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An antiserum was prepared for the determination of glibenclamide and for the estimation of other commercially available sulfonylureas. Rabbits were immunized with a glibenclamide-BSA conjugate. Tritiated glibenclamide was used as the tracer. The assay was performed in the presence of 8-anilinonaphthalenesulfonic acid to displace glibenclamide bound to serum protein, and free and antibody bound tracer were separated by dextran-coated charcoal. For glibenclamide determination in serum and plasma the limit of detection was 3 ng/ml. Sensitivity calculated for the whole determination range was 102 cpm for a 10 % concentration difference. Specificity studies showed a cross-reaction of less than 0.1 % for glibenclamide metabolite M1 and 9 % for metabolite M2. Other sulfonylurea drugs display cross-reactivities from 0.1% (chlorpropamide) to 190% (gliquidone). Both intra-assay and inter-assay imprecision were below 10 %. Accuracy was established by comparison of the present method with HPLC. The assay was applied to the specific determination of glibenclamide in clinical trials and for diagnosing factitious hypoglycemia caused by sulfonylureas.

To improve understanding of the relationships between gastric inhibitory polypeptide (GIP) and insulin secretion and food intake in obesity, immunoreactive insulin and immunoreactive GIP were measured in 5 obese children during PO glucose tolerance test carried out before and after diet. Before diet, mean insulin levels were normal at fasting and rose after glucose ingestion. The mean fasting immunoreactive GIP level was very high (1235 +- 209 pg/ml) compared to that of 8 healthy adult controls (411 +/- 44 pg/ml) and remained at this level throughout the test. There was only a short postabsorptive rise to 1515 +/- 158 pg/ml at 30 min, which was not significantly different either from the patients' basal values or from the 30-min control values (1356 +/- 67 pg/ml). After dieting for 3 to 7 months, immunoreactive insulin responses returned to normal ranges. Concomitantly, both basal and total GIP release diminished significantly (basal GIP, 343 +/- 92 pg/ml; area under the GIP curve, 3820 and 1694 pg/ml/hr before and after diet, respectively). The postabsorptive GIP increment, however, rose significantly from 180 pg/ml/hr, before diet, to 665 pg/ml/hr afterwards. These results might be compatible with the hypothesis that in obesity, hyperinsulinemia, and overactivity of the GIP cells are associated phenomena caused by overeating and reversed by reduced food intake. However, several contradictory findings remain unexplained. The discrepancy between insignificant postabsorptive GIP increments and elevated insulin responses before diet casts doubts on the causal relationship between GIP and insulin secretion. The small GIP rise might be due to a limited secretory capacity of the GIP cells or to a diminished stimulatory capacity of glucose. The constantly high level of GIP might reflect chronic hypersecretion and/or some defect in basal regulation and feedback control of GIP release. The change caused by dietary measures in the GIP secretion pattern provides evidence that in obese children, basal GIP secretion in influenced by nutritional factors.

Piretanide 60 mg was administered intravenously over 30 min to 15 men with different degrees of renal failure. The mean piretanide serum concentration at the end of the infusion period was 5.72 +/- 1.51 micrograms/ml. Serum piretanide concentration-time curves declined biexponentially and 24 hours after medication the serum level had fallen to less than twice the detection limit. The terminal half-life ranged from 1.63 to 3.44 h. A relationship to creatinine clearance was not demonstrable. The mean metabolic clearance of piretanide was 107.7 +/- 47.6 ml/min/1.73 m2 body surface area and was the same as that reported for healthy subjects. The renal clearance of piretanide ranged from 3.33 to 43.9 ml/min/1.73 m2 body surface area and very closely correlated with the creatinine clearance (p less than 0.01). Its renal clearance depended principally on active secretion of the drug into the tubule, and glomerular filtration appeared unimportant. There was a close relationship between the amount of piretanide excreted in the urine and the creatinine clearance. Because the diuretic effect of piretanide depends on the concentration of the drug in the tubule, the observed correlation might be of use in evaluating the appropriate dosage of piretanide in patients with renal failure. The present data suggest that single daily doses of piretanide will not result in accumulation, even when high doses are administered to patients with advanced renal failure.

