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K Ensing

Publications and source records attributed to K Ensing.

At least 37 records · Page 2Linked to original sources
PubMed1999

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 Ki's 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.

Filtration↗
PubMed1999

Detection of muscarinic receptors in the human lung using PET.

UNLABELLED: The characterization of pulmonary muscarinic receptors with PET is still in its infancy. Because approximately 70% of the lungs consists of air and pulmonary muscarinic receptor densities are low, ligands with high receptor affinity are required to obtain reasonable signal-to-noise ratios on PET images. Therefore, the potent 11C-labeled muscarinic antagonist N-methyl-piperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide ([R]-VC-002) was developed. We administered this radioligand to four healthy human volunteers to examine its suitability for studying pulmonary muscarinic receptors in vivo. METHODS: [11C]VC-002 (185 MBq, specific activity > 7.4 TBq/mmol) was intravenously injected on 2 separate days, with an interval of at least 1 wk. On the first day the volunteers were not pretreated, but on the second day they received the anticholinergic glycopyrronium bromide (Robinul; 2 x 0.1 mg intravenous) 25 and 30 min before the injection of the radiopharmaceutical. C[15O]O scans (approximately 740 MBq [20 mCi] by inhalation) were acquired before the receptor scan to calculate pulmonary blood volume. RESULTS: On PET images of the thorax, the lungs were clearly visible. After the volunteer was pretreated with glycopyrronium bromide, pulmonary uptake of the radioligand was reduced to 32%+/-12% of the control value at 60 min postinjection and the lungs could no longer be seen. (R)-[11C]-VC-002 was rapidly cleared from plasma and was slowly metabolized during the time course (60 min) of the PET scan. The fraction of radioligand representing parent compound decreased from 99.9% at the time of injection to 82% at 40-60 min postinjection, both in the presence and absence of Robinul. Pulmonary tissue-to-plasma ratios, calculated on a count-per-minute-per-gram basis, reached a plateau value of 17.8+/-1.2 at 40-50 min postinjection. CONCLUSION: [11C]VC-002 appears to be suitable for in vivo studies of pulmonary cholinoceptors.

Carbon Radioisotopes↗
PubMed1998

Characterisation of beta2-adrenoceptors, using the agonist [11C]formoterol and positron emission tomography.

The agonist radioligand N-[2-hydroxy-5-[1-hydroxy-2-[[2-(4-[11C]-methoxyphenyl)-1-methylethyl]am ino]ethyl]phenyl]formamide ([11C]formoterol) was synthesised in order to test its ability to visualise pulmonary beta2-adrenoceptors in vivo, with positron emission tomography (PET). Formoterol was labelled via reaction of a dibenzyl-protected precursor with [11C]CH3I. Subsequent deprotection with Pd/C and H2 yielded [11C]formoterol in 5-15% (corrected for decay) and the specific activity ranged from 5.5-22.2 TBq mmol (150-600 Ci mmol(-1)), 60-70 min after end of bombardment. Biodistribution studies with [11C]formoterol were performed in male Wistar rats which were either untreated or predosed with (D,L)-propranolol hydrochloride (2.5 mg kg(-1), beta-adrenoceptor antagonist), erythro-DL-1-(7-methylindan-4-yloxy)-3-isopropylaminobuta n-2-ol hydrochloride (ICI 118551, 0.15 mg kg(-1), beta2-adrenoceptor antagonist), isoprenaline (15 mg kg(-1), non-subtype selective beta-adrenoceptor agonist) or (+/-)-(2-hydroxy-5-[2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-i midazol-2-yl-)phenoxy)propyl)amino)ethoxy]benzamide)monomethane sulfonate (CGP 20712A, 0.15 mg kg(-1), beta1-adrenoceptor antagonist). Lungs, heart, liver and plasma were analysed for radioactive metabolites. The kinetics of [11C]formoterol in the lungs of male Wistar rats were investigated by means of a dynamic PET study. The biodistribution studies showed significant specific binding in tissues known to contain beta2-adrenoceptors (lungs, spleen, and heart). Binding in these organs was blocked by ICI 118551 and isoprenaline, but not by CGP 20712A. [11C]Formoterol was rapidly metabolised in rats but lungs and heart did not substantially take up the labelled metabolites. The binding of [11C]formoterol in various tissues of rats is consistent with the beta2-selectivity of formoterol. Whether [11C]formoterol selectively binds to the high affinity state of beta2-adrenoceptors remains to be elucidated. [11C]Formoterol is potentially useful for studying beta2-adrenoceptors with PET and this radioligand may provide new insights in the mechanisms underlying prolonged sympathomimetic action.

