Abstract
Proflavine (3,6-diaminoacridine) has potential for use as an antiinfective in fish, and its metabolism by rainbow trout was therefore studied. Fourteen hours after intraarterial bolus administration of 10 mg/kg of proflavine, three metabolites were found in liver and bile, and one metabolite was found in plasma using reversed-phase HPLC with UV detection at 262 nm. Treatment with hydrochloric acid converted the three metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with β-glucuronidase and saccharic acid 1,4-lactone, a specific β-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination of UV-VIS absorption and mass spectra, HPLC-purified metabolites were isolated from liver. Data from these experiments suggested that the proflavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuronosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. When synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix-assisted, laser desorption ionization, time-of-flight MS. In addition, they coeluted on HPLC under different mobile phase conditions. Finally, the in vitro incubation with liver subcellular preparations confirmed this characterization and provided the evidence that APG can be formed by glucuronidation of AP or acetylation of PG.
Footnotes
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Send reprint requests to: Dr. William L. Hayton, Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, the Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291.
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This work was supported by Contract 233-93-7014 from the U.S. Food and Drug Administration, Department of Health and Human Services. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
- Abbreviations used are::
- GA
- glucuronic acid
- TOF-MS
- time-of-flight mass spectrometry
- SPE
- solid-phase extraction
- CoA
- coenzyme A
- BCA
- bicinchoninic acid
- AP
- acetylproflavine
- PG
- proflavine glucuronide
- APG
- acetylproflavine glucuronide
- Met I
- metabolite I
- Met II
- metaboliteII
- Met III
- metabolite III
- Received August 2, 1996.
- Accepted January 3, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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