Studies with Induced Livers Involving Diazepam
Abstract
Microsomal protein recovery and hepatocellularity have been determined and investigated as scaling factors for interrelating clearance by hepatic microsomes, freshly isolated hepatocytes and whole liver from untreated (UT) rats and rats treated with either the cytochrome P450 inducer phenobarbital (PB) or dexamethasone (DEX). Hepatocellularity in UT rats (1.1 × 108 hepatocytes/g liver) was not significantly different after either PB or DEX induction (1.1 and 1.3 × 108 hepatocytes/g liver, respectively). However the microsomal protein recovery index, which provides a scaling factor that is inversely related to the efficiency of the microsomal preparation procedure, was 47 mg/g liver in both PB and DEX microsomes and differs from UT rats (60 mg/g liver). These contrasting findings are consistent with the interlaboratory trends in the literature, indicating that, although hepatocellularity estimates are in good accord, microsomal recovery can vary 2-fold; this has implications for scaling.
The oxidation of diazepam to its three primary metabolites was measured in PB and DEX microsomes and hepatocytes and the scaling factors were applied to these data and previously reported UT data. Marked changes in kinetics occur on induction resulting in a shift in the major pathway. In particular, 3-hydroxylation is induced over 20-fold by DEX. Diazepam CLint was determined in vivo after administration of a bolus dose into the hepatic portal vein of UT, PB, and DEX rats; values of 127, 191, and 323 ml/min/SRW (where SRW is a standard rat weight of 250 g), respectively, were obtained. Using these scaling factors, the hepatocyte predictions ofCLint were excellent (99, 144, and 297 ml/min/SRW for UT, PB, and DEX, respectively), whereas only the DEX prediction (248 ml/min/SRW) was accurate for the microsomal system, with a substantial underprediction for UT and PB (46 and 68 ml/min/SRW, respectively). Evidence is presented for product inhibition, resulting from accumulation of primary metabolites within the microsomal preparation, as the mechanism responsible for this underprediction.
These results illustrate that the scaling factor approach is applicable to induced livers in which both cytochrome P450 complement and zonal distribution are altered. These data, together with our previous studies, demonstrate that CLint in cells (2.4–297 ml/min/SRW), microsomes (2.7–248 ml/min/SRW), and in vivo (1.5–323 ml/min/SRW) are related in a linear fashion and hence inherently both in vitro systems are of equal value in predicting in vivo CLint.
Footnotes
-
Send reprint requests to: Dr. J. Brian Houston, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK.
-
↵1 Present address: Department of Anesthesia, Stanford University, Stanford, CA.
-
This study was supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Research Laboratories. A portion of this study was presented at the meeting of the British Pharmacological Society, April 5–7, 1995, Canterbury, UK, and appeared in abstract form in Br. J. Clin. Pharmacol. 40,181P (1995).
- Abbreviations used are::
- PB
- phenobarbital
- DEX
- dexamethasone
- UT
- untreated
- CYP
- cytochrome P450
- DZ
- diazepam
- 4′-HDZ
- 4′-hydroxydiazepam
- 3-HDZ
- 3-hydroxydiazepam
- NDZ
- nordiazepam
- SRW
- standard rat weight
- 3-HNDZ
- 3-hydroxynordiazepam
- CLint
- intrinsic clearance
- MRT
- mean residence time
- CL
- clearance
- VSS
- volume of distribution at steady-state
- CLIP
- intraportal clearance
- B/P
- blood:plasma ratio
- fu
- fraction of unbound drug concentration
- Received May 22, 1996.
- Accepted April 30, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
DMD 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.
|