Adrenergic beta-Agonists↗
PubMed1998

Determination of the enantiomeric purity of (-) terbutaline by capillary electrophoresis using cyclodextrins as chiral selectors in a polyethylene glycol gel.

A method was developed for determination of the enantiomeric purity of the therapeutic-pharmacological active (-)-enantiomer of terbutaline using cyclodextrins as a chiral selector dissolved in a removable liquid polyethylene glycol gel by use of capillary electrophoresis. The effect of temperature, type and concentration of polyethylene glycol and cyclodextrins was studied on the resolution between the two enantiomers. Best results were obtained with 10 mM hydroxyethyl-beta-cyclodextrin dissolved in a 10% polyethylene glycol-2000 solution at 15 degrees C. Under these conditions, an impurity of 0.1% (distomer/eutomer) can be readily detected.

Chemistry Techniques, Analytical↗
PubMed1998

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.

Administration, Cutaneous↗
PubMed1997

Size-exclusion chromatographic reconstitution of the bovine brain benzodiazepine receptor. Effects of lipid environment on the binding characteristics.

The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (Kd) of 2.7 +/- 1.2 nM and with 0.40 +/- 0.04 pmol binding sites per mg protein (Bmax). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (Kd 1.4-4 nM). Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at -20 degrees C or -80 degrees C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and Bmax over a period of 2 months.

Animals↗
PubMed1997

Stereoselective synthesis and biodistribution of potent [11C]-labeled antagonists for positron emission tomography imaging of muscarinic receptors in the airways.

Quantitation of muscarinic receptors in the lungs in vivo with positron emission tomography (PET) is of clinical interest. For that purpose we decided to develop [11C]-labeled ligands with a high affinity (KD < 0.1 nM). Three quaternary muscarinic antagonists, racemic N-methylpiperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide 1a (pKB = 10.39), its (R)-isomer 1b (pKB = 11.08), and (R,R)-quinuclidin-3-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide 2 (pKB = 11.28), were labeled by reacting [11C]CH3I with their tertiary amine precursors. The enantiomerically pure tertiary amine precursors were prepared by stereoselective synthesis starting from (R)-(-)-mandelic acid. In vitro binding assay of 1b and 2 demonstrated that both ligands bind with very high affinity to the muscarinic receptor subtypes M1, M2, and M3. They are more potent than the muscarinic antagonist (R)-N-methylquinuclidinyl benzilate ((R)-MQNB). Distribution studies with 1a, 1b, and 2 in control and atropine-treated male Wistar rats demonstrated significant specific binding (90-99% of total issue uptake) in tissues containing cholinoceptors (heart, intestine, lung, pancreas, spleen, stomach, submandibular gland). Because the tissue/plasma concentration ratios of 1b are most favorable, this ligand was used for further evaluation. Analysis of plasma samples showed a very rapid clearance (t1/2 = 0.3 min) of the radioligand 1b and a relatively slow appearance of a hydrophilic metabolite. At 15 min postinjection of 1b, analysis of heart, lungs, and liver showed that respectively 99%, 88%, and 8% of the tissue radioactivity corresponded with the parent compound. Ligand 1b appears to be an excellent candidate for PET studies of mAChR receptors in heart and lungs.

Animals↗
PubMed1997

Stability of solubilized benzodiazepine receptors.

According to the observations of other researchers, benzodiazepine receptors solubilized with sodium deoxycholate are unstable, but stability can be improved by exchanging deoxycholate for Triton X-100. In our experiments we conclude that the choice of detergent is not the restrictive factor for the stability of the solubilized receptors, but the storage conditions are. Solubilized receptors, either stored in sodium deoxycholate or in Triton X-100, were stable for at least 2 months when stored at -80 degrees C, but both preparations were strongly unstable when stored at -20 degrees C. Alternatively, sodium deoxycholate-solubilized receptors may be lyophilized and then stored at -20 degrees C.

Animals↗
PubMed1997

Characterization of pulmonary and myocardial beta-adrenoceptors with S-1'-[fluorine-18]fluorocarazolol.

