Biomedical subjects

K Ensing

Publications and source records attributed to K Ensing.

At least 55 records · Page 3Linked to original sources

PubMed1994

Application of radioreceptor assays for systematic toxicological analysis--2. Theoretical considerations and evaluation.

In this paper the applicability of radioreceptor assays for systematic toxicological analysis will be evaluated on a theoretical basis as well as on the basis of the outcomes of the analysis of a large number of urine samples collected after administration of a selected number of drugs to healthy volunteers and patients. Many drugs and other substances of toxicological relevance exert their action through an interaction with one or more receptor (sub)types. Whether the number of persons are using particular drugs intentionally or unintentionally, radioreceptor assays can be a useful tool for systematic toxicological analysis in that they can be applied to the identification of entire pharmacological classes of substances as well as pharmacologically active metabolites. In part 1 of this paper detailed procedures for radioreceptor assays for benzodiazepines, anticholinergics and antihistaminics have been described in detail in order to illustrate not only the potentials but also the limitations of assay conditions. Fifteen drugs were administered to patients and volunteers and urine samples were collected and determined with the three radioreceptor assays. The results of this study underline the theoretical applicability of receptor assays in systematic toxicological analysis though sample pretreatment procedures may contribute to an improvement in sensitivity and applicability to other biofluids.

Benzodiazepines↗

PubMed1994

Methodological aspects of quantitative receptor assays.

Receptor assays occupy a particular position in the methods used in bioanalysis, as they do not exploit the physico-chemical properties of the analyte. These assays make use of the property of the analyte to bind to the specific binding site (receptor) and to competitively replace a labelled ligand from the same binding site. The amount of labelled ligand replaced is a measure of the amount as well as the affinity of the analyte. Thus, receptor assays offer additional information about the biological (pharmacological) activity of the analyte by distinguishing the compounds on the basis of their specific binding rather than specific molecular structure (chromatographic and non-chromatographic methods). This paper, starting with the general principles of receptor-ligand interaction, focuses on the application of ligand-binding techniques to the quantitative analysis. The factors which influence the sensitivity and the specificity of quantitative receptor assays, as well as the main directions in the improvement of the receptor preparation by using the solubilized and purified receptor are discussed. In order to enhance the use of these assays in routine practice, the development of solid-phase receptor assays is considered.

Animals↗

PubMed1993

Semi-automated solid-phase extraction procedure for drug screening in biological fluids using the ASPEC system in combination with Clean Screen DAU columns.

The use of a semi-automated solid-phase extraction system (ASPEC) for the screening of drugs in plasma and urine on a single mixed-mode column (Clean Screen DAU) is described. The processes of column preconditioning, sample application, column wash, pH adjustment and elution of the drugs were accomplished by the ASPEC. After off-line evaporation, the residues were injected into a wide-bore capillary gas chromatograph. The recoveries of the tested drugs were in the range of 73-96%, with relative standard deviations less than 5% at a concentration level of 2 micrograms/ml.

Animals↗

PubMed1993

Development and registration of chiral drugs.

In this review we describe the impact of chirality on drug development and registration in the United States, Japan and the European Community. Enantiomers may have differences in their pharmacological profiles, and, therefore, chiral drugs ask for special analytical and pharmacological attention during their development. However, the registration authorities have no clear policy towards the registration of chiral drugs. The absence of a clear policy regarding chirality causes a great deal of confusion and frustration at various levels and is not in the interest of industries developing newer and more beneficial drugs.

European Union↗

PubMed1993

Identification with liquid chromatography-ionspray mass spectrometry of the metabolites of the enantiomers N-methyl dextrorphan and N-methyl levorphanol after rat liver perfusion.

To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.

Animals↗

PubMed1993

Solid phase extraction of morphine from whole blood by means of Bond Elut Certify columns.

The use of Bond Elut Certify columns for the isolation of morphine from whole blood was evaluated. In order to monitor possible losses and the elution profile of morphine, a small amount of the tritiated analogue was added to the samples. Four sample pretreatment methods, three protein precipitation methods and one sonication/dilution method, were tested. The latter one gave the best results. The blood sample was applied onto the column at pH 3.3 after sonication and dilution with 0.1 M phosphate buffer (pH 3.3). The retention of morphine was affected by the pH of the samples, and the loss of morphine during sample application was minimized at low pH (3.3). The interferences were removed by washing the column with the phosphate buffer, 0.01 M acetic acid (pH 3.3), and methanol, sequentially. Ammoniated methanol, 2 mL at 2%, was selected to elute morphine. As a result, more than 80% of 3H-morphine was recovered for concentrations of morphine ranging from 5 to 4000 ng/mL.

Forensic Medicine↗

PubMed1992

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Barbiturates↗

PubMed1989

Application of a radioreceptor assay in a pharmacokinetic study of oxitropium bromide in healthy volunteers after single i.v., oral and inhalation doses.