1 In uremia, an increased frequency of adverse drug reactions is observed at elevated plasma levels of active drug. This is a consequence of decreased renal elimination of unchanged drug or pharmacologically active metabolites. 2 To study the pharmacokinetics of nomifensine in uremia, a single dose of nomifensine maleate 50 mg was given to 10 patients with a glomerular filtration rate between 0 and 61 ml/min. In three additional patients on maintenance haemodialysis, the influence of dialysis on the elimination of nomifensine after single oral doses of 50 mg was studied. 3 Blood samples were obtained before and at regular half-hourly or hourly intervals after administration. Plasma levels of nomifensine were examined using a radioimmunological determination method and pharmacokinetic parameters were calculated by computer program. 4 Whereas in healthy subjects the half-life of nomifensine was found to be 1.8h, this parameter was found to be prolonged in renal patients. In a patient with a creatinine clearance of 0 ml/min the elimination half-life was 46 hours. Nomifensine is not eliminated by haemodialysis. 5 Our findings suggest that nomifensine should not be administered to renal patients with a glomerular filtration rate below 25 ml/minute.

The present studies on duct ligated dogs and rabbits are an attempt to demonstrate functional alterations in glucose metabolism: 1. In rabbits the insulin reserve in the pancreatic gland was reduced to about one fifth of its normal value. 2. In dogs, as well as in rabbits, the values of fasting blood glucose remained normal for a period of 12 months following the operation. In rabbits the intragastric, but in dogs both the intragastric and the intravenous glucose tolerance were impaired. 3. In duct ligated dogs, the increase in serum IRI was diminished after intra-gastric and i.v. glucose load, as well as after injection of glucagon. Attempts are being made to adapt the model for special studies concerning the early state of diabetes.

The aim of this study was to investigate the influence of glibenclamide on insulin release via insulinotropic gut factors and via a direct action on the pancreas. Maximum peripheral IRI levels appeared 1 minute after intragastric administration of the minimal effective dose of glibenclamide to rats. The corresponding drug levels were high (600 ng/g) in the duodenal mucosa, but low (2 ng/ml) in the peripheral serum. These concentrations were too low to cause insulin release by direct action on the pancreas. Intragastric glibenclamide increased the amount of “duodenal insulin releasing activity” (DIRA) in the mucosa immediately after drug administration. When glibenclamide was dissolved in plasma at a high concentration (1 μg/ml) and then injected into the coeliac trunk of an in-situ rat pancreas preparation, no additional effect on portal IRI levels was measured as compared with injection of serum alone. In contrast significant IRI release was noticed when glibenclamide was dissolved in serum (1 μg/ml) of rats pretreated with the drug intragastrically. The plasma of pretreated rats without addition of glibenclamide was ineffective. The results suggest that glibenclamide may have two effects, one releasing insulin at the pancreas directly, and the other inducing the release of a gastrointestinal factor which amplifies the first effect. Neither glibenclamide nor the factor alone can provoke an IRI release under physiological conditions. The possible importance of these findings for the regulation of insulin secretion is discussed.

A sustained release capsule formulation of piretanide (6mg) pellets was compared with a standard 6 mg tablet formulation and placebo on a double blind, double dummy basis in 33 healthy volunteers. Pharmacokinetic as well as pharmacodynamic parameters were determined. The relative bioavailability of piretanide from the pellets as compared to the tablets was 81% based on AUC and 87% based on urinary recovery comparison of unchanged drug.The sustained release formulation cuts down the peak diuresis observed with the tablets in the first 2-hour period by about 50%. The duration of the diuretic effect is more than doubled in comparison to the standard tablet without loss of overall pharmacodynamic activity (diuresis and saluresis). The potassium excretion with both formulations was in the order of magnitude of placebo.

