Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8- trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC-RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE-RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC-RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE-RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.     

Radioimmunoassay of oestrone in plasma. A comparison of different methods with respect to the relation between assay specificity, sample purification and antibody specificity

A radioimmunoassay (RIA) for oestrone (Oe1) in plasma was developed using an ether extraction, a partition between NaOH and light petroleum, and a TLC as sample purification and an antiserum cross-reacting with Oe2 for the final assay (Method A1). The reliability criteria are given in detail. In order to simplify this method a highly specific antiserum was developed by using Oe1-3-hemisuccinate-BSA as an antigen. Using this antiserum for the final assay but omitting the TLC (Method B) the Oe1 concentration in male plasma was 76% overestimated (Method B compared with Method A1). In pregnancy plasma Oe1 could specifically be determined after a simple ether extraction (Method C). It was concluded that the use of a highly specific antiserum (as determined by cross-reaction studies) for the final assay does not necessarily imply that a chromatographic sample purification can be omitted without loss in assay specificity. This appears to be true mainly in cases where the steroid concentration of the sample is low. Normal values of Oe1 from 80 healthy adult males were ascertained by Method A1. Age group I (22-61 years, n = 48) ranged from 1.22-5.60 ng/100 ml, median 2.81; age group II (67-90 years, n = 32) from 1.55-6.40, 100 median 3.41. The small increase of Oe1 with age was highly significant.     

Time-resolved fluoroimmunoassay for the determination of lisinopril and enalaprilat in human serum

A solid-phase immunoassay with detection based on time-resolved fluorescence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to the wells of microtitration strips. At the end of the first incubation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with the biotin already bound to the solid-phase antibody. After a washing step, the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence from each sample is inversely proportional to the concentration of the drug in the sample. The assay demonstrates good accuracy, reproducibility and specificity at serum concentrations down to 0.5 ng ml-1. However, the useful concentration range of TR-FIA is much narrower than that obtained by double antibody radioimmunoassay (RIA).     

Development of a sensitive ELISA for human leptin, using monoclonal antibodies

A new, sensitive ELISA for human leptin in plasma and cerebrospinal fluid (CSF) was developed, using monoclonal antibodies. The lower limit of detection of this ELISA was 0.78 pg/assay. Both intra- and interassay imprecision values were <7%. The dilution curves of plasma and CSF showed good linearity, and the recovery was 83.2-95.6%. There was good correlation between plasma leptin concentrations by the ELISA and a commercially available RIA (r = 0.99). Our ELISA is advantageous because it does not require radioisotopes, it produces results in hours rather than days, and more importantly, it improves on the detection limit and plasma interference of the RIA kit. The new ELISA enables measurement of low concentrations of leptin, as are seen in CSF and in plasma of patients with anorexia nervosa.     

Absorption, distribution and excretion of 14C-cefatrizine (14C-S-640 P) in rat (author''s transl)

The reliability and rapidity of methods utilizing radioimmunoassay ( RIA) for detecting chorionic gonadotropin (CG) and estrogens in plasma, competitive protein binding assay for plasma progestins, and the hemagglutination inhibition test for urinary CG in the diagnosis of early pregnancy was evaluated in baboons. The hemagglutination inhibition test for detection of urinary CG did not give satisfactory results as late as Day 25 of pregnancy. Confirmation of pregnancy could not be established on Days 8 and 12 of pregnancy by RIA of estrogens, progestins, or CG. However, detection of plasma CG by RIA was 96.6% successful by Day 16, though the method was time-consuming. However, the determination of plasma estrogens and progestins by RIA was determined more quickly on Day 16, and gave equally successful results as the RIA for CG.     

The porphyromonas gingivalis prtP/kgp homologue exists as two open reading frames in strain 381

Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels increase in patients with heart failure with the progression of clinical symptoms and with the deterioration of hemodynamics; consequently, assay methods for these peptides may be useful in the follow-up of cardiac patients. Non-competitive immunoradiometric assay (IRMA) methods for ANP or BNP do not generally require preliminary extraction and/or purification of the plasma sample, and so may be more suitable than competitive immunoradiometric assay (RIA) methods for the routine assay of plasma peptide concentrations. We evaluated the analytical characteristics and clinical usefulness of two IRMAs for plasma ANP and BNP, to verify whether these methods may be considered suitable for the follow-up of patients with heart failure. Both methods are based on the solid-phase sandwich IRMA system, which uses two monoclonal antibodies prepared against two sterically remote epitopes of peptide molecule; the first antibody was coated on the beads solid-phase and the second was radiolabeled with 125I. Blood samples were collected from a brachial vein in ice-chilled disposable polypropylene tubes containing aprotinin and EDTA after the patient had rested for at least 20 min in the recumbent position. Plasma samples were immediately separated by centrifugation and stored at -20 C until assay. The IRMA methods showed a better sensitivity and a wider working range sensitivity (about 2 ng/l) than those of RIA methods. Moreover, the normal range found with these methods (ANP = 16.1 +/- 8.6 ng/l, 5.2 +/- 2.8 pmol/l, BNP = 8.6 +/- 8.2 ng/l, 2.5 +/- 2.4 pmol/l) was similar to that generally reported using the most accurate methods, such as the other IRMAs or RIAs, using a preliminary extraction and purification of plasma samples with chromatographic procedures. Our results obtained in patients with different degrees of heart failure indicate that plasma ANP and BNP increase with the progression of clinical symptoms (NYHA class) (ANOVA p < 0.0001). Indeed, circulating levels of ANP (R = -0.701, no. = 86) and BNP (R = -0.745, no. = 55) were significantly (p < 0.0001) and negatively correlated with the left ventricular ejection fraction values. Furthermore, a close curvilinear regression (R = 0.960, no. = 215) was found between ANP and BNP values, because plasma BNP progressively increases more than plasma ANP in patients with different stages of heart failure. In conclusion, IRMA methods are preferable for the measurement of plasma ANP and BNP for experimental studies and routine assay because they are more practicable, sensitive and accurate than RIA procedures. Finally, BNP assay appears to be better than ANP for discriminating between normal subjects and patients with different degrees of heart failure.     

Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.     

The pathophysiology of the blood-brain barrier dysfunction induced by severe hypercapnia and by epileptic brain activity

Antiserum to fentanyl was obtained in rabbits repeatedly injected with carboxyfentanyl conjugated to bovine serum albumin. Using the antiserum, a highly sensitive radioimmunoassay has been developed, based on the dextran-coated charcoal method. It proved possible to assay the drug directly in plasma, in amounts as small as 30 picogram in 0.5 ml. The antibody was highly specific for fentanyl and no cross-reaction was observed with its major metabolites. This sensitive and specific radioimmunoassay method was employed to determine fentanyl in plasma from six volunteers after an intravenous bolus of 0.2 mg, and in plasma from dogs treated both intravenously and subcutaneously with 0.02 mg/kg. The plasma level of fentanyl could be followed for up to 6 h after a therapeutic dose in dogs and man.     

Dose-dependent plasma clearance of MK-826, a carbapenem antibiotic, arising from concentration-dependent plasma protein binding in rats and monkeys

After intravenous administration of MK-826, a new carbapenem antibiotic, the compound exhibited nonlinear pharmacokinetics in rats and monkeys. In both species, time-averaged plasma clearance (based on total concentrations) increased about 5-fold over the 10- to 180-mg/kg dose range. MK-826 was extensively plasma protein bound in rat and monkey plasma, and the extent of binding was concentration dependent at plasma concentrations achieved after administration of these doses. Rosenthal analysis of the plasma protein binding indicated that there were two classes of binding sites. The binding capacity of the primary site was comparable to the plasma albumin concentration, which suggested that this primary site consisted of a single site on albumin. The extent of binding of MK-826 to rat albumin was similar to that in whole plasma. Clearance values based on unbound concentrations appeared independent of dose from 10 to 180 mg/kg, which is consistent with saturation of protein binding as the primary cause of the nonlinear pharmacokinetic behavior.     

Dunham Treatment Including Effects of the Born-Oppenheimer Breakdown for Open-Shell Diatomic Molecules in 2Sigma States

Two high-performance liquid chromatographic (HPLC) methods are described for determination of (+/-)-ethopropazine (ET) in rat plasma. After deproteination and liquid-liquid extraction, assay of (+/-)-ET was performed using either a C18 column (non-stereospecific assay) or an (alpha-R-naphthyl)ethylurea column (stereospecific assay). The UV detection was at 250 nm. Mean recovery was >85%. Both assays demonstrated excellent linear relationships between peak height ratios and plasma concentrations; quantitation limits were < or =25 ng/ml, based on 100 microl rat plasma. Accuracy and precision were <17% with both methods. Both methods were applied successfully to the measurement of ET plasma concentrations in rats given the drug intravenously.     

