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Inhibition of cytochromes P-450 (CYP) is a principal mechanism for metabolism-based drug interactions. In vitro methods for quantitatively measuring the extent of CYP inhibition are well-established. Classical methods use drug molecules as substrates and HPLC-based analysis. However, methodologies, which do not require HPLC separations for data acquisition generally offer higher throughputs and lower costs. Multiwell plate-based, direct, fluorometric assays for the activities of the five principal drug-metabolizing enzymes are available and parameters for the use of these substrates to measure CYP inhibition have been established. This methodology is quantitative, rapid, reproducible, and compatible with common high throughput screening instrumentation. This article describes approaches to establishing this methodology in a drug-discovery support program.
online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11274899&dopt=Abstract
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Biochemistry. 2001 Oct 9;40(40):12112-22.
Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19.
Ha-Duong NT, Dijols S, Macherey AC, Goldstein JA, Dansette PM, Mansuy D.
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Paris V, 45 Rue des Saints-Peres, 75270 Paris Cedex 06, France.
Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibitor of CYP 2C19. The present studies demonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily. UV-visible studies on the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K(s) value of 2.8 +/- 1 microM. Derivatives that do not involve either the o-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did not lead to such spectral interactions and failed to inhibit CYP 2C19. Ticlopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V(max) = 13 +/- 2 and 0.4 +/- 0.1 min(-1)). During this oxidation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This process meets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation. It occurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidine metabolite(s) to proteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site. The effects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivation of CYP 2C9 by tienilic acid. This suggests that a similar electrophilic intermediate, possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively. The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K(I) = 87 microM, k(inact)/K(I) = 37 L.mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitor although somewhat less efficient than tienilic acid for CYP 2C9. Importantly, ticlopidine is the first selective mechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.
online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11580286&dopt=Abstract
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Gastroenterology. 1991 Sep;101(3):650-6.
Observations on a proposed measure of genotoxicity in rat gastric mucosa.
Holt S, Zhu ZH, Powers RE.
Department of Internal Medicine, University of South Carolina School of Medicine, Columbia.
Current methods for the study of the toxicological effects of antisecretory medications on the gastric mucosa possess disadvantages or limitations. A novel assay has been proposed to assess gastric mucosal genotoxicity in which the proton-pump inhibitor omeprazole has been reported to induce direct damage to cellular DNA, raising questions about the safety of this drug. To define the applicability of this proposed measure of genotoxicity and to examine the effects of omeprazole in this assay, control agents, known carcinogens, and omeprazole in various doses and formulations were administered to rats by gavage, followed by [3H]thymidine labeling of DNA in vivo approximately 14 hours later. The incorporation of the [3H]thymidine label into DNA of gastric mucosal cells liberated by limited pronase digestion was in close agreement with published results for negative and positive controls. Omeprazole, administered in doses ranging from 10 mg/kg to 300 mg/kg, did not increase [3H]thymidine incorporation into cellular DNA in this assay. The gastric carcinogen 1-methyl-2-nitro-1-nitrosoguanidine at 20 and 50 mg/kg increased [3H]thymidine incorporation. Pretreatment in vivo with hydroxyurea before [3H]thymidine labeling to inhibit replicative DNA synthesis suppressed [3H]thymidine incorporation more than 97% in negative controls and MNNG and more than 93% in omeprazole treatments. This indicates that replicative DNA synthesis was almost totally responsible for the [3H]thymidine incorporation and the contribution of unscheduled DNA synthesis to the total [3H]thymidine incorporation is minor. Flow cytometric analysis of the cell cycle of the gastric mucosal cells liberated by the limited pronase digestion indicated significant contamination of the preparation with dividing cells (4% in negative controls and 14% in MNNG-treated positive controls). These findings indicate that the proposed screening assay for genotoxicity in rat gastric mucosa is not a reliable measure of unscheduled DNA synthesis in its present form, and conclusions about genotoxic effects of any drug using this assay as initially proposed appear questionable.
online pharmacy ref source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1860629&dopt=Abstract
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