ABSTRACT:Apparent intrinsic clearance (CL int,app ) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL int,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL int,app . This could partly be attributed to the differences in the free fraction (f u ). The f u in hepatocyte incubations (f u,hep-inc ) was influenced not only by the free fraction of compounds in the incubation buffer (f u,buffer ) but also by the rate constants of uptake (k up ) and metabolism (k met ). This report provides a new derivation for f u,hep-inc , which can be expressed as, where the C hep , C buffer , V hep , and V buffer represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the f u,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.The determination of in vitro intrinsic clearance (CL int ) for drug candidates in the early discovery stage is a common practice in the pharmaceutical industry (Houston, 1994;Lave et al., 1997;Obach et al., 1997). The CL int values of drug candidates can help to confirm whether metabolism is the main clearance pathway when it is compared with the total body clearance in vivo. It is also helpful in rank-ordering drug candidates based on their metabolic stabilities, assessing species and gender differences in metabolic clearance, and projecting the metabolic clearance of drug candidates in humans. The in vitro CL int may be derived from enzyme kinetic data such as V max /K m (Lin et al., 1996;Tan and Pang, 2001;Griffin and Houston, 2004) or from the in vitro t 1/2 values where subK m substrate concentrations are used (Lave et al., 1997, Obach, 1999Lau et al., 2002;Jones and Houston, 2004). The CL int can be calculated from the experimental apparent intrinsic clearance, CL int,app , by correcting for free fraction of test compounds in the incubations. To further predict the in vivo hepatic clearance from the in vitro intrinsic clearance, a well stirred model is often used (Naritomi et al., 2001;Ito and Houston, 2004). A survey of literature revealed that in hepatocyte incubations, the free fraction of test compound has not been well defined. Simply assuming a steady state where the intracellular free concentration equals the extracellular free concentration may allow one to roughly estimate CL int for some compounds. However, clearance, after a dose in vitro or in vivo, is actually a dynamic system such that at any given time the amount of compound getting into a cell typically equals the amount of compound leaving the cell by diffus...

ABSTRACT:VELCADE (bortezomib, PS-341), reversibly inhibits the 20S proteasome and exhibits cytotoxic and antitumor activities. Pretreatment of cancer cells with bortezomib increases the chemosensitivity of these cells, suggesting that bortezomib may be used in combination chemotherapy. The relative contributions of the five major human cytochromes P450 (P450s), 1A2, 2C9, 2C19, 2D6, and 3A4 (the focus of the present study), to the metabolism of bortezomib are an important aspect of potential drug interactions. Relative activity factor (RAF), chemical inhibition, and immunoinhibition using monoclonal antibodies were three approaches employed to determine the relative contributions of the major human P450s to the net hepatic metabolism of bortezomib. RAFs for the P450 isoform-selective substrates were determined; the ratio of the rate of metabolism of bortezomib with cDNA-expressed P450s versus rate of metabolism with human liver microsomes was normalized with respect to the RAF for each P450 isoform to determine the percentage contributions of the P450s to the net hepatic metabolism of bortezomib. CYP3A4 followed by CYP2C19 were determined to be the major contributors to the metabolism of bortezomib. Chemical inhibition and immunoinhibition confirmed that CYP3A4 and CYP2C19 were the major P450s responsible for the hepatic metabolism of bortezomib. The studies were conducted with 2 M bortezomib, and the disappearance of bortezomib, rather than appearance of a specific metabolite, was quantified to determine the contributions of the P450s to the overall hepatic metabolism of bortezomib in humans.Boronic acids as protease inhibitors were first synthesized in the early 1970s and were demonstrated to act as potent transition state analogs of serine proteases (Koehler and Leinhard, 1971;Philipp and Bender, 1971;Kettner and Shenvi, 1984). Throughout the 1980s, peptide boronic acids were shown to be effective inhibitors of trypsin, chymotrypsin, ␣-lytic protease, pancreatic elastase, leukocyte elastase, thrombin, and ␤-lactamases (Kettner and Shenvi, 1984;Crompton et al., 1988) and have been explored for use as therapeutic agents in various disease states (Snow and Bachovchin, 1995;Groziak, 2001). More recently, peptidyl boronic acids were demonstrated as potent proteasome inhibitors, and antitumor and anti-inflammatory efficacy was observed both in vitro and in animal models (Adams et al., 1998). The approval of VELCADE (bortezomib, PS-341; Fig. 1), a dipeptidyl boronic acid, by the United States Food and Drug Administration in 2003 for the treatment of relapsed refractory multiple myeloma made it the first boronic acid and the first in a new class of drugs, proteasome inhibitors, to be marketed as a therapeutic agent.Bortezomib, an N-pyrazinylcarbonylated derivative of the dipeptide boronic acid Phe-Leu-B(OH) 2 , is a potent, selective, and reversible inhibitor (K i ϳ 0.62 nM) of the 26S proteasome in mammalian cells (Adams et al., 1998). The molecular mechanisms by which bortezomib exerts its effects include inhibition...

The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/- 27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.

Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinibinduced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (K m and k cat ) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 k cat / K m = 6.8 mM 21 min 21 versus CYP3A5 k cat /K m = 1.3 mM 21 min 21). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by k cat (maximum relative GSH adduct levels)/K m (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A-and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation.