Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.

Ocular examination in nine-month-old infants with very low birthweights.

In the period 1980-1987 ocular examinations were performed on 185 infants with a very low birthweight (less than 1500 g) at the age of nine months corrected for the duration of pregnancy. The mean gestational age of the infants was 30 weeks, while the mean birthweight was 1160 g. The mean spherical refraction was 0.9 D (S.D. 1.3 D), the mean astigmatism was C-0.6 D (S.D. 1.0 D), while convergent strabismus was found in 10% of the patients. The purpose of the investigation was to see if there was a relation between spherical equivalent of refraction, astigmatism and strabismus on the one hand and 11 perinatal parameters and cerebral palsy diagnosed at the age of nine months on the other hand. Statistical analysis was performed with the Student t-test. No significant correlation between the findings of ocular examination and the perinatal parameters could be detected.

Pharmacodynamic dependent disposition of the angiotensin converting enzyme inhibitor, libenzapril.

Libenzapril, an angiotensin converting enzyme inhibitor, was administered to healthy male volunteers in a randomized, two-phase pharmacokinetic study. One phase compared the pharmacokinetics of a 4 mg intravenous infusion and 20 mg oral solution, and the other phase provided two additional intravenous infusions of 1.7 and 12 mg for comparison. The intravenous model-independent pharmacokinetic parameters MRTiv, Vss, CL, and CLr all exhibited dose dependence. The concentration dependent renal clearance was maximal at 83 mL/min and minimal at 32 mL/min following intravenous administration. The mechanism of libenzapril's self-inducible clearance appears to have a pharmacodynamic basis. The absolute bioavailability was estimated at less than 10% and the renal clearance following oral administration exhibited additional route dependency.

Cleavage of synthetic peptides by purified poliovirus 3C proteinase.

Synthetic peptides, 14-16 residues in length, were used as substrates for purified recombinant poliovirus proteinase 3C. The sequences of the substrates correspond to the sequences of authentic cleavage sites in the poliovirus polyprotein, all of which contain Gln-Gly at the scissile bond. Specificity of cleavages was demonstrated by analysis of 3C digests of synthetic peptides. Relative rate constants for the cleavages were derived by competition experiments. The rate constants roughly correlated with the estimated half-life of the homologous precursor proteins detected in poliovirus-infected cells. The peptide most resistant to cleavage corresponded to the 3C/3D junction, a site known to be cleaved very slowly by 3C in vivo. Substitution of threonine for alanine in P4 position of this peptide, however, resulted in significant cleavage. This observation supports the hypothesis that the residue in P4 position, in addition to the Gln-Gly in P1 and P1', respectively, contributes to substrate recognition. Ac-Gln-Gly-NH2 was not a substrate for 3C.

Effects of food on the bioavailability of CGS 16617, an angiotensin-converting enzyme inhibitor, in healthy subjects.

CGS 16617, a direct-acting angiotensin-converting-enzyme inhibitor, was administered as a single dose of 20 mg in aqueous solution to 12 healthy male volunteers on two occasions in a randomized, cross-over design study. On one occasion, the dose was administered after an overnight fast; on the other occasion, it was administered after subjects ate a standard breakfast. Administration of CGS 16617 after food was associated with statistically significant decreases in peak plasma concentrations (58%) and areas under the plasma concentration-time curves (23%) compared with drug administration in the fasted state. Also, the time to peak plasma concentration was increased (57%) in a statistically significant manner when CGS 16617 was administered after food. Thus, the ingestion of food decreased both the rate and extent of absorption of this drug, but the mechanism of the interaction is unknown at present.

Direct determination of angiotensin-converting enzyme inhibitors in plasma by radioenzymatic assay.

We describe a simple, rapid, specific radioenzymatic assay for "CGS 16617," a new, potent inhibitor of angiotensin-converting enzyme (ACE; EC 3.4.15.1) in human plasma. This assay is based on the principle that the inhibitor (i.e., the drug) binds to the ACE in plasma and hence the amount of free ACE in plasma is inversely related to the amount of active inhibitor present. Free enzyme is reacted with a radiolabeled substrate, and the radioactive product is selectively extracted into the scintillation cocktail for quantification. Fivefold-diluted plasma samples are incubated with [3H]hippuryl-glycyl-glycine enzyme substrate at 37 degrees C for 30 min and the liberated [3H]hippuric acid is selectively extracted into scintillation cocktail. The radioactivity is counted in a liquid scintillation counter. Both within-run and between-run, the variability (CV) of the assay is less than 10%. As little as 200 ng of the drug per liter can be quantified in 50-microL plasma samples. The method can also be used to assay two other ACE inhibitors, pentopril and CGS 14831, demonstrating that the method can be readily adapted to any ACE inhibitor having a single active component in plasma. The ester prodrug pentopril can also be assayed after ester hydrolysis. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the concentrations of active ACE inhibitors in blood.