UNLABELLED: S-1'-[18F]fluorocarazolol was administered to healthy volunteers to assess its potential for noninvasive measurement of regional pulmonary and myocardial beta-adrenoceptor densities. METHODS: High-specific activity fluorocarazolol was intravenously injected on two separate occasions within a 1-wk interval. The initial injection was without pretreatment, but before the second injection, the volunteers either inhaled salbutamol (2 x 200 micrograms aerosol) or they ingested pindolol (3 x 5 mg during a 12-hr interval). Twenty-eight PET time frames of 31 planes were acquired over a period of 60 min after each injection. Blood samples were drawn and analyzed for the presence of fluorocarazolol and radioactive metabolites. RESULTS: Uptake of fluorocarazolol in the target tissues was hardly affected by salbutamol but was strongly depressed by pindolol. Pulmonary and myocardial tissue-to-plasma concentration ratios of fluorocarazolol reached plateau values of 11.6 +/- 0.6 (lungs) and 18.1 +/- 1.0 (heart) at 45-50 min postinjection. These values were reduced to 2.0 +/- 0.4 and 2.0 +/- 0.6 after treatment with pindolol. CONCLUSION: These data indicate that: 1. Pulmonary and myocardial uptake of radioactivity after intravenous administration of S-1'-[18F]fluorocarazolol represents radioligand binding to beta-adrenoceptors. 2. Pulmonary binding occurs mainly in alveoli rather than in airway smooth muscle under these conditions. 3. Binding kinetics do not preclude quantification of receptors with compartment models.

Adrenergic beta-Agonists↗
PubMed1997

Imaging beta-adrenoceptors in the human brain with (S)-1'-[18F]fluorocarazolol.

UNLABELLED: We evaluated the suitability of fluorocarazolol for in vivo studies of cerebral beta-adrenoceptors because (S)-1'-[18F]fluorocarazolol has a higher affinity to beta-adrenoceptors than to serotonergic receptors (pKi beta 1 9.4, beta 2 10.0, 5HT1A 7.4, 5HT1B 8.1) and rapidly crosses the blood-brain barrier. METHODS: The (S)-[18F]fluorocarazolol (74 MBq, > 37 TBq/mmol) was intravenously administered to healthy volunteers on two separate occasions with an interval of at least 1 wk. The initial injection was without pretreatment, but before the second injection, the volunteers received the beta blocker (+/-)-pindolol (3 x 5 mg orally, during 18 hr). The brain was studied with a PET camera in dynamic mode. RESULTS: Uptake of radioactivity delineated gray matter and was particularly high in the posterior cingulate, precuneus and striatum. Low uptake occurred in the thalamus, whereas the lowest uptake was observed in the white matter of the corpus callosum. After pindolol pretreatment, uptake was reduced and its distribution became homogeneous throughout the brain. The ratio of total-to-nonspecific binding was about 2 at 60 min, increasing to 2.5-2.75 at longer intervals. CONCLUSION: Fluorocarazolol is the first radioligand that can visualize cerebral beta-adrenoceptors and may enable monitoring of these binding sites during disease.

Adrenergic beta-Antagonists↗
PubMed1996

Immobilization of antibodies as a versatile tool in hybridized capillary electrophoresis.

Hybridization of capillary electrophoresis (CE) and immunoassays (IA) can theoretically lead to highly sensitive and selective assays. Immobilization of antibodies in the capillaries employed for CE can be achieved either by adsorption to the capillary wall, which was coated prior to use in order to improve the adsorption, or by covalent binding to modified capillaries. For the evaluation of the concept, a fluoroimmunoassay for the herbicide atrazine was used. Antibodies were immobilized by adsorption, and the specificity of the binding of the labeled ligand was confirmed by saturation and competition experiments. For this particular assay the use of a C8-modified capillary was shown to be preferable over C18- and mercaptodimethylsilane-modified capillaries. The first part of the C8 capillary wall was partially covered by antibodies and the remainder was covered by adsorbed bovine serum albumin to eliminate non-specific binding of the labeled ligand. In the present approach the antibody-bound fraction of the labelled ligand was quantitated, which means that after removal of the free fraction of the labeled ligand from the capillary, the binding of the labeled ligand and the analyte to the antibodies, should be broken. By changing the chemical environment such as pH, salts and organic solvents, this dissociation process can be facilitated. Addition of 25% methanol to the assay buffer increased the dissociation rate by 50% without inactivation or mobilization of the antibodies. On the other hand, these chemical tools should not interfere with the requirements for CE and fluorescence detection. Moreover, the methanol caused stacking of fluorescein-labeled atrazine (FA) in the sample plug by a factor of 30, which was very advantageous for the quantitation of FA. The results of this study imply that combination of antibodies and fluorescent labels with CE opens the way to multi-analyte immunoassays and forms a valuable tool for the selective preconcentration of analytes originating from complex biological matrices.

