This article is available online at http://dmd.aspetjournals.org ABSTRACT:We investigated hepatic in vitro intrinsic clearance (CL int,in vitro ) in freshly isolated or cryopreserved hepatocytes and compared with CL int,in vivo by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDPglucuronosyltransferase, sulfotransferase, and/or esterase. CL int,in vitro was determined from substrate disappearance rate at 1 M in hepatocytes. CL int,in vivo was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL int,in vitro in freshly isolated hepatocytes, the rat scaling factor values (CL int,in vivo /CL int,in vitro ) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL int,in vitro values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL int,in vitro were determined by use of cryopreserved hepatocytes. When human CL int,in vitro was regarded as the predicted CL int,in vivo , the observed and predicted CL int,in vivo for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL int,in vitro corrected with the rat scaling factors yielded better predictions of CL int,in vivo that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.Recently, pharmacokinetic investigation has played an increasingly important role in drug discovery. In particular, it is very important to predict human hepatic metabolic clearance because most drugs are eliminated from the body predominantly by hepatic metabolism. For predicting hepatic clearance, theoretical aspects of in vitro/in vivo scaling based on a physiological model and clearance concepts have been developed (Roberts and Rowland, 1986). Application of this method has been successful in predicting in vivo hepatic clearance in rats for many drugs metabolized by P450 1 from in vitro metabolism data using rat liver microsomes and isolated hepatocytes (Sugiyama et al., 1988;Houston and Carlile, 1997). Because human liver samples have become more readily available, it would also be very useful to predict in vivo outcomes from in vitro data in humans. However, there has been relatively limited application of this approach (Hoener, 1994), and there have been some failed attempts at the prediction of human hepatic clearance. For example, Iwatsubo et al. (1997) and Houston and Carlile (1997) reported that CL int,in vitro generally exhibited a positive correlation with CL int,in vivo , but in some cases animal or human clearance values were not well predicted from in vitro studies. To improve the predict...
1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug. 2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CL(int)) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively. 3. CL(int) of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL(int) of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CL(int) of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4. 4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.
) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type 1 MMP (MT1-MMP/MMP-14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP-14, on a rat collagen-induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti-IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg À1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT-MMP, gelatinases are also involved in joint destruction in arthritis.
1. We used chimeric mice (PXB mice®), which were repopulated with human hepatocytes, to evaluate their predictabilities of human pharmacokinetics. 2. The relationships of total clearance (CLt) and the volume of distribution at steady state (Vdss) between that predicted from single-species allometric scaling (SSS) of PXB mice and the observed human values indicated good correlations for various drugs metabolized by cytochrome P450s (CYPs) and non-CYPs. 3. We examined the Dedrick plot with which the plasma concentration-time curves can exhibit superimposability using SSS of PXB mice for CLt and Vdss. The predicted plasma concentration-time curves using the complex Dedrick plot from PXB mice were generally superimposed with the observed human data. 4. However, the predicted curve of diazepam was not superimposable with the observed profile. Residual mouse hepatocytes in the livers of PXB mice may affect predictability of CLt of diazepam because significant discrepancy of in vitro intrinsic clearance in PXB mouse liver microsomes consisted of low and high replacement of human hepatocytes were observed. 5. The complex Dedrick plot with SSS from PXB mice is useful for predicting the plasma concentration-time curve in drug discovery, although there are some limitations.
In this study, a simple in vitro method for detecting human P450 (CYP) quasi-irreversible and irreversible inhibitors was evaluated. For the method, cDNA-expressed CYPs were applied to microtiter plate assays, CYP inhibitors were co-incubated with fluorometric substrates, and IC(50) were continuously measured (without stopping enzyme reactions). The typical reversible inhibitors (sulfaphenazole, tranylcypromine, quinidine, ketoconazole) showed constant IC(50) throughout the reaction. In contrast, the typical quasi-irrversible inhibitors (isosafrole, erythromycin, troleandomycin, diltiazem) and the typical irreversible inhibitors (furafylline, propranolol, mifepristone) showed time-dependent decreases in IC(50). For CYP3A4 inhibition studies, two substrates, 7-benzyloxyresorufin (BzRes) and 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), were used. The IC(50) of the CYP3A4 inhibitors were dependent on the substrate. However, the quasi-irreversible and irreversible inhibitors could be detected by examining changes in the IC(50), regardless of the substrate. Further, the detection method was applied to josamycin and bergamottin. Josamycin did not show definite time-dependent decreases in IC(50) for CYP 3A4, suggesting that josamycin is neither a quasi-irrversible nor an irreversible inhibitor of CYP3A4. On the other hand, bergamottin showed time-dependent decreases in IC(50) for CYP1A2, CYP 2C9, CYP 2C19, CYP 2D6 and CYP 3A4, suggesting that bergamottin is a quasi-irrversible or an irreversible inhibitor of the 5 CYP isoforms. This method provides more rapid and reliable detection of quasi-irreversible and irreversible inhibitors and may be useful in drug discovery.
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