Development and implementation of an electrochemiluminescence immunoassay for the determination of an angiogenic polypeptide in dog and rat plasma.

A quantitative method based on electrochemiluminescence immunoassay for the determination of the angiogenic agent aFGF-S117 has been developed and validated. Two polyclonal antibodies specific to aFGF-S117 and a wild-type aFGF antibody were selected for the analysis. The assay was based on the non-competitive sandwich immunoassay principle in which the drug is trapped with a biotinylated antibody that is immobilized on a streptavidin magnetic particle. The drug is then sandwiched with a ruthenium chelated second antibody. The assay demonstrates good accuracy and reproducibility at plasma concentration of 0.5 ng/ml.

Determination of an echinocandin, MK-0991, in mammalian plasma by radioimmunoassay.

A quantitative method based on radioimmunoassay for the determination of the antifungal agent, CANCIDAS (MK-0991) has been developed and validated. The immunogen was prepared by coupling MK-0991 to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to MK-0991 was selected for RIA. The assay was based on the competitive immunoassay principle in which the drug competes with iodinated drug for a limited quantity of specific antibody. The bound tracer was separated via goat anti-rabbit globulin. The assay demonstrates good accuracy and reproducibility at plasma concentration down to 10 ng/ml. The specificity of the RIA method was confirmed by cross-validating against an established HPLC method.

Enalapril in RAPIDISC (wafer formulation): pharmacokinetic evaluation of a novel, convenient formulation.

BACKGROUND: Enalapril in RAPIDISC* (wafer), a new easy-to-administer formulation of enalapril, may improve the convenience of enalapril therapy, thereby helping patients adhere to antihypertensive treatment. SUBJECTS AND METHODS: To determine whether 20 mg enalapril wafer is bioequivalent to the conventional 20 mg enalapril tablet, an open-label, two-period crossover study was performed in 16 healthy male volunteers. Cumulative urinary recovery of free enalaprilat (active metabolite of enalapril) and the serum maximum concentration of free enalaprilat (Cmax) were the primary pharmacokinetic parameters used to determine bioequivalence in this study. Bioequivalence was defined as the geometric mean ratio (wafer: tablet) falling within the equivalence limits of 0.80 to 1.25 for both parameters. RESULTS: Cumulative urinary recovery of free enalaprilat (0 - 72 hours) was similar between the wafer and conventional tablet formulations (arithmetic mean 5.13 vs. 5.03 mg, about 36% of dose). The geometric mean ratio of the urinary recovery of free enalaprilat (wafer: tablet) was 1.03 (90% CI: 0.93, 1.15). Cmax of serum enalaprilat was also similar between the wafer and conventional tablet formulations (arithmetic mean 85.7 vs. 76.3 ng/ml). The geometric mean Cmax ratio (wafer: tablet) was 1. 10 (90% CI: 1.00, 1.22). Both enalapril formulations were well tolerated. CONCLUSION: This study demonstrates that 20 mg enalapril in RAPIDISC is bioequivalent to 20 mg enalapril conventional tablet.

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).

The development and cross-validation of methods based on radioimmunoassay and LC/MS-MS for the quantification of the class III antiarrhythmic agent, MK-0499, in human plasma and urine.

An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired student's t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.

Determination of MK-383, a non-peptide fibrinogen receptor antagonist, in human plasma and urine by radioimmunoassay.

MK-383 is a novel, non-peptide fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via the N-hydroxysuccinimide ester from which the radioligand was also prepared by reaction with [I125]iodotyrosine. The method was specific and no immunoreactive material other than the parent drug was detectable in plasma and urine from dosed volunteers. This direct assay, using 5 microliters of plasma or 0.5 microliter of urine, is sensitive to 1 and 10 ng ml-1, respectively, without matrix interference and has sufficient sensitivity, specificity, accuracy, and precision for the analysis of clinical samples.

Disposition of MK-852, a fibrinogen receptor antagonist, in rats and dogs.

MK-852, an antagonist of the platelet fibrinogen receptor GPIIb/IIIa, is the cyclic disulfide N-acetyl-cys-asn-(5,5-dimethyl-4-thiazolidine-carbonyl)- (4-aminomethyl-phe)-gly-asp-cys, monoacetate (all L-amino acids). Radiolabeled MK-852 was synthesized with either a 3H label in the N-acetyl group of the cystine residue or a 14C label in the aminomethyl group. Plasma concentrations of unchanged MK-852 in five rats declined with a mean terminal half-life of 0.92 hr after a 2.5 mg/kg i.v. dose of MK-852; plasma clearance and Vd were 23.1 ml/min/kg and 1.81 liters/kg, respectively. More label was excreted in urine (74-76%) than in feces (7-15%) when either [3H]MK-852 or [14C]MK-852 was given intravenously to groups of four rats (2.5 mg/kg). High concentrations of 3H in rat kidney were consistent with high renal clearance of MK-852, and MK-852 accounted for virtually all of the urinary 3H (and 14C) label. Following a 0.6 mg/kg i.v. dose, the half-life, plasma clearance, and Vd of MK-852 in four dogs were 0.84 hr, 3.93 ml/min/kg, and 0.28 liters/kg, respectively. In dogs, the excretion patterns of radioactivity were similar to those of rats, except that 14C urinary recoveries (79%) were higher than 3H (63%). Unchanged MK-852 represented essentially all of the urinary 3H label. Fractionation of dog 14C urinary radioactivity yielded one major and several minor polar labeled species. The major species was unchanged [14C]MK-852 (quantitated by radioimmunoassay as approximately 80% of the label).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of MK-852, a new fibrinogen receptor antagonist, in plasma and urine by radioimmunoassay.

MK-852 is a novel fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via a dinitrophenylene bridge and the radioligand by reaction of the drug with the 125I-labelled Bolton-Hunter reagent. The method was specific and no immunoreactive material other than parent drug was detectable in plasma from dosed volunteers. The direct assay using 0.05 ml of plasma is sensitive to 0.2 ng ml-1 without matrix interference and has sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples. The lower quantifiable limit in (diluted) urine is 50 ng ml-1.

Determination of L-365,260, a new cholecystokinin receptor (CCK-B) antagonist, in plasma by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

L-365,260 is a novel cholecystokinin receptor antagonist. A sensitive and specific liquid chromatographic/mass spectrometric assay has been developed for the determination of the drug in plasma using the CD3-labeled species as the internal standard. Plasma extracts were separated on a 3 cm C18 reverse-phase high-performance liquid chromatography column. The column eluate passed, by means of a heated nebulizer interface, into a corona discharge atmospheric chemical ionization source. The mass spectrometer was operated in the positive ion tandem mass spectrometric mode. The method has sufficient sensitivity, specificity, precision, accuracy and selectivity for the determination of drug concentrations in clinical samples. The chromatographic run time is less than 2 min.