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.     

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.     

Determination of nuvenzepine in human plasma by a sensitive [3H]pirenzepine radioreceptor binding assay

A sensitive method for the quantitation of small amounts of nuvenzepine, a new M1-selective antimuscarinic drug, in plasma is described. The analytical method involves the use of a radioreceptor binding assay based on [3H]pirenzepine displacement in rat cerebral cortex homogenates; no previous extraction is required. The method is reliable, with an interassay CV ranging from 5 to 10%, and allows the analysis of greater than 100 samples/experiment. The limit of detection is approximately 0.1 ng/assay. Using this method we have determined the plasma levels of nuvenzepine in eight healthy volunteers treated PO with 15 or 25 mg of nuvenzepine.HCl. The pharmacokinetic parameters obtained were (for 15 and 25 mg): Cmax, 64 and 131 ng/mL; AUC0-infinity, 851 and 1379 ng.h/mL; t1/2, 8.6 and 7.2 h. These values are in good agreement with those obtained using an HPLC method. Therefore, this radioreceptor binding assay proved to be simple, rapid, and specific for the determination of low levels of nuvenzepine in human plasma.     

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.     

高效液相色谱-质谱联用法测定人血浆中奥昔布宁的浓度

目的 建立一种用高效液相色谱-电喷雾离子化质谱联用技术测定奥昔布宁血药浓度的方法。方法 以0．0lmol／L乙酸铵水溶液-甲醇(15：85)为流动相，盐酸非洛普为内标，血浆样品经用环己烷萃取后上样，经C18柱分离后，以质谱为检测器，采用选择性离子检测(SIM)测定人体血浆中奥昔布宁的浓度。结果 线性范围0．2～50ng／mL(r=0．9948)，平均相对回收率在90％～110％之间，日内和日间精密度的RSD均小于10％，奥昔布宁的定量限为0．2ng／mL，提取回收率大于90％。结论 该方法快速、准确、灵敏，可用于奥昔布宁的药代动力学研究。     

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 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.     

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.     

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.     

Simple and reliable radioreceptor assay for beta-adrenoceptor antagonists and active metabolites in native human plasma

Summary A radioreceptor assay (RRA) for the assay of beta-adrenoceptor antagonists in native human plasma is described. The hydrophilic antagonist³H-CGP 12177 was used as the radioligand. In contrast to the hydrophobic radioligand³H-dihydroalprenolol, which was investigated in parallel, the beta-adrenoceptor binding of³H-CGP 12177 by rat reticulocyte membranes was found not to be affected by inclusion of increasing proportions (0–66% of incubation volume) of human plasma in the assay. Thus, solvent extraction of drug and/or active metabolites was not necessary to avoid binding of the radioligand tracer to plasma added in the RRA. The assay of unprocessed samples was possible. Drug concentrations in plasma after oral administration of propranolol (240 mg) or carteolol (30 mg) to 6 healthy volunteers were measured by the RRA and in parallel by a chemical method. The results from both methods agreed when the plasma concentration kinetics of propranolol were investigated (elimination half-life: 3.9 h). In contrast, plasma concentrations of carteolol were consistently higher according to the RRA after oral administration of the drug. Identical concentrations, however, were found by the RRA and chemical method using plasma samples spiked with carteolol. Plasma concentrations of carteolol detected by the chemical method decline monoexponentially (elimination half-life: 5.4 h). A similar half-life of elimination for parent drug was found by the RRA (5.9 h), but an additional term describing the appearance of an active metabolite was necessary to account for the biphasic drug elimination (elimination half-life of metabolite: 17.3 h). The latter result is in agreement with the appearance of 8-hydroxy-carteolol as an active metabolite, which shows similar affinity for beta-adrenoceptors as the parent drug. The active metabolite, with a 3-fold longer elimination half-life than the parent drug, will prolong the duration of the clinical effects of orally administered carteolol. In conclusion, the RRA permits the determination of beta-adrenoceptor antagonistic activity in native human plasma at concentrations as low as 0.1-fold the IC₅₀-value of the drug or an active metabolite.     

Quantitation of human chorionic gonadotrophin (hCG) by radioreceptor assay

A sensitive radioreceptor assay was developed for pharmaceutical preparations of human chorionic gonadotrophin with the use of rat testicular membranes as receptor preparation and human 125I-chorionic gonadotrophin as tracer. The addition of unlabelled human chorionic gonadotrophin or luteinizing hormone inhibited the binding of 125I-chorionic gonadotrophin to the receptors in a concentration dependent way. Concentrations of human chorionic gonadotrophin between 30-300 mIU ml(-1) were normally used for a three-dose assay fulfilling pharmacopoeial statistical requirements for assay validity. The relative standard deviation for five assays was 7%. Estimates of potency of commercial preparations of human chorionic gonadotrophin obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. The proposed radioreceptor assay, however, provides a considerable saving in the number of animals required, requires less technical support, and is more precise than the in vivo method.     

