Extrapolation of Diclofenac Clearance from in Vitro Microsomal Metabolism Data: Role of Acyl Glucuronidation and Sequential Oxidative Metabolism of the Acyl Glucuronide
- Sanjeev Kumar,
- Koppara Samuel,
- Ramaswamy Subramanian,
- Matthew P. Braun,
- Ralph A. Stearns,
- Shuet-Hing Lee Chiu,
- David C. Evans and
- Thomas A. Baillie
- Dr. Sanjeev Kumar, Department of Drug Metabolism, Merck, P.O. Box 2000, RY80E-200, Rahway, NJ 07065. E-mail: sanjeev_kumar{at}merck.com
Abstract
Diclofenac is eliminated predominantly (∼50%) as its 4′-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (∼50%) and dogs (>80–90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4′-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CLint =Vmax/Km) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4′-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ∼75% of the estimated hepatic CLint of diclofenac is apparently inconsistent with the 4′-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4′-hydroxy diclofenac AG, was observed. The estimated hepatic CLint of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4′-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4′-hydroxylation of diclofenac. These findings may have general implications in the use of total (free + conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4′-hydroxy diclofenac urinary excretion.
Footnotes
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DOI: 10.1124/jpet.102.038992
- Abbreviations:
- P450
- cytochrome P450
- UGT
- UDP glucuronosyltransferase
- ER
- endoplasmic reticulum
- AG
- acyl glucuronide
- UDPGA
- uridine diphosphoglucuronic acid
- LC-MS/MS
- liquid chromatography with tandem mass spectrometry
- HPLC
- high-performance liquid chromatography
- HLM
- human liver microsome
- RT
- retention time
- CID
- collision-induced dissociation
- MS
- mass spectrometer
- B/P
- blood-to-plasma concentration ratio
- CLint
- intrinsic clearance
- CL
- microsomal intrinsic clearance
- CL
- whole liver intrinsic clearance
- CL
- microsomal intrinsic clearance of oxidative metabolism of diclofenac acyl glucuronide
- CL
- whole liver intrinsic clearance of oxidative metabolism of diclofenac acyl glucuronide
- fu
- unbound fraction of the drug in plasma
- Km
- Michaelis constant or substrate concentration required for half-maximal enzyme activity
- Qh
- hepatic blood flow
- Vmax
- maximal velocity of the enzymatic reaction
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- Received May 14, 2002.
- Accepted July 2, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