This paper describes a specific radioimmunological method for determining the diuretic agent piretanide (4 phenoxy-3-(1-pyr-rolidinyl)-5-sulfamoyl benzoic acid) in human serum, plasma and urine. The antiserum was raised in rabbits against an immunogen of piretanide coupled to bovine serum albumin. The iodinated hydroxy derivative of piretanide was used as tracer. The separation of free and antibody-bound piretanide was performed by precipitating the antibody-tracer complex by polyethylene glycol. The limit of detection was 4 ng/ml. Studies on specificity showed less than 0.5 % cross-reactivity of an identified metabolite. Intraassay reproducibility showed an average coefficient of variation of 7.3%, the interassay variation was 5 %. A recovery experiment yielded 100.9 % recovery. There is good agreement between parallel determinations of piretanide by RIA and HPLC in both human serum and urine.

Penecillium camemberti var. candidum wurde auf einem synthetischen Nahrboden gezuchtet und der14CO2-Einbau in das Mycel, speziell in die Aminosauren, innerhalb der „logarithmic phase” in Abhangigkeit von der Zeit verfolgt. Asparaginsaure, Glutaminsaure, Threonin, Isoleucin, Arginin, Glycin und Prolin waren stark markiert und wurden zur Ermittlung der Aktivitatsverteilung auf die einzelnen C-Atome abgebaut. Die Ergebnisse zeigen, das14CO2 durchβ-Carboxylierung der Brenztraubensaure und im Gange der Synthese von Citrulllin aus Ornithin aufgenommen wird. Die nach dem erstgenannten Mechanismus fixierte Aktivitat geht schnell in Asparaginsaure und uber die Glieder des Citronensaurecyclus in Glutaminsaure uber. Asparaginsaure ist Ausgangsverbindung fur die Synthese von Threonin und Isoleucin. Glutaminsaure geht in Prolin uber und ist weiterhin am Aufbau des Arginins beteiligt.

The role of CO2 and the influence of pH on the insulin-stimulated glucose metabolism of cell suspensions from rat adipose tissue were investigated in a Trisbuffered system. CO2 was assimilated in presence of glucose, fructose or pyruvate in the medium. The fixed CO2 is mainly localized in the C-1 position of lactate. Insulin enhances the turnover rates only when cells are incubated in glucose-containing medium. The influence of the extracellular CO2 concentration on CO2 fixation, on glucose oxidation, and on the transfer of glucose-6-14C to lipid compounds follows saturation kinetics. The same metabolic reactions show a defined maximum at an extracellular pH of 7.5. On both sides of the pH-peak the turnover rates decrease very rapidly.

Aus Zellsuspensionen von Pankreasgewebe jugendlicher Ratten und Meerschweinchen sowie menschlicher Feten zuchteten wir Monolayer Cultures groser fibroblastoider Zellen. Diese farben sich mit Aldehydfuchsin ebenso wie B-Zellen der Langerhansschen Inseln an; mit Pseudoisocyanin geben sie eine positive Reaktion auf Insulin. Mit Hilfe einer immunologischen Methode konnten wir sowohl in der gesammelten Nahrlosung als auch in den Zellen nach Ausbildung eines Zellrasens „Insulin“ (=IRI=immuno reactive insulin) feststellen.

We describe in the present paper the results of the influence of normal and low-molecular-weight heparin on the interaction of human fibrinogen and thrombocytes in human whole blood cotting ex vivo. During the coagulation process sequential measurements of fibrinopeptide A reflect fibrin formation and determination of platelet factor 4 indicate activation of thrombocytes. The data show that low-molecular-weight heparin inhibits plasma thrombin generation in vivo for longer than normal heparin and it affects the fibrinogen platelet binding less. There is good evidence that a lonely factor Xa inhibition mediates this anticoagulant mechanism. Therefore, these data favor the hypothesis that antifactor Xa activity prevents indeed blood clotting.

Experiments were performed in order to study a possible participation of gastrointestinal factors in the insulinotrophic action of glibenclamide in man. Six healthy volunteers received 5 mg glibenclamide in 50 ml saline orally. Biopsies were taken from the duodenal mucosa before and after administration of the drug. The duodenal insulin-releasing activity (DIRA) was assayed in the extracts of the biopsy material by using an in situ pancreas preparation of rat. The corresponding drug, IRI and glucose levels were measured in peripheral blood. The values of IRI correlated with both the prior elevation of DIRA and the increasing levels of the drug in the blood. These data indicate that glibenclamide might stimulate the release of gut factor(s) which, in turn, could possibly sensitize the pancrease response to the drug.

