Date of Award:

5-1-1979

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Life Sciences:Biology

Committee Chair(s)

Rex S. Spendlove

Committee

Rex S. Spendlove

Committee

William B. Barnett

Committee

Raghubir P. Sharma

Abstract

The minimum viral-inhibitory concentration (MIC) of ribavirin (1-ß-D-ribofuranosy1-1,2,4-triazole-3-carboxamide) was determined in the presence of human serum and urine for use as a bioassay for the drug. Since ribavirin's antiviral activity varies with the cell line and virus used, the sensitivity of four viruses to ribavirin dissolved in Eagle's medium containing serum or urine was evaluated using at least two different cell lines for each virus. The viruses studied included type 3 parainfluenza, type 1 herpes, type 1 reovirus, and measles. To eliminate the viral inhibitory effects of measles anti- body, the serum was filtered through immersible molecular separators (Millipore). Reovirus was assayed using immunofluorescence; the other viruses were studied using cytopathic effect inhibition. Initial cell tolerance to the urine- or serum-containing medium was determined: MDBK, MDCK, MA104, MK2 and BS-C-1 cells tolerated 10% urine and 50% serum, whereas toxic effects were seen with KB and HeLa cells at much lower concentrations. The MIC of the drug in serum or urine against parainfluenza and herpes viruses in MA104 and BS-C-1 cells was 0.32-1.0 μg/ml; reovirus inhibition in MDBK and MDCK cells occurred at 3.2 μg/ml in serum and 0.32 μg/ml in urine. Measles in BS-C-1 cells was the most sensitive virus studies, with an MIC of 0.0032 μg/ml seen in repeated experiments using 10 percent urine. With both measles and reovirus, a lower MIC was observed in urine compared with serum. Urine was therefore added to serum samples for subsequent bioassay of unknown fractions containing ribavirin. Using measles virus and BS-C-1 cells for biassay, serum and urine ribavirin levels were determined in rats receiving doses of 1000 mg/kg and 100 mg/kg. In the 1000 mg/kg study, peak serum levels of 24 μg/ml were observed at 2 hours. Maximum urine concentrations of 640-700 μg/ml were measured between 8 and 12 hours. In the 100 mg/kg study, peak serum levels of 20 μg/ml were evident at 1 hour, and peak urine levels (64 μg/ml) were observed between 8 and 12 hours. Considering maximum concentrations of rat serum (filtered) and urine on cells, the bioassay was sensitive to 0.0064 μ g/ml for serum and 0.064 μg/ml for urine.

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