Oxitropium bromide (OXBR) is a new anticholinergic drug, which is expected to be useful in the treatment of nocturnal asthma. The only pharmacokinetic data were obtained with the 14C-labelled compound. A sensitive radioreceptor assay for the determination of unlabelled OXBR in plasma was developed, based on competition between OXBR and 3H-N-methylscopolamine for binding to muscarinic receptors. OXBR was isolated from plasma by ion-pair extraction and re-extraction. Active metabolites present in significant amounts might interfere in the assay, but this was not the case for OXBR metabolites. Detection limits were 300 pg.ml-1 and 3 ng.ml-1 for plasma and urine, respectively. For the latter no extraction step was required. The single dose pharmacokinetics of OXBR was studied following inhalation (3 mg), oral (2 mg) and i.v. (1 mg) administration to 12 men, following an open, cross-over design. After i.v. administration the kinetic parameters were: Vc 38.41; t1/2 alpha 5.3 min; t1/2 beta 142 min; AUC 8.9 h.ng.ml-1; renal excretion 50.2%, k10 3.5 l.h-1 and total clearance 1874 ml/min. The apparent bioavailabilities were 0.48% and 12.4% by the oral and inhalation routes, respectively, based on the cumulative renal excretion. There were moderate adverse reactions due to the anticholinergic properties of the drug.

Administration, Inhalation↗

PubMed1989

Pharmacokinetics of ipratropium bromide after single dose inhalation and oral and intravenous administration.

Single doses of ipratropium bromide were administered intravenously, orally and by slow inhalation to ten healthy male volunteers. The plasma level after oral administration followed a low but broad plateau persisting for several hours. After i.v. administration the kinetic parameters were: Vc = 25.9 l, V alpha = 13.1 l, V beta = 3.38 l, t1/2 alpha = 3.85 min, t1/2 beta = 98.4 min, AUC = 15.0 h.ng/ml, kel = 11.8 l/h and total clearance is 2325 ml/min. The bioavailability was 3.3% (range 0.9-6.1%) on comparing the plasma AUCs following i.v. and 20 mg oral administration. The cumulative renal excretion (0-24 h) after i.v. administration was compared with that after oral administration and inhalation. Following oral administration, the apparent systemic availability was around 2%, and after inhalation it was 6.9%. In comparison with oral placebo administration, only after i.v. administration was there a significant change in heart rate (from 63.7 to 90.2 beats/min). The systolic blood pressure rose from 115.1 to 119.6 mm Hg and the diastolic blood pressure from 68.3 to 78.3 mm Hg.

Administration, Inhalation↗

PubMed1989

Comparison of intravenous and endobronchial atropine: a pharmacokinetic and -dynamic study in pigs.

In an experimental animal study using adolescent pigs we compared the pharmacokinetics and -dynamics of atropine following intravenous injection (0.25 mg, n = 6) or endobronchial instillation (2 mg, n = 6). Results showed that endobronchial atropine is rapidly absorbed by the pulmonary circulation, resulting in a peak plasma concentration of 48.8 +/- 25.9 ng ml-1 (mean +/- SD) after 2 min, compared to 46.3 +/- 16.7 ng ml-1 in the first min after intravenous injection. A first increase in heart rate could be observed within 1 min after both routes of drug administration. Significant changes in heart rate were found 9-30 min after endobronchial and 12-15 min after intravenous medication, with a maximum after 9 min (+57%) and 12 min (+24%), respectively. Mean bioavailability of atropine following the endobronchial route reached only 23% during the first 6 h when compared to intravenous administration. In light of this reduced bioavailability, we suggest an adult dosage in humans of atropine 2 mg diluted in 5-10 ml of saline administered endobronchially to attain a reliable increase in heart rate during a cardiac emergency when, in an intubated individual, no intravenous line is readily available.

Animals↗

PubMed1988

Development of a radioreceptor assay for the D2-selective dopamine agonist N-0437.

N-0437 is a recently developed dopamine (D2) agonist, theoretically attractive in the therapy of Parkinson's disease and glaucoma. Since its high potency allows small doses of the compound in clinical use and as extensive metabolism occurs in animals, a highly sensitive assay method was required for drug-monitoring purposes. To this end we developed a radioreceptor assay (RRA), a sensitive tool for the assessment of the sample's (dopaminergic) bioactivity. The RRA is based on competition between N-0437 and its tritium-labeled analogue for binding to dopamine receptors. The assay has been optimized for the preparation of the receptor suspension and the incubation conditions. Direct application of the assay for biological samples was impossible because of matrix interferences. Therefore, a solid-phase extraction method was developed in which the combination of a polar Si column and dichloromethane as eluent resulted in an effective elimination of the interferences. Recoveries were better than 90 and 95% for plasma and urine, respectively, even at concentrations at the determination limit of the method (300 pg/ml). Relative standard deviations were less than 15%. Because RRAs are stereoselective, the method discriminates between active and inactive species.

Animals↗

PubMed1988

Discrepancies in the specific activity of 3H-N-methylscopolamine as determined by mass spectrometry and radioreceptor assay due to racemisation.