Radioimmuno assays for routine analyses of hormones were developed in clinical laboratories in the early sixties. The importance of this procedure has increased permanently. The method bases upon the reaction of an antigen with an antibody to an antigen-antibody complex. A labelled antigen is used to quantify the formation of the complex. To demonstrate the development of a drug radioimmuno assay nomifensine was used, that has a complex pattern of metabolism. High sensitivity, high specificity, low interference in samples of patients with multiple therapy, simple handling of a great number of samples per person, good interlaboratory reproducibility, and low costs are the advantages of this procedure justifying the relatively high expense for introducing a control area for isotopes.

Twelve healthy male volunteers participated in an open cross-over study designed to test whether two fixed-dose combination formulations of penbutolol (40 mg) and furosemide (20 mg), an ordinary and a film-coated tablet, are bio-equivalent. Penbutolol capsules (40 mg) and 20 mg furosemide tablets (Lasix; Hoechst) served as reference formulations. The maximum concentration of furosemide was significantly depressed in the case of fixed-dose combinations, reflecting a possible pharmacokinetic interaction between penbutolol and furosemide. However, the area under the concentration versus time curve for furosemide and its cumulative urinary excretion and diuretic effect were not influenced by penbutolol. On the basis of plasma concentration versus time data and urinary parameters, the two fixed-dose formulations are bio-equivalent.

1. Among the numerous possibilities of drug interactions, pharmacokinetic interactions may cause mutual changes in absorption, distribution, metabolism and elimination of either drug. In the present study this approach was used to investigate pertinent effects of nomifensine and clobazam. 2. Ten normal subjects participated in an intra-individual comparison of nomifensin 75 mg alone and in combination with clobazam 15 and 30 mg. The study design was carried out according to a Latin square in double-blind conditions. One-week wash-out periods were used between the trial days. Serum levels of nomifensine were measured by radioimmunoassay (RIA) and of original clobazam by gas chromatography (GC). Classical criteria for bioavailability (peak serum levels, time of peak, area under the serum level time curve) and the half-life of elimination from the serum were used for retrieval of pharmacokinetic information. 3. Results showed no relevant differences in the criteria mentioned above, after administration of each drug alone or in combination. Therefore, extrapolations were made to multiple dose kinetics based on assumptions derived from practical therapy. They showed comprehensive agreement with therapeutic results in depressed patients. 4. The use of the classical criteria for bioavailability, in addition to the calculation of the half-time of elimination from serum, provides sufficient information for the decision whether pharmacokinetic drug interaction is present or absent. There was no such interaction after single doses of nomifensine or clobazam.

1 The absorption, excretion, distribution and metabolism of a new antidepressant agent, nomifensine, was examined in rats, dogs and monkeys. After oral administration of 14C-nomifensine 1 mg/kg body weight, the compound was absorbed rapidly, and as shown in rats and dogs, virtually completely. Nomifensine was predominantly found in a conjugated form in the plasma of rats, dogs and monkeys. Nomifensine was bound to serum proteins (approximately 60%) and the degree of binding was not species specific. 2 The maximum levels of total radioactivity (unchanged drug and metabolites) in the blood were 0.50 ± 0.14 μg equiv./ml (n = 5) in dogs and 0.34 ± 0.17 μg equiv./ml (n = 6) in monkeys. These leves were markedly higher than those in rats, with values of 0.034 ± 0.014 μg equiv./ml at 0.5-0.75 h and 0.048 ± 0.018 μg equiv./ml at 2-8 h after dosing (second peak; n = 19). 3 The ratio of radioactivity in organs and tissues relative to plasma was small and similar to that in blood. The highest concentrations of radioactivity were measured in the excretory organs; the lowest concentrations were found in the brain. When multiple doses of 14C-nomifensine were administered accumulation was minimal. 4 After single oral doses, the plasma levels of radioactivity were higher than those of blood. Accumulation of unconjugated nomifensine in plasma could not be demonstrated in dog, and multiple doses of the drug had no influence on the elimination of either conjugated or unconjugated nomifensine. 5 Rats excreted about 50% and dogs excreted about 70% of the compound in urine, predominantly as conjugates. The metabolites found in man were also found in monkey, these being 4'-hydroxy nomifensine, 4'-hydroxy-3'-methoxy nomifensine and 3'-hydroxy-4'-methoxy nomifensine.

