R.A. de Zeeuw

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.

Novel diffusion cell for in vitro transdermal permeation, compatible with automated dynamic sampling

The development of a new diffusion cell for in vitro transdermal permeation is described. The so-called Kelder cells were used in combination with the ASPEC system (Automatic Sample Preparation with Extraction Columns), which is designed for the automation of solid-extractions (SPE). Instead of SPE columns, 20 Kelder cells were placed in the racks. This allowed automatic sampling of up to 20 cells for 24 h in a dynamic mode. The cells consist of an inlet compartment, a donor compartment and a receptor compartment. The size and the depth of the inlet compartment were important to avoid entrapment of air bubbles in the receptor compartment. The Kelder cells mimic blood flow beneath the skin by replacement of the permeating drug every 2 min. Hence sink condition are more easily maintained than with the static Franz diffusion cell. The performance of the cells was tested with permeation experiments using atropine as a model drug permeating through an artificial membrane (Silastic). The use of this skin model minimized the variability in permeation of atropine as compared with human skin.

RADIORECEPTOR ASSAYS OF IPRATROPIUM BROMIDE IN PLASMA AND URINE

Ipratropium bromide (Ipbr) is a frequently used quaternary anticholinergic administered by inhalation in the treatment of chronic obstructive lung diseases. Hardly any pharmacokinetic data are available, which can be useful in the optimisation of anticholinergic therapies. Hence, a radioreceptor assay (RRA) for Ipbr has been developed. The RRA is based on the competition between (3)H-N-methylscopolamine chloride ((3)H-NMS) and Ipbr for binding to lyophilised muscarinic receptors from calf brains. The assay has been optimised in respect of incubation conditions and extraction by ion-pair formation with sodium picrate. Detection limits of the drug were 5 ng ml(-1) in urine and 500 pg ml(-1) in plasma, after extraction of 2-ml samples. The method is applicable to monitoring the drug in plasma and urine after therapeutic dosing.

APPLICATION OF RADIORECEPTOR ASSAYS FOR SYSTEMATIC TOXICOLOGICAL ANALYSIS .1. PROCEDURES FOR RADIORECEPTOR ASSAYS FOR ANTIHISTAMINICS, ANTICHOLINERGICS AND BENZODIAZEPINES

Radioreceptor assays can be a useful tool for systematic toxicological analysis in that they can be applied for the detection of an entire pharmacological class of drugs. In the present paper procedures for radioreceptor assays for benzodiazepines, anticholinergics and antihistaminics have been described in detail. The development of the assay for antihistaminics in urine is given in order to illustrate the prerequisites for these types of assays with regard to the incubation conditions. In part 2 the applicability of the three assays for systematic toxicological analysis will be evaluated on the basis of testing a large number of urine samples after administration of a selected number of drugs to healthy volunteers and patients.

DETERMINATION OF CARBISOCAINE, HEPTACAINE AND PENTACAINE IN PLASMA BY CAPILLARY GAS-CHROMATOGRAPHY WITH NITROGEN-SELECTIVE DETECTION

A gas-liquid chromatographic method is described for the determination of the local anaesthetics carbisocaine, heptacaine and pentacaine in plasma. A C(18) solid-phase extraction was used in a modification to increase selectivity. Following on-column derivatization with trimethylanilinium hydroxide, the analytes were determined by means of capillary gas chromatography and nitrogen-phosphorus selective detection. In comparison with flame ionization detection, the sensitivity of NPD was 20 times higher with a limit of determination in plasma of 10 ng ml(-1).

