactobacillus casei, to a compound tentatively identified as dihydroaminopterin diglutamate. Methotrexate or aminopterin at a concentration of 25 muM did not inhibit the uptake of 5-formyl-tetrahydrofolic acid in Pediococcus cervisiae, but inhibited the conversion of this compound to polyglutamate forms almost completely at a level of 2.5 mu M.