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Comparison of the Stimulatory and Inhibitory Effects of Steroid ←確認用doi （左上Y座標：－17.647 pt） Vol. 44, No. 4 Biol. Pharm. Bull. 44, 579–584 (2021) 579 Regular Article Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies Toshiro Niwa,* Manami Toyota, Hinako Kawasaki, Risa Ishii, and Shoko Sasaki School of Pharmacy, Shujitsu University; 1–6–1 Nishigawara, Naka-ku, Okayama 703–8516, Japan. Received December 6, 2020; accepted January 14, 2021 The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6β-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphi- cally expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the pre- dominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for proges- terone 6β-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6β-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activ- ity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6β-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6β-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6β-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6β-hydroxylation mediated by all CYP3A subfam- ily members. The inhibitory/stimulatory pattern of steroid–steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated. Key words CYP3A4; CYP3A5; CYP3A7; steroid hormone; progesterone; α-naphthoflavone INTRODUCTION the U.S. Food and Drug Administration, European Medicines Agency, and Japanese Ministry of Health, Labour, and Wel- Cytochrome P450 (CYP or P450) 3A4, one of most impor- fare.14–16) Progesterone, a progestational hormone, is hydroxyl- tant human P450 enzymes, accounts for approximately 30% ated not only at the C17 and C21 positions by steroidogenic of all P450 enzymes in the human liver microsomes,1,2) and CYP17A1 and CYP21A2, respectively, but also at the C2β, C6, is responsible for more than 50% of the oxidation of clini- C16α, and C21 positions by drug-metabolizing P450s, includ- cally used pharmaceuticals.3) CYP3A4 and polymorphically ing CYP3A4.12,13) Because cortisol is a stress hormone bio- expressed CYP3A54) are 83% homologous in terms of amino transformed by the adrenal glands and is released for various acid sequences, however, we previously demonstrated that reasons including the physiological response to stress,17) this some differences in the reported parameters including the Mi- steroid hormone is used as a biomarker of psychophysiological 18,19) chaelis constant (Km), maximal velocity (Vmax or kcat), intrinsic stress. Furthermore, the ratio of 6β-hydroxycortisol to cor- clearance (CLint, calculated by dividing kcat by Km) and inhibi- tisol in urine or plasma is proposed as an in vivo endogenous 20–22) tion constant (Ki) values for reactions mediated by CYP3A4 marker of CYP3A4 metabolic activity. CYP3A5 and and CYP3A5 can be identified, although CYP3A4 and CYP3A7 as well as CYP3A4 catalyze the oxidation of various CYP3A5 have overlapping substrate specificities5,6); findings compounds, including the 6β- and 16α-hydroxylation of endo- that are also consistent with those reported in other reviews.7,8) genous steroid hormones such as testosterone, cortisol, and CYP3A7 is the major form of P450 expressed in the human dehydroepiandrosterone (DHEA).10,23,24) We recently compared fetal liver (>36% of the total P450 in fetal liver); however, the enzymatic kinetic parameters, such as Km and kcat values, CYP3A7 presents in only trace amounts in the adult liver.9,10) for the 6β-hydroxylation of progesterone, testosterone, and The amino acid sequence of CYP3A7 shows 88% similarity to cortisol mediated by CYP3A4, CYP3A5, and CYP3A7.25,26) that of CYP3A4.11) We previously found that steroid hormones, such as proges- Various endogenous steroid hormones are metabolized terone, testosterone, and α-naphthoflavone (ANF), stimulate by drug-metabolizing P450s such as CYP2C, CYP2D, and CYP3A4-mediated metabolism, including nifedipine oxidation CYP3A subfamilies as well as steroidogenic P450s, including and/or 7-benzyloxyresorufin O-debenzylation.27) In addition, CYP11B1, CYP11B2, CYP17A1, CYP19A1, and CYP27A1.12,13) many studies have demonstrated the activation of CYP3A4- In terms of 6β-hydroxylation, testosterone is predominantly mediated metabolic reactions, including the hydroxylation of metabolized by the CYP3A subfamily12,13) and is recommended steroid hormones such as progesterone.28,29) ANF is a famous as a preferred reaction of CYP3A for in vitro studies in accor- activator of various CYP3A4-mediated reactions including dance with the guidelines for new drug applications regarding aflatoxin B1 8,9-epoxidatin, 17β-estradiol 2-hydroxylation, and in vitro experiments of drug–drug interactions regulated by phenanthrene 9,10-dihydrodiol formation in addition to the * To whom correspondence should be addressed. e-mail: [email protected] © 2021 The Pharmaceutical Society of Japan 580 Biol. Pharm. Bull. Vol. 44, No. 4 (2021) reactions described above.28) The activation has similarly been Aldrich (St. Louis, MO, U.S.A.) and Steraloids Inc. (Newport, demonstrated for other P450s, including CYP1A2, CYP2C9, RI, U.S.A.), respectively. Progesterone, pregnenolone, DHEA, CYP2D6, and CYP3A7,28,29) However, few studies have re- and ANF were obtained from Tokyo Chemical Industry ported the activation of metabolic activities mediated by (Tokyo, Japan). Cortisol and deoxycorticosterone (21-hydroxy- CYP3A5 and CYP3A7, although it has been reported that the progesterone) were purchased from FUJIFILM Wako Pure activation of sulfate conjugates of steroid hormones, such as Chemical Corporation (Osaka, Japan), and testosterone was DHEA and pregnenolone, stimulates CYP3A7-mediated carba- obtained from Nacalai Tesque (Kyoto, Japan). All other chemi- mazepine 10,11-epoxidation.29) In addition, we recently found cals used were of the highest quality commercially available. that testosterone stimulated progesterone 6β-hydroxylation Hydroxylation Activities of Steroid Hormones Proges- mediated by CYP3A5 and CYP3A7 as well as CYP3A4, al- terone and testosterone 6β-hydroxylation activities were deter- though progesterone inhibited testosterone 6β-hydroxylation.26) mined as described previously.25,26,31–33) Briefly, the incubation Pregnenolone is biotransformed from cholesterol by mixtures (final volume, 0.5 mL) contained 10 nM CYP3A4, CYP11A1, and DHEA is formed from pregnenolone via CYP3A5, or CYP3A7; 1 mM NADPH; and 100 mM phosphate 17α-hydroxypregnenolone by CYP17A1.13) As mentioned buffer (pH 7.4). In the preliminary experiments, the linearity above, ANF is a reported activator of CYP3A4.27) Thus, in the of the reaction with incubation time and P450 concentration present study, we compared the effects of these steroid hor- was confirmed for each CYP3A subfamily member.25,26,31–33) mones and ANF on the 6β-hydroxylation of progesterone and All data were analyzed using the average of duplicate or trip- testosterone mediated by CYP3A4, CYP3A5, and CYP3A7. licate measurements. Km, kcat, and Ki values were estimated by These results would be a useful resource for clarifying the fitting to Michaelis–Menten kinetics by means of nonlinear effect of CYP3A5 polymorphism and fetal CYP3A7 expres- least squares regression analysis (MULTI34)). sion on the drug–drug interaction (especially, cooperation) with various compounds including steroid hormones. RESULTS MATERIALS AND METHODS The effects of steroid hormones and ANF on progesterone and testosterone 6β-hydroxylation activities by CYP3A4, Materials Easy CYP CYP3A4R, CYP3A5R, and CYP3A7R, CYP3A5, and CYP3A7 were estimated at substrate con- expressed in recombinant Escherichia coli, which were co- centrations of 10 µM (for CYP3A4 and CYP3A5) or 20 µM expressed with reduced form of nicotinamide adenine dinu- (for CYP3A7) for progesterone and 100 µM for testosterone cleotide phosphate (NADPH)-P450 reductase (high reductase (Table 1). The substrate concentrations were approximately Bactosomes) but not with cytochrome b5, were obtained from equal to or below the Km values for each CYP3A subfamily Cypex Limited (Dundee, U.K.).30) 6β-Hydroxyprogesterone member.25,26) Interestingly, DHEA markedly increased pro- and 6β-hydroxytestosterone were purchased from Sigma- gesterone 6β-hydroxylation activities mediated by CYP3A5 Table 1. Effect of Steroid Hormones and ANF on the 6β-Hydroxylation of Progesterone and Testosterone Mediated by CYP3A4, CYP3A5, and CYP3A7 % of the velocity in the absence of effector Substrate Effector CYP3A 1 µM* 10 µM* 25 µM* 100 µM* 250 µM* Progesterone DHEA CYP3A4 109.3 120.4 — 51.1 — CYP3A5 115.1 136.0 — 156.0 — CYP3A7 100.3 63.0 — 33.2 — ANF CYP3A4 90.8 59.0 — 27.7 — CYP3A5 140.3 66.1 — 12.9 — CYP3A7 83.1 38.8 — 22.4 — Pregnenolone CYP3A4 87.5 63.4 — 40.5 — CYP3A5 120.9 107.4 — 118.4 — CYP3A7 90.2 64.7 — 55.7 — Testosterone
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