A method for assessing dihydrofolate reductase inhibition in vivo |
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| Authors: | L C Mishra A S Parmar J A Mead |
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| Affiliation: | 1. Microbiological Associates, Inc., National Institutes of Health, Bethesda, Maryland 20014 USA;2. National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 USA;1. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA;2. Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA;3. Department of Biostatistics, College of Medicine, University of Florida, Gainesville, FL 32610, USA;4. Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA;5. Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32610, USA;6. Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA;7. UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA;1. National Engineering Research Center for Strategic Drugs, Beijing Institute of Pharmacology and Toxicology Institution, Beijing, 100850, China;2. National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing, 102206, China;1. Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK;2. Protein Degradation Group, Medicines Research Centre, GSK, Gunnels Wood Road, Stevenage, UK;3. Chemical Biology, Medicines Research Centre, GSK, Gunnels Wood Road, Stevenage, UK |
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| Abstract: | A method for studying inhibition of dihydrofolate reductase activity in vivo has been described. Mice were given 500 μCi (25 mg) 3H-H2F/kg intraperitoneally, the tetrahydrofolate (H4F) formed in liver and gut were separated by DEAE-cellulose column chromatography of tissue homogenates, and the radioactivity in peaks corresponding to H4F and H2F was determined. A linear relationship was observed in the inhibition of H2F-reductase by methotrexate in both liver and intestine when the mice were treated with methotrexate 1 hr before H2F injection and the tissues were assayed 2 hr later. The recovery of enzyme activity from inhibition by methotrexate in gut and liver was biphasic: a rapid recovery (45% in gut and 72% in liver) within 6 hr was followed by a slower recovery (92% in gut and 98% in liver) in 2 to 3 days. The data presented indicate greater inhibition and slower recovery of H2F-reductase in intestine than in liver and illustrate the feasibility of assessing H2F reductase activity in vivo. |
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