Solubilized benzodiazepine receptors for use in receptor assays

In the development of non-radioactive receptor assays for benzodiazepines, employing fluorescent ligands, it was observed that the fluorescence measurements were hampered by the background fluorescence of the receptor preparation. This receptor preparation is a brain tissue homogenate in which the benzodiazepine receptors are membrane-bound. To minimize the influence of the receptor material on the fluorescence detection, the benzodiazepine receptors were solubilized with 0.5% sodium deoxycholate. The binding characteristics of the receptors were examined after solubilization and compared with membrane-bound receptors. The Kd and Bmax values for membrane-bound receptors were 1.20 nM and 1.01 pM mg-1 protein and for solubilized receptors they were 4.1 nM and 0.54 pM mg-1 protein respectively. Inhibition curves with the benzodiazepine antagonist flumazenil and the agonist lorazepam revealed that their affinities for the solubilized receptor as compared to the membrane-bound receptor were also reduced from 0.67 nM to 3.2 nM and from 1.49 nM to 8.4 nM respectively. The detection limits for the two benzodiazepines, however, were not affected by the solubilization. Furthermore, three different methods to separate the fraction of free labelled ligand and the fraction bound to the solubilized receptor were compared, namely polyethylene glycol precipitation/filtration, ion exchange filtration and charcoal adsorption. Polyethylene glycol precipitation/filtration gave the highest yield for the bound fraction and the best reproducibility.

Improved benzodiazepine radioreceptor assay using the MultiScreen® Assay System

In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time. The filtration in this method was performed by using the MultiScreen Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom, which allows both the incubation and the filtration of the specimen in the same plate. After the filtration, the filters were punched out for quantitation of the bound labeled ligand [3H]flunitrazepam. The results obtained with the MultiScreen Assay System did not differ significantly from the data obtained with the conventional filtration manifold (48S): The Kis of lorazepam were 2.4 +/- 0.30 and 1.9 +/- 0.15 nM, respectively. In case a radioactive label is replaced by a fluorescent label, the bound labeled-ligand usually cannot be determined in the presence of the receptor material. Here, the bound labeled-ligand has to be dissociated after the filtration step. To dissociate the ligand-receptor complex, Tris- HCl buffer, containing 10 microM flumazenil, was added to the filters and the second filtrates were collected containing the previously bound fractions in the absence of receptor material. This approach showed the same Ki for lorazepam, 2.5 +/- 0.04 nM as without dissociation, when using the radio-labeled benzodiazepine [3H]flunitrazepam.

Evaluation of a novel diffusion cell for in vitro transdermal permeation: effects of injection height, volume and temperature

The objective of this study was to evaluate the performance of a new, compact, dynamic diffusion cell for in vitro transdermal permeation. These so-called Kelder-cells were developed as an automated alternative to the static Franz diffusion cells. The new cells were used in combination with the ASPEC-system (automatic sample preparation with extraction columns) which was initially designed for the automation of solid-phase extractions. Three variables were tested to optimize the performance of the new cell system: injection height into the inlet compartment, volume flowing through the receptor compartment and temperature. Experiments were performed using the tritium labelled anticholinergic [3H]dexetimide permeating through an artificial membrane (Silastic). The injection height of the needle into the inlet compartment of the cell should be programmed at -34 mm to ensure complete air tightness, thus forcing the buffer to flow through the cell. The volume of buffer flow through the receptor compartment is important in maintaining sink conditions: a volume of 117 microliters was chosen to replace the total content of the cell (84 microliters) every 2 min. The temperature was precisely controlled in a thermostatic cabinet to minimize variations in experimental conditions. For [3H]dexetimide, an increase in temperature of 20 degrees C reduced the lag time by a factor of approximately two, however the influence on the flux was negligible. The data for the Kelder-cells were comparable with static Franz diffusion cells at a pseudo-steady state, however Kelder-cells have the advantage of automatic sampling, continuous replacement of the receptor solution, and unattended operation over at least 24 h.

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.

Impact of urine matrix and isolation procedure on retention behaviour of basic drugs in thin-layer chromatography.

The influence of three different isolation procedures, namely liquid-liquid extraction, Extrelut column extraction and XAD-2 column extraction, and of the urine matrix on the standardised Rf values and variability of Rf values of some selected basic drugs was investigated. It appears that the liquid-liquid extraction may give a significant deviation of standardised Rf values in respect of pure drugs, which is dependent on the TLC system. For all three isolation procedures, the search window for substance identification by means of data collection based on standardised Rf values of pure drugs should be slightly wider after extraction than when using pure drugs. The TLC system cyclohexane-toluene-diethylamine (75:15:10, v/v/v) showed the best accuracy and precision of Rf values.