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.

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.

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.

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.

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.

Use of molecularly imprinted solid-phase extraction for the selective clean-up of clenbuterol from calf urine.

A feasibility study was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for clean-up of clenbuterol from urine. A binding study of clenbuterol in several solvents was performed on a clenbuterol imprinted polymer as well as on a blank polymer. These binding experiments were used to find suitable loading, washing and elution solvents for the MISPE procedure. Extraction of clenbuterol from calf urine was performed by directly loading a 10-ml urine sample onto the MIP column. Thereafter the column was washed with 10 ml of acetonitrile containing 1% acetic acid, and finally clenbuterol was eluted with 6 ml of methanol containing 10% acetic acid. A recovery of 65% was obtained. This recovery could be increased up to 75% if a sample volume of 1 ml was used or up to 100% if urine was freeze-dried and the residue was dissolved in acetonitrile and spiked with clenbuterol prior to analysis. Chromatograms of the wash and eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of clenbuterol from calf urine.

Dynamically coated capillaries improve the identification power of capillary zone electrophoresis for basic drugs in toxicological analysis.

In systematic toxicological analysis (STA), analytical methods should have a high identification power. This can be suitably expressed by parameters such as mean list length (MLL) or discriminating power (DP). The reproducibility of a method has a great impact on its identification power, and should be as high as possible. In this study, two separation methods based on capillary zone electrophoresis (CZE) were evaluated towards STA applications. Besides a normal phosphate buffer, the commercially available buffer CElixir was used, which is a double-layer dynamic coating system. The coating stabilizes the endoosmotic flow, is independent of the pH, and is claimed to be more reproducible and faster at low pH than with normal buffers. A test set of 73 basic pharmaceutical compounds was analyzed by the two CZE methods. The total analysis time, including rinsing steps, was 8 min when the coating was used and 18 min without the coating. Effective mobilities were calculated and the reproducibilities were a factor of 2 better when the coating was used (between-days SD 0.020 and 0.040 m2/V s with and without the coating, respectively). MLL and DP were calculated for the two CZE methods and for combinations with standardized liquid and gas chromatography systems. CZE with CElixir coating clearly has a high potential for STA applications, as it was shown to have a higher identification power and shorter analysis times than normal CZE.

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.

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis.

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine.

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

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.

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.

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.

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.

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.

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.

Modelling of conditions for the enantiomeric separation of beta2-adrenergic sympathicomimetics by capillary electrophoresis using cyclodextrins as chiral selectors in a polyethylene glycol gel.

A two-factor central composite design was used to determine a mathematical model for prediction of the optimal conditions for the separation of the enantiomers of some widely used beta2-sympathicomimetic drugs (beta2-agonists) by capillary electrophoresis using cyclodextrins (CD) as a chiral selector in a polyethylene glycolgel. The effects of the chemical structure of these drugs along with the addition of polyethylene glycol to the cyclodextrin solution on the resolution of their enantiomers were studied. To allow impurity studies down to 0.1% (distomer eutomer) a resolution of 2.5 should be warranted. Those beta2-agonists containing two hydroxylic groups in the aromatic ring structure show the highest enantiomeric separation, due to the fact that one of their enantiomers has a better geometric structure to fit into the beta-cyclodextrin cavity.

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 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.

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.