Abstract
The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritonavir was determined over a range of concentrations (0.02–20 μM) in both rat liver microsomes and fresh isolated rat hepatocytes in suspension. Clearance values were found to be concentration dependent for both systems, and at low concentrations, microsomal clearance was much greater (7–14-fold) than in hepatocytes. Kinetic parameters showed substantially lower microsomal Km values (5–42 nM) compared with suspended rat hepatocytes (34–270 nM) but similar scaled Vmax values (2–26 nmol/min/g liver). In the absence of metabolism (achieved by pretreating hepatocytes with a mechanism-based inhibitor of cytochrome P450), saquinavir, nelfinavir, and ritonavir were actively and rapidly taken up into hepatocytes (cell/medium concentration ratios of 306-3352), and intracellular unbound drug concentrations between 5- and 12-fold higher than extracellular unbound concentrations were achieved. Comparison of the rate of uptake into hepatocytes with the rate of metabolism in hepatocytes and microsomes indicates that the former is the rate-limiting step at low concentrations. The rate of metabolism saturates at lower concentrations (100–400-fold) than the rate of uptake; hence, at the high concentrations metabolic rate-limited clearance occurs. In conclusion, the clearance of saquinavir, nelfinavir, and ritonavir is extremely rapid, and it is proposed that in the case of hepatocytes and by inference in vivo, the rate of uptake limits the metabolic clearance of these three drugs.
Footnotes
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A.J.P. was financially supported by a Biotechnology and Biological Sciences Research Council Collaborative Award in Science and Engineering (CASE) studentship with Roche Products Limited.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.020917.
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ABBREVIATIONS: PI, protease inhibitor; BSA, bovine serum albumin; Kp, tissue-to-medium total drug concentration ratio; fu, fraction(s) of unbound drug; LC, liquid chromatography; M1, 3′methoxy-4′hydroxynelfinavir); CLint, intrinsic clearance; Perm, linear permeability parameter; Kpu, tissue-to-medium unbound drug concentration ratio; M8, nelfinavir hydroxy-t-butamide; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; OATP, organic anion transporter polypeptide; P-gp, P-glycoprotein.
- Received February 7, 2008.
- Accepted April 17, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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