This article is available online at http://dmd.aspetjournals.org ABSTRACT:Recently, we reported that several endogenous steroids affect CYP3A4-mediated drug metabolism, using human adult liver microsomes as an enzyme source. Especially, carbamazepine (CBZ) 10,11-epoxidation is activated by androstenedione (AND). In the present studies, we investigated the effects of endogenous steroids on the activity of CBZ 10,11-epoxidation by expressed CYP3A4 and CYP3A7. When expressed CYP3A4 was used as an enzyme source, the addition of AND to the reaction mixture also caused a drastic increase in the activity of CBZ 10,11-epoxidase, and resulted in a change in the kinetics from sigmoid to MichaelisMenten type. On the other hand, expressed CYP3A7-mediated CBZ 10,11-epoxidation was activated by sulfate conjugate steroids, such as pregnenolone 3-sulfate, 17␣-hydroxypregnenolone 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEA-S), whereas the unconjugated form corresponding to these three steroids did not activate the reaction. Especially, DHEA-S was found to be a potent activator of CBZ 10,11-epoxidation by expressed CYP3A7. The kinetic character of CBZ 10,11-epoxidation by CYP3A7 is Michaelis-Menten type regardless of the presence of DHEA-S. The presence of DHEA-S caused a decrease in K m and increase in V max for CYP3A7-mediated CBZ 10,11-epoxidation, whereas DHEA-S 16␣-hydroxylation was not affected by the coexistence of CBZ. In conclusion, CYP3A4 and CYP3A7-mediated CBZ 10,11-epoxidations are activated by different types of endogenous steroids. This is the first report regarding CYP3A7 cooperativity.

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.

ABSTRACT:CYP3A7 is a member of the human CYP3A family and a major form of P450 expressed in human fetal livers. Although CYP3A7 shares nearly 90% base sequence with CYP3A4, CYP3A7 shows striking functional differences in the catalytic preference for several substrates, such as dehydroepiandrosterone (DHEA) or dehydroepiandrosterone 3-sulfate (DHEA-3S). First, to clarify the reason for the differences between CYP3A7 and CYP3A4, a homology model of CYP3A7 was constructed using the CYP3A4 crystal structure. Because these two structures were similar, four kinds of chimeric enzymes were constructed to determine which sequences are important for exhibiting the characteristics of CYP3A7. The results of kinetic analysis of DHEA and DHEA-3S 16␣-hydroxylations by CYP3A7, CYP3A4, and CYP3A chimeras suggested that the amino acid residues from Leu 210 to Glu 279 were important to express the specificity for substrates as CYP3A7. This region was on the F and G helices of the modeled CYP3A7. Furthermore, to assess which amino acid in this sequence is important for the substrate specificity of CYP3A7, a one-point mutation of CYP3A7 to CYP3A4 was made by site-directed mutagenesis. The mutants of K224T and K244E had lost DHEA and DHEA-3S 16␣-hydroxylation activities. The mutants also greatly decreased the metabolism of testosterone, erythromycin, nevirapine, and triazolam relative to those activities of CYP3A7 wild-type enzyme. From these results, it is expected that CYP3A7 can recognize specific substrates using the lysines in F-G loops.Cytochromes P450s (P450s) comprise a superfamily of monooxygenases that metabolize a wide variety of exogenous chemicals as well as endogenous substrates such as steroids, fatty acids, and prostaglandins. Multiple forms of P450 have been shown to be present in the liver (Nelson et al., 1996). Among the forms of P450s, CYP3A is the most abundant form in human livers (Kitada et al., 1985a;Shimada et al., 1994), and the human CYP3A subfamily consists of four isoforms: CYP3A4 (Beaune et al., 1986), CYP3A5 (Aoyama et al., 1989), CYP3A7 (Komori et al., 1989), and CYP3A43 (Domanski et al., 2000.CYP3A7 accounts for 30 to 50% of total P450s in the fetal liver (Kitada et al., 1985b;Shimada et al., 1996). CYP3A4 is the major form of P450 expressed in the adult liver, and the expression of CYP3A4 mRNA was not seen in human fetal livers and began to increase after birth (Lacroix et al., 1997). It has been reported that a 5Ј-flanking region from 1 kilobase to the transcriptional start site of the CYP3A7 gene was 91% identical to that of the CYP3A4 gene (Itoh et al., 1992;Hashimoto et al., 1993). Recently, an experiment using HepG2 cells showed that Sp1 and Sp3 bound to the nuclear factor-B-like element of the CYP3A7, but not the CYP3A4 gene, and that the expression of the CYP3A7 gene was cooperatively regulated by Sp1, Sp3, hepatocyte nuclear factor 3␤, and upstream stimulatory factor 1 (Saito et al., 2001).The nucleotide and amino acid sequences of CYP3A7 (Komori et al., 1989) share 94 and 88% identities, r...

Aliskiren is a substrate for P-glycoprotein (P-gp) and is metabolized via cytochrome P450 3A4 (CYP3A4). The aim of the present study was to assess whether P-gp influenced the pharmacokinetics of aliskiren and also if drug-drug interactions (DDIs) mediated through P-gp could be reproduced in cynomolgus monkeys. The study investigated the pharmacokinetics of aliskiren in mdr1a/1b gene-deficient (P-gp KO) and wild-type (WT) mice. The area under the plasma concentration-time curve (AUC) following the oral administration of aliskiren was 6.9-fold higher in P-gp KO mice than in WT mice, while no significant differences were observed in the AUC or total plasma clearance following the intravenous administration of aliskiren to P-gp KO mice. Then the pharmacokinetics of aliskiren were evaluated and DDIs between aliskiren and P-gp inhibitors, such as cyclosporin A (CsA) and zosuquidar, examined in cynomolgus monkeys. The AUC for aliskiren were 8.3- and 42.1-fold higher after the oral administration of aliskiren with the concomitant oral administration of zosuquidar and CsA at doses of 10 and 30 mg/kg, respectively. In contrast, the AUC after the intravenous and oral administration of aliskiren was not significantly affected by the oral administration of zosuquidar or intravenous administration of CsA, respectively. These results indicated that P-gp strictly limited the intestinal absorption of aliskiren in mice and monkeys, and also that the effects of intestinal P-gp inhibition by CsA or zosuquidar on the pharmacokinetics of aliskiren were sensitively reproduced in monkeys. In conclusion, aliskiren can be used as a sensitive substrate to evaluate intestinal P-gp inhibition in monkeys.

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