CYP induction

LC/MS/MS with selected reaction monitoring (SRM) offers a fast and simple means to analyze biological matrices, which is a key factor in high-throughput CYP inhibition screens using liver microsomes. Potentially, the LC/MS/MS technique is suitable for analyses of cocktail substrates in other in vitro drug metabolism evaluations such as CYP induction/activation assays, rapid analysis of pooled liver microsomes, rapid reaction phenotyping of tissue (hepatic and extrahepatic) samples, as well as evaluation of hepatocytes/tissue slice CYP activity. ° ... 

Table 3.3 Description of CYP induction recepto rs and physiochemical parameters that describe the active binding site. 

Mechanism and Genetics of Induction in Mammals. Many different mechanisms may be involved in CYP induction. These include increased transcription of DNA, increased mRNA translation to protein, mRNA stabilization, and protein stabilization. Induction can only occur in intact cells and cannot be achieved by the addition of inducers directly to cell fractions such as microsomes. It has been known for some time that in most cases of increase in monooxygenase activity there is a true induction involving synthesis of new enzyme, and not the activation of enzyme already synthesized, since induction is generally prevented by inhibitors of protein synthesis. For example, the protein synthesis inhibitors such as puromycin, ethionine, and cyclo-heximide inhibit aryl hydrocarbon hydroxylase activity. A simplified scheme for gene expression and protein synthesis is shown in Figure 9.7. 

Induction of CYP expression by xenobiotics has been reported in mainly three ways (1) induction potential (fold induction over control), (2) EC50 (effective concentration for 50% maximal induction), and (3) potency index (the ratio of induction response of the test compound compared to that of a gold standard). In our laboratory, we have defined CYP induction as a potency index or a percentage of a classic inducer rather than as fold increase over a control (induction potential). The reason for this is twofold. First, the basal levels of some CYPs may be low and therefore difficult to accurately quantitate. Second, we, and others, have found that basal CYP levels in culture may be highly... 

In regards to culturing hepatocytes in a 96-well plate format, we have adopted the same conditions that we used when culturing cells in 60-mm dishes and 24-well plates (12) and simply scaled them down to a 96-well plate format. The 96-well plates are precoated with Matrigel and are commercially available (Collaborative Biomedical Products, Boston, Massachusetts, U.S.) or, alternatively, normal plates can be coated with diluted Matrigel and dried overnight (27). Hepatocytes cultured on collagen-coated 96-well plates have also been reported to be suitable for CYP induction (28). 

The application of this technology for determining CYP induction in primary hepatocytes was first described by Strong et al. (40). To further demonstrate the potential of this technology, a study was conducted in our laboratory using primary human primary hepatocytes cultured on a 96-well plate precoated... 

These type of assays have great potential as screening tools for CYP induction without having to use valuable human hepatocytes. The human hep-atocyte model could be reserved to confirm results of a lead compound after exhaustive screening with such reporter gene construct models. 

Figure 17 Strategies for dealing with CYP induction in drug discovery. Abbreviation CYP, cytochrome P450.

Lin JH. CYP induction-mediated drug interactions In vitro assessment and clinical implications. Pharm Res 2006 23 1089-1116. 

The quantitative extent of CYP induction depends on the dosage (concentration) of the inducer and on the duration of exposure. However, the induction process, in contrast to inhibition, is not as straightforward to study in vitro, since induction requires intact cellular protein synthesis mechanisms as available in cell culture models (57-62). 

Sex differences in POP toxicokinetics have also been noted. Female rats initially metabolized tra 5-chlordane to racemic oxychlordane, which later became enriched in the (—)-enantiomer post-exposure [272]. On the other hand, male rats immediately degraded tra 5-chlordane enantioselectively to (—)-oxychlordane [272]. Both sexes preferentially depleted (—)-tra s-chlordane, as well as (+)-oxychlordane in organisms individually exposed to oxychlordane, trara-chlordane, or achiral tra 5-nonachlor. These results supported the hypothesis that although CYP3A induction in rats was not sex-dependent, CYPlAl induction was [272]. More research remains to be done to elucidate the stereoselectivity of CYP induction and its effects on the metabolism of POPs. 

A number of chemicals and drags, such as phenobarbital, strongly induce CYP activity and increase their own metabolism. In vivo assessment of CYP induction is reserved for the later phase of drag development if indicated. It requires multiday treatment, an effort which is not justified for a compound in the early phase. A number of... 

From experiments conducted with OPZ administered before, during, and after AFBl, it was concluded that OPZ acts as a blocking rather than a suppressive chemoprotective agent [29]. Such an anti-initiating agent can, in principle, act at several levels, including inhibition or induction of CYPs, induction of phase 2 enzymes, scavenging of electrophilic metabolites and ROS, and induction of DNA repair [30]. 

Although undesirable, CYP induction isn t quite as big a problem as CYP inhibition, so there s less of a focus on it at the early stages of drug discovery. This is largely because, as shown in Table 10.2, while a clinical CYP inhibitor might boost levels of co-administered drugs to toxic concentrations by preventing their breakdown, CYP inducers would increase... 

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