In radioligand binding studies, the accuracy of the specific activity of the radiolabeled ligand has a large impact on the calculated receptor concentration and on the calculated affinities of both labeled and competitive ligands. A radioreceptor assay for anticholinergic drugs was employed to determine the specific activity of 3H-N-methylscopolamine in which the assumption was made that the labeling does not affect the interaction between the ligand and the muscarinic receptor. With this method a specific activity of 48 Ci/mmole was determined which was about 60% of the manufacturer's specification. However, the specific activity as determined by mass-spectrometry with field desorption showed 2.8 tritium-atoms per molecule NMS corresponding with an specific activity of 84.5 Ci/mmole. The only explanation for this discrepancy was a racemisation of the label, which could be proven by titration of the label with an increasing receptor concentration showing that 40% was not bound at all. This was attributed to the formation of the (+)-isomer.

Animals↗

PubMed1988

Development and application of a radioreceptor assay for scopolamine.

6 beta,7 beta-Epoxy-3a(1aH,5aH)-tropanyl-(S)-tropate (scopolamine) has proved to be a very effective drug in the prevention of motion sickness, however, the drug has a small therapeutic window, a low bioavailability and a short half-life. A transdermal drug delivery system (Scopoderm TTS) was developed to circumvent these problems as well as the variability in gastric absorption. In order to study the pharmacokinetics of the drug and its glucuronide, a highly sensitive radioreceptor assay with an absolute detection limit of 15 pg scopolamine was developed. In children undergoing minor surgery, a Scopoderm TTS patch of different sizes (according to the age of the children) was applied to the retro-auricular skin. Urine samples were collected and assayed for free and conjugated scopolamine. Furthermore possible anticholinergic effects on the pupil reaction, salivation, blood pressure and heart rate were monitored. The urine excretion of free and glucuronidated scopolamine showed large intra- and interindividual variations. However, in all age groups relatively high percentages of free scopolamine were found, namely 47.5% (3-6 years), 48.3% (7-12 years) and 36.0% (13-18 years) of the sum of free plus glucuronidated scopolamine. During the application of the patch, a prolonged plateau of scopolamine excretion could be found. Although in general the patches were well tolerated, both locally and systematically, moderate anticholinergic effects were observed in patients.

Adolescent↗

PubMed1987

Determination of atropine in plasma by a direct radioreceptor assay.

A highly sensitive radioreceptor assay for the anticholinergic atropine was developed and could be applied directly to plasma samples obtained from mini-pigs without any clean-up. Plasma samples were collected during 6 h after atropine was administered intravenously or endobronchially. The endobronchial plasma concentration-time curves were characterized by a very rapid rise of the concentration but with a subsequent much slower decrease than after intravenous administration. This indicates an initially high as well as prolonged uptake from the lungs.

Animals↗

PubMed1986

Centrifugation or filtration in quantitative radioreceptor assays.

In quantitative radioreceptor assays the amount of a drug present in the medium to be assayed is inversely related to the amount of receptor-bound radiolabelled ligand. Usually, separation of the bound and free fractions of radiolabelled ligand is done by filtration, in which the bound fraction can easily be collected. However, the filtration disturbs the equilibrium between bound and free fractions, which may lead to erroneous results. Because the decrease in bound radiolabelled ligand is accompanied by an increase in free labelled ligand, we decided also to measure this free fraction after separation by centrifugation and to compare these data with the filtration data. In these experiments a radioreceptor assay for anticholinergics was employed. The results indicate that both methods are compatible in precision when appropriate conditions are used whereas each method has its specific features.

Animals↗

PubMed1984

Does the tissue concentration in receptor binding studies change the affinity of the labelled ligand?

When the tissue concentration in a radioreceptor assay for anticholinergic drugs was varied in order to obtain optimum conditions, and the receptor concentration Cr and the equilibrium dissociation constant KD were determined by Scatchard analysis, the KD increased with increasing tissue concentrations. This phenomenon was considered as an artefact caused by non-specific binding of the labelled ligand to constituents of the receptor preparation which were not completely retained on the glass-fibre filters used for the separation of bound and free fraction of radio-labelled ligand. The increase in KD in these experiments could be described with a mathematical model of the binding experiments.

Animals↗

PubMed1984

Development of a sensitive radioreceptor assay for oxyphenonium in plasma and urine.

A radioreceptor assay (RRA) for oxyphenonium has been developed. It is based on competition between [3H]dexetimide and oxyphenonium for binding to muscarinic receptors from calf striata. The RRA is optimized towards incubation medium and to extraction by ion pair formation with sodium picrate. At least 4 X 10(-10) M of oxyphenonium is necessary to permit a reliable assay. This corresponds to a detection limit of drug of 2 ng ml-1 urine. After extraction, drug at 100 pg ml-1 of plasma can be estimated using 4 ml samples. The method is applicable to monitoring the drug and to the determination of its pharmacokinetics after therapeutic dosing. Urine levels can also be monitored.

Animals↗

PubMed1984

Radioreceptor assay for oxyphenonium.

The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium.

Animals↗