ABSTRACT:Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6␤-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

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ABSTRACT:Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), ␤-naphthoflavone (33 M), phenobarbital (100 or 250 M), isoniazid (100 M) and/or rifampin (20 or 50 M), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by ␤-naphthoflavone (on average 13-fold, n ‫؍‬ 28 preparations), and weakly induced by phenobarbital (1.9-fold, n ‫؍‬ 25) and rifampin (2.3-fold, n ‫؍‬ 22); CYP2A6 activity tended to be increased with phenobarbital (n ‫؍‬ 7) and rifampin (n ‫؍‬ 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n ‫؍‬ 13) and rifampin (13-fold, n ‫؍‬ 14); CYP2C8 was induced by phenobarbital (4.0-fold, n ‫؍‬ 4) and rifampin (5.2-fold, n ‫؍‬ 4); CYP2C9 was induced by phenobarbital (1.8-fold, n ‫؍‬ 14) and rifampin (3.5-fold, n ‫؍‬ 10); CYP2C19 was markedly induced by rifampin (37-fold, n ‫؍‬ 10), but relatively modestly by phenobarbital (7-fold, n ‫؍‬ 9); CYP2D6 was not significantly induced by phenobarbital (n ‫؍‬ 5) or rifampin (n ‫؍‬ 5); CYP2E1 was induced by phenobarbital (1.7-fold, n ‫؍‬ 5), rifampin (2.2-fold, n ‫؍‬ 5), and isoniazid (2.3-fold, n ‫؍‬ 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n ‫؍‬ 42) and rifampin (10-fold, n ‫؍‬ 61), but not by ␤-naphthoflavone. Based on these observations, we generalize that ␤-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily. Drugs and NMEs5 are often screened for their ability to induce P450 and other drug-metabolizing enzymes with the aim of predicting or explaining drug-drug interactions and pharmacokinetic tolerance.

Protein expression of major hepatic uptake and efflux drug transporters in human pediatric (n=69) and adult (n=41) livers was quantified by LC-MS/MS. Transporter protein expression of OCT1, OATP1B3, P-gp and MRP3 was age-dependent. Particularly, significant differences were observed in transporter expression (p <0.05) between the following age-groups: neonates vs. adults (OCT1, OATP1B3, P-gp), neonates or infants vs. adolescents and/or adults (OCT1, OATP1B3 and P-gp), infants vs. children (OATP1B3 and P-gp) and adolescents vs. adults (MRP3). OCT1 showed the largest increase, of almost 5-fold, in protein expression with age. Ontogenic expression of OATP1B1 was confounded by genotype and was revealed only in livers harboring SLCO1B1*1A/*1A. In livers > 1 year, tissues harboring SLCO1B1*14/*1A showed 2.5-fold higher (P<0.05) protein expression than SLCO1B1*15/*1A. Integration of these ontogeny data in physiologically based pharmacokinetic (PBPK) models will be a crucial step in predicting hepatic drug disposition in children.

Cytochrome P450 3A4 (CYP3A4), an enzyme that is highly expressed in the human liver and small intestine, plays a major role in the metabolism of a large variety of xenobiotics, including an estimated 50% of therapeutic drugs, as well as many endogenous compounds. The expression of CYP3A4 can be induced by xenobiotics. Such induction leads to accelerated metabolism of the xenobiotics themselves (autoinduction) or of concomitantly administered CYP3A4 substrates/drugs, thereby significantly altering their pharmacokinetic and pharmacodynamic profiles. During the past decade, much progress has been made in our understanding of the biological mechanisms responsible for regulation of CYP3A4 expression. It is now known that many xenobiotics induce CYP3A4 expression via the pregnane X receptor (PXR) pathway, while others are thought to act through the constitutive androstane receptor (CAR) and the vitamin D receptor (VDR). As a result, most pharmaceutical companies have recognized that it is important to evaluate CYP3A4 induction potential preclinically and are using primary cultures of human hepatocytes and/or PXR reporter gene assays. In general, the results from these two assay methods correlate well. The reporter gene assays in particular can be used to rapidly screen hundreds of drug candidates, whereas methods using primary human hepatocyte cultures may more accurately assess the potential for CYP3A4 induction in vivo. Although it is important to consider CYP3A4 induction in the early stages of the drug development process, it should be recognized that the assessment of induction potential preclinically is a difficult and imprecise endeavor and can be complicated by many factors.