A radioreceptor assay for the measurement of cyclosporine activity: a preliminary report

Cyclosporine A (CsA) is extensively metabolized in the liver. Some of the known metabolites share immunosuppressive properties with the parent drug. Furthermore, CsA therapy is used in an increasing number of clinical conditions, some of which affect the pharmacokinetics of the drug. At this time, it is not yet known if CsA or its metabolites are responsible for the nephrotoxicity or hepatotoxicity observed in some individuals. Some high-performance liquid chromatography (HPLC) and monoclonal immunoassay procedures measure parent drug and not the pharmacologically active metabolites, while polyclonal immunoassays and nonspecific monoclonal antibody immunoassays measure both parent drug and metabolites. However, it is unlikely that the degree of cross-reactivity with metabolites correlates with their immunosuppressive effect. To overcome these drawbacks, we have developed a method of measuring CsA activity in whole blood using specific receptors from the cytosol of human mononuclear leukocytes that have been semipurified through ultracentrifugation. The basis of the procedure is the competitive binding at specific receptor(s) between a constant concentration of [3H]dihydrocyclosporine and the variable concentrations of cyclosporine and its pharmacologically active metabolites in whole blood. Comparisons between six different assays (three specific, two nonspecific, and the receptor assay) were made. Overall, the two specific radioimmunoassay procedures correlated well to HPLC, while correlation of the two nonspecific immunoassay procedures to HPLC was poor. Poor correlation was also found to exist between the radioreceptor assay and the nonspecific assays, indicating that the cytosolic binding of CsA and its metabolites cannot be correlated to currently available assays.     

A radioreceptor assay to study the affinity of benzodiazepines and their receptor binding activity in human plasma including their active metabolites.

1 A radioreceptor assay has been established to measure the receptor affinities of numerous benzodiazepines in clinical use. 2 The time course of receptor binding activity was studied by this method in the plasma of eight healthy subjects randomly treated with 1 mg lormetazepam (Noctamid, 2 mg flunitrazepam (Rohypnol, and 10 mg diazepam (Valium, and placebo on a cross-over basis. Blood samples were collected up to 154 h after treatment. 3 Receptor affinities of numerous benzodiazepines on vitro show good correlation with therapeutic human doses (r = 0.96) and may be predictive of drug potency in man. 4 Mean peak plasma levels of lormetazepam binding equivalents were 4.8 +/- 1 ng/ml at 2 h after lormetazepam, 7.2 +/- 1.8 ng/ml at 8 h after flunitrazepam, and 17.9 +/- 2.7 ng/ml at 15 h after diazepam. Plasma elimination half-lives of benzodiazepine binding equivalents were 9.3, 23 and 63 h, respectively. 5 Slow elimination of benzodiazepine binding equivalents following flunitrazepam and diazepam may be due to persistent active metabolites.     

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.     

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.     

Triazolam pharmacokinetics in rats using a radioreceptor assay

The time course of the activities arising after oral (0.33 and 1 mg/kg) and intravenous (0.33 mg/kg) administration of triazolam to rats was assessed in different tissues using a radioreceptor assay. In almost all cases the activities in plasma were lower than in liver, brain, kidney and heart showing that the drug and possibly some active metabolites distribute extensively and rapidly in all the sampled tissues. Peak plasma levels were 34 pmol/ml, 10.2 pmol/ml and 34.9 pmol/ml after 0.33 mg/kg intravenously, 0.33 mg/kg per os and 1 mg/kg p.o. respectively. The area under curve in plasma was proportional to the administered oral dose and it was much smaller after intravenous injection than after the equivalent oral dose (13.8 pmol.h/ml after the i.v. dose and 47.3 pmol.h/ml after the oral dose).     

A sensitive radioreceptor assay for determining scopolamine concentrations in plasma and urine

A sensitive and reliable procedure for the quantitation of low picogram levels of scopolamine in plasma and urine is described. The method consists of two steps, a preparative extraction step using C18 columns (Sep-Pak), followed by an analytical quantitation step involving a muscarinic radioreceptor assay. The extraction efficiency of the C18 columns was 85-95% for both plasma and urine over a wide concentration range. When [3H]methyl scopolamine is used as a tracer, the assay can detect picogram concentrations (greater than 25 pg) of scopolamine (base) in plasma and urine. The applicability of the procedure for therapeutic drug monitoring of scopolamine was demonstrated by using the method to determine plasma levels in humans after transdermal administration.