Abstract
Diltiazem (DTZ) N-demethylation occurs by cytochrome P-450 (CYP) 3A based on the following observations: 1) a single enzyme Michaelis-Menten model of metabolite formation, 2) high correlations of DTZ N-demethylation activity to other CYP3A activities, 3) inhibition of DTZ N-demethylation activity by triacetyloleandomycin, and 4) DTZ N-demethylation activity by expressed CYP3A enzymes only. The meanKms for DTZ N-demethylation in human liver microsomes and expressed CYP3A4(+b5) were 53 and 16 μM, respectively. A 30-min preincubation of DTZ in expressed CYPs inhibited CYP3A4(+b5) by 100%, of which 55% was due to formation of a metabolite intermediate complex (MIC), which is an inactive form of CYP. MIC was observed in human liver microsomes and cDNA-expressed CYP3A only. In experiments to assess simultaneous MIC formation and loss of CYP3A activity, DTZ caused greater than 80% inhibition of midazolam hydroxylation after a 60-min preincubation in human liver microsomes. The rate constants for MIC formation and loss of midazolam hydroxylation activity were equivalent for the line of best fit for both data sets, which illustrates that MIC formation causes the inhibition of CYP3A activity. The mechanistic inhibition was characterized in expressed CYP3A4(+b5), which exhibited a concentration-dependent formation of MIC by DTZ (1–100 μM) with an estimated kinact of 0.17 min−1 and KI of 2.2 μM. The partition ratio for expressed CYP3A4(+b5) was substrate concentration dependent and varied from 13 to 86. This study showed that DTZ inhibition of CYP3A substrate metabolism occurs primarily by MIC formation.
Footnotes
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Send reprint requests to: David R. Jones, Ph.D., Indiana University School of Medicine, Division of Clinical Pharmacology, Wishard Memorial Hospital, OPW 320, 1001 W. 10th St., Indianapolis, IN 46202. E-mail: drjones1{at}iupui.edu
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↵1 This work was supported in part by a grant (AG 13718) from the National Institutes of Health (Bethesda, MD).
- Abbreviations:
- DTZ
- diltiazem
- α-NF
- α-naphthoflavone
- AUC
- area under the plasma concentration time curve
- Clint
- intrinsic clearance
- CYP
- cytochrome P-450
- kcat
- rate constant for catalysis
- kinact
- rate constant for inactivation
- Ki
- dissociation constant for reversible inhibition
- KI
- inactivator concentration that produces half the maximal rate of inactivation
- λ
- pseudo first-order rate constant
- MA
- N-desmethyl diltiazem
- MIC
- metabolite intermediate complex
- MICmax
- maximum metabolite intermediate complex
- r
- partition ratio
- Vmax
- maximum velocity of metabolite formation
- Received December 7, 1998.
- Accepted May 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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