Abstract
The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Qformation complied with Michaelis-Menten kinetics (mean values ofVmax and Km: 74.4 nmol/mg/h and 74.2 μM, respectively).Q-N-OX formation followed two-site kinetics with mean values of Vmax,Km andVmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 μM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OXformations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q orQ-N-OX formation, withKi values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q andQ-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OXformation, but CYP2C9 and 2E1 also catalyzed minor proportions of theN-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.
Footnotes
-
Send reprint requests to: Torben Leo Nielsen, Department of Clinical Pharmacology, Institute of Medical Biology, Odense University, Winsløwparken 19, DK-5000 Odense C, Denmark. E-mail:t.nielsen{at}winsloew.ou.dk
- Abbreviations:
- CYP
- cytochrome P-450
- HL
- human liver
- 3-OH-Q
- (3S)-3-hydroxyquinidine
- Q-N-OX
- quinidine N-oxide
- 4-CPBG
- 4-chlorophenylbiguanide
- [S]
- concentration of substrate
- [I]
- concentration of inhibitor
- IC50
- inhibitor concentration causing a 50% reduction in velocity of a reaction
- n
- Hill coefficient describing sigmoidicity of cooperative binding of an inhibitor
- rs
- correlation coefficient using Spearman’s rank correlation
- Received May 5, 1998.
- Accepted October 22, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
