CDNA-expressed CYPs

Crespi, C. L., Greenblatt, D. J., Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes ratios of accessory proteins as sources of discrepancies between the approaches, Drug Metab. Dispos. 2000, 28, 1493-1504. 

A number of different approaches, have been designed to characterize the metabolic clearance of compounds, namely antibody inhibition, correlation analysis, chemical inhibitors and cDNA-expressed CYPs. As expected the science underlying these tools has developed over the past two decades reducing the need to use several approaches simultaneously to make unequivocal conclusions regarding the CYP isoform responsible for the metabolism of a drug of interest. For the purposes of the current chapter, only the latter two are discussed. The readers are referred to recent reviews that describe in detail the science behind antibody inhibition and correlation analysis [87-89]. 

A recent paper by Chauret et al. described the discovery of a novel fluorescent probe that is selectively metabolized by CYP3A in human liver microsomes (32). This probe, DFB [3-[(3,4-difhiorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl) phenyl] furan-2(5F/)-one], is metabolized to DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5//)-one], which has fluorescent characteristics (Fig. 7). In vitro CYP reaction phenotyping studies (cDNA-expressed CYP proteins and immunoinhibition experiments with highly selective anti-CYP3A4 antibodies) demonstrated that DFB was metabolized primarily by CYP3A4 (Fig. 8). Furthermore, metabolism studies performed with human liver microsomes obtained from different donors indicated that DFB dealkylation and testosterone 6P-hydroxylation correlated well (Fig. 9). 

Over the past decade there has been a substantial improvement in the ability to predict metabolism-based in vivo drug interactions from kinetic data obtained in vitro. This advance has been most evident for interactions that occur at the level of cytochrome P450 (CYP)-catalyzed oxidation and reflects the availability of human tissue samples, cDNA-expressed CYPs, and well-defined substrates and inhibitors of individual enzymes. The most common paradigm in the prediction of in vivo drug interactions has been first to determine the enzyme selectivity of a suspected inhibitor and subsequently to estimate the constant that quantifies the potency of reversible inhibition in vitro. This approach has been successful in identifying clinically important potent competitive inhibitors, such as quinidine, fluoxetine, and itraconazole. However, there is a continuing concern that a number of well-established and clinically important CYP-mediated drug interactions are not predictable from the classical approach that assumes reversible mechanisms of inhibition are ubiquitous. 

Direct evidence that irreversible inhibition is the principle mechanism underlying in vivo drug-drug interactions (DDIs) is often lacking because of the requirement for either direct tissue sampling to reveal inactivated enzyme or in vivo inhibition of activity after drug is essentially eliminated from the body. Nevertheless the steady-state plasma concentrations of several clinically important CYP inhibitors are well below the in vitro estimated competitive inhibition constant, Kv This suggests that competitive inhibition is unlikely to occur in vivo, yet these compounds inhibit CYP activity in a time and concentration-dependant manner when cDNA-expressed CYPs or HLMs are used as an enzyme... 

Metabolism by purified or recombinant (cDNA-expressed) human CYP enzymes, which can establish whether a particular CYP enzyme can or cannot metabolize a drug candidate, but it does not address whether that CYP enzyme contributes substantially to reactions catalyzed by human liver microsomes. The information obtained with purified or recombinant human CYP enzymes can be improved by taking into account large differences in the extent to which the individual CYP enzymes are expressed in human liver microsomes, which is summarized in Table 8, and by determining the... 

Kobayashi, K., K. Urashima, N. Shimada, and K. Chiba (2002). Substrate specificity for rat cytochrome P450 (CYP) isoforms Screening with cDNA-expressed systems of the rat. Biochem. Pharmacol. 63, 889-896. 

CYP inhibition assays include ones that utilize liver microsomes, isolated/cultured hepatocytes, and human cDNA-expressed enzymes. Typically, LC-MS/MS quantification is used for the microsome and hepatocyte methods while fluorimetric assays are used for the human cDNA-expressed enzymes. Typically, a weak inhibitor is defined as having a fei>20 rmolN, whereas a potent inhibitor has a ki[Pg.882]

The catalytic activity of CYP enzymes requires functional coupling with its redox partners, cytochrome P450 NADPH oxidoreductase (OR) and cytochrome bs. Measurable levels of these two proteins are natively expressed in most cell lines. Therefore, introduction of only the CYP cDNA is generally needed for detectable catalytic activity. However, the levels of expression of the redox partner proteins may not support maximal CYP catalytic activity, and therefore enhancement of OR levels may be desirable. This approach has been used successfully with an adenovirus expression system in LLC-PKi cells [12],... 

MANGOLD, U EICHEL, J., BATSCHAUER, A., LENZ, A., KAISER, T SPANGENBERG, G., WERCK-REICHHART, D, SCHRODER, J., Gene and cDNA for plant cytochrome P450 proteins (CYP 72 family) from Catharanthus roseus and transgenic expression in tobacco and Arabidopsis thaliana., Plant Sci., 1994, 96, 129-136. 

---