Preliminary animal pharmacokinetics of the parenteral antifungal agent MK-0991 (L-743,872)

MK-0991 (L-743,872) is a potent antifungal agent featuring long half-life pharmacokinetics. The pharmacokinetics of MK-0991 administered intravenously to mice, rats, rhesus monkeys, and chimpanzees is presented. Unique to MK-0991 is its consistent cross-species performance. The range of values for the pharmacokinetic parameters were as follows: clearance, 0.26 to 0.51 ml/min/kg; half-life, 5.2 to 7.6 h; and distributive volume, 0.11 to 0.27 liters/kg. The level of protein binding of MK-0991 was determined to be 96% in mouse and human serum. The compound exhibited high affinities for human serum albumin and at least two lipid components. The rationale for the selection of MK-0991 as a drug development candidate was based on its two- to threefold superior pharmacokinetic performance in chimpanzees over the performance of an otherwise equivalent analog, L-733,560. Once-daily dosing for MK-0991 is indicated by a graphical comparison of levels in the circulations of chimpanzees and mice. In a study of the pharmacokinetics of MK-0991 in mouse tissue, the organs were assayed following intraperitoneal administration. The area under the concentration-versus-time curves (AUC) segregated the tissues into three exposure categories relative to plasma. The tissues with greater exposure than that for plasma were liver (16 times), kidney (3 times), and large intestine (2 times). The exposure for small intestine, lung, and spleen were equivalent to that for plasma. Organs with lower levels of exposure were the heart (0.3 times that for plasma), thigh (0.2 times), and brain (0.06 times). Kinetically, drug was cleared more slowly from all tissues than from plasma, indicating that terminal-phase equilibrium had not been achieved by 24 h. Thus, some measure of accumulation is predicted for all tissues. Single daily doses of MK-0991 should provide adequate systemic levels of fungicidal activity as a result of its long half-life pharmacokinetics, wide distribution, and slowly accumulating concentrations in tissue.     

Novel enzyme-linked immunoassay to determine nanogram levels of pneumocandins in human plasma

A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4 degrees C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4 degrees C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.     

GLC determination of methotrimeprazine and its sulfoxide in plasma

A GLC method, based on flame-ionization detection, was developed for the assay of methotrimeprazine and its sulfoxide in plasma. For a 6-ml aliquot, the sensitivity was 2-3 ng/ml for the unchanged drug and 4-5 ng/ml for the sulfoxide. The coefficient of variation, calculated from duplicate analyses of plasma samples, was 8-15% for concentrations between 10 and 100 ng/ml. Patients treated with orally administered methotrimeprazine had higher plasma levels of the sulfoxide than of unmetabolized drug. The method also was applied to the analysis of promazine and chlorpromazine in patient plasma.     

Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis

The in vivo activity of the Merck antifungal echinocandin drug candidate MK-0991 (L-743,872) was evaluated in mouse models of disseminated candidiasis, aspergillosis, and cryptococcosis. The echinocandins are potent inhibitors of 1,3-beta-D-glucan synthase. Two models of disseminated candidiasis were used. In a Candida albicans mouse survival model with both DBA/2N and CD-1 mice, estimates of the 50% effective doses (ED50s) of MK-0991 were 0.04 and 0.10 mg/kg of body weight/dose at 21 days after challenge, respectively. In a C. albicans target organ assay (TOA) with DBA/2N mice, MK-0991 at levels of > or =0.09 mg/kg/dose significantly reduced the numbers of C. albicans CFU/g of kidneys compared to the numbers in the kidneys of control mice from 1 to 28 days after challenge. Even when given as a single intraperitoneal dose either 30 min or 24 h after challenge, MK-0991 was effective and significantly reduced the numbers of C. albicans CFU/g of kidney compared to those in the controls. MK-0991 was >300-fold less active when it was administered orally than when it was administered parenterally. MK-0991 was efficacious in mouse TOAs against other C. albicans strains and Candida species including Candida tropicalis, Candida (Torulopsis) glabrata, Candida lusitaniae, Candida parapsilosis, and Candida krusei. MK-0991 was ineffective against disseminated Cryptococcus neoformans infections. In the model of disseminated aspergillosis in mice, MK-0991 at doses of > or =0.02 mg/kg/dose significantly prolonged the survival of DBA/2N mice, with estimates of the ED50 and ED90 of MK-0991 being 0.03 and 0.12 mg/kg/dose, respectively, at 28 days after challenge. MK-0991 is a potent, parenterally administered therapeutic agent against disseminated candidiasis and aspergillosis that warrants further investigation in human clinical trials.     

Comparative plasma concentrations of quinidine following administration of one intramuscular and three oral formulations to 13 human subjects

A GLC method, based on flame-ionization detection, was developed for the assay of methotrimeprazine and its sulfoxide in plasma. For a 6-ml aliquot, the sensitivity was 2-3 ng/ml for the unchanged drug and 4-5 ng/ml for the sulfoxide. The coefficient of variation, calculated from duplicate analyses of plasma samples, was 8-15% for concentrations between 10 and 100 ng/ml. Patients treated with orally administered methotrimeprazine had higher plasma levels of the sulfoxide than of unmetabolized drug. The method also was applied to the analysis of promazine and chlorpromazine in patient plasma.     

Diploma of continuing education from the Society of Physician of Thüringia

A rapid and sensitive high-performance thin-layer chromatographic assay has been developed for the measurement of nimesulide in human plasma. Its use for pharmacokinetic studies has been evaluated. The method includes a single-stage extraction procedure without the use of an internal standard. Analysis was performed on plasma containing known amounts of the drug, on drug-free plasma, and on plasma containing an unknown quantity of the drug. Known amounts of extract and nimesulide (100 and 200 ng, as external standard) were spotted on precoated silica-gel 60F254 plates by means of a Camag Linomat IV autosampler. Quantification was achieved using a Camag TLC scanner 3. The recovery of the method was 97.10 +/- 2.22%. The method was applied for the determination of plasma levels and pharmacokinetic parameters of nimesulide after oral administration of two formulations (100 mg) in healthy volunteers. The method is a sensitive, economical, rapid and specific assay for nimesulide in human plasma, and is suitable for pharmacokinetic studies after therapeutic doses.     

Development and validation of a sensitive and specific radioimmunoassay for the determination of cicaprost in biological samples

Cicaprost is a potent, chemically and metabolically stable PGI2-mimetic. Pharmacodynamic effects were observed after oral administration of approximately 10 micrograms in man when plasma levels were in the low pg-range. The present report describes the development of a selective antiserum and a tracer with high specific activity and their use for the RIA determination of Cicaprost in biological samples. Cicaprost-[3H]-methylester with a specific activity of 819 GBq/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH 2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound Cicaprost was achieved by the charcoal method. Extraction recovery of Cicaprost was approximately 90% at pH approximately 2. The detection limit of the assay was 10-20 pg/ml plasma. Coefficients of variations were 6, 3 and 9% (within-day, n = 5) and 25, 12 and 10% (day-to-day, n = 11) at 50, 100 and 200 pg/ml. HPLC-chromatograms of plasma extracts did not reveal any peak apart from Cicaprost, demonstrating the specificity of the method. The present RIA for Cicaprost exhibits high specificity and sensitivity and will be used for further bioanalyses in pharmacokinetic study.     

Association of periampullary diverticula with primary choledocholithiasis but not with secondary choledocholithiasis

The precision and the diagnostic performance of the Boehringer Mannheim CEDIA DAU LSD assay was evaluated. The assay was performed in the semi-quantitative mode on a Hitachi 917 analyzer. Within-run coefficients of variation (CVs) of the semiquantitative values for 0.25 and 1.0 ng/mL were 11.2 and 6.2%, respectively. Day-to-day CVs for the same concentrations were 12.6 and 8.6%. We analyzed 318 urine samples by CEDIA, DPC Coat-A-Count RIA and Behring EMIT II. Confirmation was performed by GC-MS, after extraction on Bond Elut Certify columns. Two hundred sixty-three samples were negative by all methods. Twenty-five samples were positive by all immunoassays, 19 of which were confirmed by gas chromatography-mass spectrometry (GC-MS). One sample was falsely negative by CEDIA. Three samples were positive by EMIT and CEDIA, but negative by RIA and GC-MS. Twenty-six samples were positive by EMIT alone, but they were not confirmed by GC-MS. The LSD CEDIA assay seems to be less specific than DPC RIA but more specific than the EMIT LSD assay.     

A sensitive assay of 5-fluorouracil in plasma by gas chromatography-mass spectrometry

1 A gas chromatographic-mass spectrometric method was developed for determining 5-fluorouracil in plasma, using methylated thymine as an internal standard. 2 5-fluorouracil was extracted from plasma by a novel procedure which removed plasma components interferring with the sensitivity of the assay. The method included heating the plasma, washing with ether and extracting the drug under optimum conditions. 3 The sensitivity of the assay was 10 ng/ml plasma, sufficient to determine the low concentrations of 5-fluorouracil found in plasma during continuous infusion of the drug in patients receiving chemotherapy for cancer.