Investigations in vitro on the time-dependent increase in thrombin activityand platelet function have been used tocharacterize the kinetics of the clotting process in nonanticoagulated blood. The test procedures described involve great effort and expense and therefore are not suitable for routine tests in pharmacology and clinical pharmacology. The present contribution describes the determination of clotting times in ATP secretion in the Chrono-Log Whole Blood Aggregometer.Blood was taken from healthy donors who had not used any drug in the two weeks before the trial. 0.5 ml blood wereimmediately transfered into siliconizedglass cuvettes containing 0.4 ml salineand 0.1 ml luciferin-luciferase cocktail prewarmed to 37°C. Impedance and luminescence were recorded continuously. Clotting at the electrodes is indicated by an immediate steep rise in both impedance and luminescence. Clotting time is defined as the time from diluting the blood in the cuvettes until the point at which marked elevation of these variables begins.In the blood of twelve subjects the mean clotting time was 3.8 min and intersubject variation (SD) was 0.45 min. Drastic interindividual differences in response to collagen and ADP in citratedwhole blood were observed in the study group.In vitro addition of 20 μl Fibraccel(Behringwerke AG, Marburg, FRG),a platelet factor 3 containing plateletextract decreased clotting time by 35 %(n=10). In the presence of 0.2 U heparin a slow and lona-lasting increase in impedance was seen. 1 g oral AspirinR didnot influence clotting time measured ex vivo.The results indicate that whole blood aggregometry is a simple, fast, and precise method of determining blood clotting and the effects of drugs in a medium reflecting almost physiological conditions.

Metabolic and pharmacokinetic studies of nomifensine maleate, a tetrahydroisoquinoline derivative with antidepressant properties, are reviewed. Results of pharmacokinetic studies indicate that nomifensine has a short distribution phase and a large volume of distribution. It is rapidly metabolized to its N-glucuronide. Plasma levels of nomifensine-N-glucuronide are up to 100-fold higher than those of nomifensine, obviously because of a smaller volume of distribution. As nomifensine-N-glucuronide is extremely unstable and cleaved to nomifensine, determinations of nomifensine are easily falsified. It is therefore recommended only to determine the sum of nomifensine and its N-glucuronide (total nomifensine) in clinical trials. Kinetics of total nomifensine can best be described by the open two-compartment model: Maximum plasma levels are obtained 1-2 hours postadministration; mean elimination half-life is 2 hours. Excretion is almost entirely by the kidneys, with approximately 88% of an oral dose excreted within 24 hours.

1 A radioimmunoassay (RIA) has been developed for determination of both nomifensine and total nomifensine (nomifensine + conjugated nomifensine) in serum, plasma, and urine. 2 Antibodies were prepared in rabbits by immunization with N-(8-Nomifensine) succinamic acid-bovine serum albumin. 3H-labelled drug was used as tracer. Separation of free from antibody-bound nomifensine was carried out using dextran-coated charcoal. For determination of total nomifensine, the acid-labile conjugate was split by acidification. 3 The limit of detection for nomifensine is 300 pg/ml plasma and the cross-reactivity of the metabolites is less than 1%. The influence of conjugated nomifensine on the results of nomifensine can be corrected. 4 Pharmacokinetics of nomifensine were determined in healthy volunteers after oral administration of 100 mg 14C-labelled drug. Peak levels of 14C radioactivity (2,150 ng/ml), total nomifensine (1,252 ng/ml) and nomifensine (53 ng/ml) appeared within 1.5-2 h; the half-life of elimination from plasma was 1.5-2 hours. The advantages of this routine method are high sensitivity, the requirement of small amounts of plasma, and simple handling.