Pharmacokinetics of flunitrazepam in rats studied by a radioreceptor assay

In rat the kinetics of flunitrazepam (FNZ) was evaluated by a radioreceptor assay (RRA) after i.v. administration of 1 mg/kg and after oral administration of 1 and 3 mg/kg. The i.v. kinetics is biexponential and the g.i. absorption is very rapid (with a plasma peak at 0.25 hour) with a good bioavailability (69%); the apparent distribution volume is high, 4.8 L/kg; the half-life is equal to 3.5 hours; the elimination constant is equal to 0.8 h-1; the urinary excretion of FNZ-equivalent is negligible; the plasma total clearance is equal to 3.9 (L/kg)h-1. The concentrations of FNZ-equivalents after oral administration of 1 mg/kg show a peak at the 2-nd hour with a very high concentration in the following organs (in decreasing order): brain, kidneys, heart, liver; after 8 hours no FNZ-equivalents are present in these organs except in the brain, which shows detectable concentrations at the 32-nd hour. The peak concentrations of FNZ-equivalent in brain, kidneys and heart are higher than the corresponding peak concentration in plasma.     

Plasma kinetics of dihydroergotoxin in rat by using a radioreceptor assay

A radioreceptor assay (RRA) (with brain dopamine receptors) has been developed to determine the levels of dihydroergotoxin-equivalent (DHT-equivalent) material in rat plasma and its kinetics after oral and i.v. administration (5 mg/kg). After i.v. administration the plasma kinetics follows a two-exponential equation, with an apparent distribution volume of 7-9 1/kg and a long half-life (about 36 hours). The kinetics of DHT after oral administration shows two peaks; this could indicate a biliary recycling of the drug and/or of its metabolites. The low bioavailability (19%) and the biliary recycling of DHT-equivalent material suggest a first pass effect.     

Enkephalin: radioimmunoassay and radioreceptor assay in morphine dependent rats

A sensitive and specific radioimmunoassay for enkephalins is described which discriminates between leucineenkephalin and methionine-enkephalin. Total opioid peptide activity was assayed by the ability of brain extracts to compete for opiate receptor binding. In rats treated with morphine and/or naloxone, opiates were separated from opioid peptides by Dowex AG-50W column chromatography prior to radioreceptor assay. Chronic administration of morphine failed to alter immunoreactive enkephalin levels or total opioid activity in radioreceptor assays.     

Measurement of beta-adrenoceptor antagonists in biological fluids using a radioreceptor assay.

A new assay for plasma levels of beta-adrenoceptor antagonists is described. The assay depends upon the ability of the drug to compete with a labelled beta-adrenoceptor antagonist (-)-[3H]dihydroalprenolol for beta-adrenoceptor binding sites on lung membranes. The assay is simple to perform, very sensitive and does not require prior extraction of plasma. The assay can also detect bioactive metabolites of these agents and is clearly not limited to a single beta-adrenoceptor antagonist.     

Determination of the benzodiazepine plasma concentrations in suicidal patients using a radioreceptor assay

Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the interrelationship between the neurochemical and memory processes. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. To correlate the loss of memory with the benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct radioreceptor assay using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to high-performance liquid chromatographic (HPLC) analysis, and the HPLC data were converted to diazepam equivalents using cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the radioreceptor assay in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed an x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using this radioreceptor assay. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the cross-reactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantitated in the HPLC analysis, but were measured in the radioreceptor assay. Therefore, the radioreceptor assay proved to be a valuable tool for the assessment of clinical effects, such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose.     

Tricyclic antidepressant radioreceptor assay

A radioreceptor assay for tricyclic antidepressants described here is based on the ability of these drugs to compete with [3H]-3-quinuclidinyl benzilate (3H-QNB) for binding to muscarinic cholinergic receptors in rat brain membranes. The assay is sensitive, in that in can detect, for example, 2 ng/ml nortriptyline in plasma. Seven plasma samples from depressed patients treated with nortriptyline were assayed with the radioreceptor and gas liquid chromatographic methods, and the results from these two methods were almost identical. This assay should be used cautiously, if at all, in patients treated with other drugs that have potent anticholinergic effects.     

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.     

Bioavailability of two oral formulations of triazolam using radioreceptor assay

The radioreceptor assay (RRA) was used to quantitate plasma triazolam concentrations in eight female volunteers following single 0.5 mg doses of two tablet formulations in a cross-over study. Bioavailability in terms of area under the plasma concentration versus time curve (AUC0 infinity), maximum plasma concentration (Cmax), time to maximum (tmax), and mean residence time (MRT) was not statistically significantly different from one formulation to the other.     

A radioreceptor assay for benzodiazepines

A simple, rapid and sensitive radioreceptor assay for determining benzodiazepines in serum is based on the displacement by the drug of specific [³H]diazepam binding to a membrane fraction from rat brain. The limit of detection of the more active benzodiazepines is about 0.5 ng. Diazepam, nitrazepam, clobazam and HR 458t have been assayed in human serum after a single oral clinical dose. The results can be used for determining pharmacokinetic parameters. The technique measures not only the parent benzodiazepine but also clinically active metabolites.     

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.     