Antigen-Antibody Reactions↗
PubMed1996

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.

Adsorption↗
PubMed1996

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.

Administration, Cutaneous↗
PubMed1996

Effect of purification followed by solubilization of receptor material on quantitative receptor assays for anticholinergic drugs.

In order to optimize quantitative receptor assays for anticholinergics, the different receptor preparations resulting from the purification and the solubilization of the P2 pellet from the calf striatum were evaluated. The dissociation constants for two chemically different anticholinergics, the tertiary amine scopolamine and the quaternary amine oxyphenonium, were calculated from inhibition studies of 3H-NMS binding in buffer and plasma. The Kd values for both anticholinergics were similar for all the membrane-bound receptor preparations (unpurified and the purified P2 pellet) either in buffer or in plasma. More pronounced differences were observed between the membrane-bound and solubilized receptors. By introducing the solubilized receptor as well, differences between the individual anticholinergics appeared. On the one hand, for scopolamine, a gain in sensitivity of 1.5-2.8 in plasma was observed for the solubilized receptor. On the other hand, in the case of oxyphenonium, a dramatic loss in sensitivity (by a factor of about 24) was observed with the solubilized receptor, as compared to the membrane-bound receptor, in buffer. Very interestingly, however, when the solubilized receptor was used in plasma, a lowering of the Kd value was found for both anticholinergics, i.e. the assays became more sensitive. Such an effect (not observed for the membrane-bound receptor) could be obtained only when the percentage of digitonin present in the assay was at least 0.12% (w/v) or higher.

Animals↗
PubMed1995

Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part A: The membrane-bound receptor.

The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary 3H-dexetimide (3H-DEX) and quaternary 3H-N-methylscopolamine (3H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet. The overall yield of receptors--80%, when labelled with 3H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of 3H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with 3H-DEX. The total yield of 3H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85. We did not succeed in the improvement of the ratio specific/non-specific binding, neither for 3H-DEX nor for 3H-NMS for the purified receptor preparations. The use of 3H-NMS is preferable to 3H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with 3H-DEX.

Animals↗
PubMed1995

Evaluation of a purification procedure for the muscarinic receptor for the purpose of quantitative receptor assays of anticholinergics. Part B: The solubilized receptor.

For the purpose of quantitative receptor assays, a three-step solubilization procedure including three optimization sets for muscarinic receptor from calf striatum was developed. The first step includes the extraction of the P2-pellet with n-hexane and consequently with 2 M NaCl. By the latter, 39% of non-receptor proteins was extracted. The resulting pellet (NaCl-pellet), enriched in muscarinic receptors by a factor of 1.5-1.7, was solubilized with 1% digitonin. The binding parameters of the solubilized receptor were determined for the tertiary 3H-dexetimide (3H-DEX) and the quaternary 3H-N-methylscopolamine (3H-NMS). The resulting receptor density measured with 3H-dexetimide was lower (43.3% of that for the NaCl-pellet) than that for 3H-N-methyl-scopolamine (56.7%). The treatment with digitonin preserved the high affinity for 3H-N-methylscopolamine (Kd = 0.645 nM), however the affinity of 3H-dexetimide decreased after solubilization (Kd = 8.526 nM). The use of solubilized receptors in combination with hydrophilic 3H-NMS allows to increase the ratio specific/non-specific binding, since the non-specific binding for this ligand to the solubilized preparation is lower when compared with membrane-bound receptors. The above solubilization procedure was found preferable over directly solubilizing the P2-pellet since (a) the receptor density for 3H-NMS was higher for the solubilized NaCl-pellet by a factor of about 1.7, and (b) the treatment of the P2-pellet with digitonin resulted in a lowering of the Kd to 2.422 nM. However, with respect to the plasma effect on the ligand binding, both solubilized preparations give similar results. The use of the solubilized NaCl-pellet or the P2-pellet can considerably improve the quantitative receptor assays of plasma samples. Unlike the membrane-bound receptor, a high volume of plasma, such as 400 microliters, can be added to the assay without any influence on the 3H-DEX binding when solubilized preparation is used.

Animals↗
PubMed1994

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.

Animals↗