Evaluation of a radioreceptor assay for beta-adrenoceptor antagonists in plasma

Summary A radioreceptor assay (RRA) recently developed in this laboratory for beta-adrenoceptor antagonists in plasma was evaluated in normal volunteers and compared with a radioimmunoassay (RIA) for propranolol. The RRA depends upon the ability of beta-adrenoceptor antagonists to compete with a radiolabelled ligand for beta-adrenoceptor binding sites on lung membranes. Unlike other assays, it measures biologically active drugs including active metabolites of the parent compound. In volunteers given a single oral dose of (±)-propranolol, considerable differences between the two assay methods were demonstrated. In other experiments this difference was shown to relate to the RIA's sensitivity to the inactive (+)-isomer of propranolol and possibly to inactive metabolites. The facility of the RRA in measuring plasma levels of several other non-selective beta-adrenoceptor antagonists was also demonstrated. By employing (–)-propranolol as the standard in the RRA, all of these drugs can be directly compared with a single and relatively simple assay technique.     

Development of a radioreceptor assay to measure glucocorticoids.

We have developed a radioreceptor assay to measure glucocorticoids. The assay employs the partially purified 95-kDa receptor isolated from human liver and purified by size fractionation on high-performance liquid chromatography (HPLC). In the assay [3H]prednisolone competes with steroids (endogenous and exogenous) for binding to the receptor. Bound and free are separated by treatment with charcoal. The between-day precision [% coefficient of variation (CV)] at concentrations of 9.4, 18.7, and 69.9 micrograms/L prednisolone is 16.6, 9.3 and 4.5%, respectively. Specificity studies revealed that hydrocortisone, deoxycorticosterone, 4-pregnene-17 alpha,21-diol-3,20-dione, 17 alpha-hydroxyprogesterone, corticosterone and beta-hydroxyprogesterone all compete with [3H]prednisolone for binding to the receptor. Prednisone and 6 alpha-methyl prednisolone displace [3H]prednisolone to only a minor degree. The assay has been used to assess "glucocorticoid activity" in children with rheumatic diseases treated with prednisolone.     

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.     

A radioreceptor assay for benzodiazepines.

A simple, rapid and sensitive radioreceptor assay for determining benzodiazepines in serum is based on the displacement by the drug of specific [3H]diazepam binding to a membrane fraction from rat brain. The limit of detection of the more active benzodiazepines is about 0.5 ng. Diazepam, nitrazepam, clobazam and HR 458 have been assayed in human serum after a single oral clinical dose. The results can be used for determining pharmacokinetic parameters. The technique measures not only the parent benzodiazepine but also clinically active metabolites.     

A sensitive radioreceptor assay for atropine in plasma

The development of a sensitive, relatively simple radioreceptor assay for atropine in plasma is reported. It is based on the ability of anti-muscarinic compounds to compete with tritiated quinuclidinyl benzilate for muscarinic receptor binding sites. As little as 300fmol (3 × 10^?10M) atropine could be reliably assayed, the extraction procedure permitting the estimation of 3 pmol (0.9 ng) atropine per ml of plasma. The method has been applied to the determination of plasma concentration-time profiles after i.m. administration of atropine to man, and could probably be extended to other anti-muscarinic drugs.     

Determination of idazoxan in plasma by radioreceptor assay

A radioreceptor assay to determine the plasma concentration of idazoxan, a potent, highly selective antagonist for the ₂-adrenoreceptor, is described. The assay is based upon a technique in which plasma extracts containing idazoxan compete with radiolabelled ligand for binding sites on receptor-rich tissue prepared from beef brain cortex. Using a logistic data-fit the limit of detection is of the order of 1 ng ml⁻¹ and represents a 10-fold increase in sensitivity over that from an established HPLC procedure. Comparison of human plasma data from the two assays indicates a correlation coefficient of 0.92 (N = 27) although the chromatographic method gave consistently higher values than the binding assay. The binding assay requires no sample extraction or pretreatment of plasma and its accuracy, precision and inherent specificity are such that the method represents a useful alternative to HPLC for therapeutic drug monitoring.     

Antipsychotic radioreceptor assay: a modification identifying selective receptor effects

Radioreceptor assays offer the advantage of a single assay that can assess uniform exposure to multiple chemical compounds. The advent of atypical antipsychotic agents has led to new awareness of the multiple receptor subtypes through which antipsychotic agents may exert their effects, and a renewed interest in comparative drug trials of antipsychotics. The objective of this study was to show the development and validation of antipsychotic radioreceptor assays using clonal cell lines stably expressing isolated human receptors. Model assays were developed using the dopamine(2) (D(2)) and D(4) receptors. D(2) and D(4) activities measured by radioreceptor assay in plasma of antipsychotic-treated subjects were highly correlated with high-performance liquid chromatography determinations of antipsychotic concentrations. Similarly, for a variety of typical and atypical antipsychotic agents, the quotients of D(4)/D(2) activity in plasma of antipsychotic-treated subjects were highly correlated with the quotients of D(4)/D(2) affinities of these agents. Valid receptor-selective antipsychotic assays can be established and may have utility for dissecting the in vivo activity of atypical antipsychotics in relation to specific outcomes in clinical trials.     

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.     

Solubilities of adenosine antagonists determined by radioreceptor assay

The practical use of many adenosine receptor antagonists is limited by poor aqueous solubility. In some cases, solubilities are so low that they are difficult to measure by conventional means. To determine solubilities of adenosine antagonists, a sensitive radioreceptor method has been developed. Solubilities in Tris buffer (pH 7.7) ranged from 141 nM for 8-(2-amino-4-chlorophenyl)-1,3-dipropylxanthine to 945 microM for the amino-substituted xanthine PD 113,297. Ratios between solubility and adenosine receptor affinity varied from 15.8 for the A2-selective antagonist HTQZ to 169,000 for PD 113,297. From literature data on functional activity, it is apparent that useful adenosine antagonist activity in-vivo is only seen in compounds with solubility/affinity ratios greater than 100.