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Regular Article Cytochrome P450 Is Responsible for Nitric Oxide Generation from NO-Aspirin and Other Organic Nitrates

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Regular Article Cytochrome P450 Is Responsible for Nitric Oxide Generation from NO-Aspirin and Other Organic Nitrates Drug Metab. Pharmacokinet. 22 (1): 15–19 (2007). Regular Article Cytochrome P450 is Responsible for Nitric Oxide Generation from NO-Aspirin and Other Organic Nitrates Yukiko MINAMIYAMA1,2,*,ShigekazuTAKEMURA2, Susumu IMAOKA3, Yoshihiko FUNAE4,andShigeruOKADA1 1Department of Anti-aging Food Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, Departments of 2Hepato-Biliary-Pancreatic Surgery and 4Chemical Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan, 3School of Science and Technology Kwansei Gakuin University, Sanda, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Nitric oxide (NO) biotransformation from NO-aspirin (NCX-4016) is not clearly understood. We have previously reported that cytochrome P450 (P450) plays important role in NO generation from other organic nitrates such as nitroglycerin (NTG) and isosorbide dinitrate (ISDN). The present study was designed to elucidate the role of human cytochrome P450 isoforms in NO formation from NCX-4016, using lymphoblast microsomes transfected with cDNA of human P450 or yeast-expressed, puriˆed P450 isoforms. CYP1A2 and CYP2J2, among other isoforms, were strongly related to NO production from NCX-4016. In fact, these isoforms were detected in human coronary endothelial cells. These results suggest that NADPH-cytochrome P450 reductase and the P450 system participate in NO formation from NCX-4016, as well as other organic nitrates. Key words: human cytochrome P450; nitric oxide; NO-aspirin; organic nitrates ated ‰uorescence, indicating that NCX-4016 penetrates Introduction cell membranes and is metabolized to release free Nitric oxide (NO)-aspirin [NCX-4016; 2-acetoxy- NO.11) Grosser et al.12) reported that NO generation benzoate 2-(2-nitroxymethyl)-phenyl ester] is an NO- from NCX-4016 is mediated through a pathway similar donating derivative of acetyl salicylic acid (ASA), and is to that responsible for NO generation from nitroglyce- currently in phase II clinical trials for the treatment of rin (NTG) and other organic nitrates. They suggest that cardiovascular events including thrombosis,1–4) resteno- these bioactivation pathways of organic nitrates, which sis5) and endothelium-related complications in dia- have been shown to involve cytochrome P450 (CYP or betes.6–10) In cell culture, NCX-4016, but not aspirin P450), might also be responsible for NO release from increases intracellular NO content in a time-dependent NO-aspirin. The present study observed NO formation manner, as measured by 4,5 diamino‰uorescein-gener- from NCX-4016 in the microsomes of lymphoblasts transfected with cDNAs for various human CYP isoforms, and yeast-expressed, puriˆed P450 isoforms This work was supported by grants from Osaka City University, Okayama University Medical Research Fund for Medical Research, which are present in the human heart. and the Special Coordination Funds of the Ministry of Education, Materials and Methods Culture, Sports, Science, and Technology, Japan. Reprint requests: Yukiko Minamiyama, Ph.D., Department of Anti-Aging Food Chemicals: Glucose-6 phosphate (G6P), G6P phos- Sciences, Graduate School of Medicine, Dentistry, and Pharmaceuti- phate dehydrogenase (G6PDH) and NADPH were cal Sciences, Okayama University, Shikata-cho, Okayama 700–8558, Japan. purchased from Oriental Yeast (Tokyo, Japan). Micro- Received; March 9, 2006, Accepted; August 4, 2006 *To whom correspondence should be addressed: Yukiko MINAMIYAMA,Ph.D,Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. Tel. ＋81-6-6645-3841, Fax. ＋81-6-6646-6057, E-mail: yukiko＠med.osaka-cu.ac.jp Abbreviations used are: ISDN, isosorbide dinitrate; NO, nitric oxide; NO-ASA, NO-aspirin, 2-acetoxybenzoate-2-(1-nitroxy-methyl)-phenyl- ester; NSAID, non-steroidal anti-in‰ammatory drug; NTG, glyceryl trinitrate 15 16 Yukiko MINAMIYAMA, et al. somes from human lymphoblasts transfected with CYP1A2, CYP2A6, CYP2D6, CYP2E1 or CYP3A4 cDNA were obtained from Gentest (Woburn, MA, USA). Other reagents used were of analytical grade from Wako Pure Chemicals (Osaka, Japan). NO- aspirin (NCX-4016) was kindly provided by Nicox, Nice, France. Isosorbide dinitrate (ISDN) and NTG (500 mgWmL ethanol solution) were gifts from Eisai (Tokyo, Japan) and from Nihon Kayaku (Tokyo, Japan), respectively. ISDN (100 mM) was dissolved in 100z methanol. － － Plasma levels of NO2 ＋NO3 (NOx)afterISDNor NCX-4016 administration: Male Wistar rats, 200–230 g, were purchased from SLC (Shizuoka, Japan), and fed laboratory chow and water ad libitum.ISDN 24 mgWkg (0.05 mmolWkg) or NCX-4016 82.5 mgWkg (0.25 mmolWkg) was administered orally. At the indicat- ed times, 300 mL blood was collected via the tail vein with a heparinized syringe under diethylether anesthe- Fig. 1. Plasma levels of NOx after ISDN and NCX-4016 administra- sia. Plasma was immediately separated and treated with tion. ISDN 24 mgWkg (0.05 mmolWkg) or NCX-4016 82.5 mgWkg (0.25 100z methanol (1:1 volume) and centrifuged at 10 000 mmolWkg) was administered orally to rats. At the indicated times, g for 2 min. The supernatant was applied to an auto- plasma was collected and NOx levels were measured. Values are means mated NO detector-HPLC system (ENO-10, Eicom, ±SE (n＝4). Open circles, ISDN; closed circles, NCX-4016. Kyoto, Japan).13) The investigation conformed to the Guide for the added, and samples were centrifuged at 10000 g for 2 Care and Use of Laboratory Animals approved by the min at 49C. Since NO released from organic nitrates is － authorities of Osaka City University Medical School. immediately converted to NO2 under aerobic condi- － NCX-4016-induced NO formation in CYP-expressing tions, supernatants were used for the analysis of NO2 microsomes of human lymphoblast cells: ACYP-ex- by HPLC. pressing microsome preparation (100 pmol) was sus- Immunohistochemistry of human heart blood vessels: pended in 0.5 mL (total volume) PBS (pH 7.4) and Samples were obtained at autopsy from a patient incubated with 3.3 mM G6P, 0.5 UWmL G6PDH, without heart failure at Okayama University Medical 1 mM NADPH, 3.3 mM MgCl2･6H2O, and 100 mM School. Heart tissue samples were ˆxed with 10z NCX-4016 for 15 min at 379C. After incubation, sam- buŠered formalin and embedded in para‹n. The 4 mm ples were immediately kept on ice and centrifuged at thin sections were depara‹nized with xylol and ethanol. 100000 g for 60 min at 49C. Since NO released from They were treated with 0.3z hydrogen peroxide in － NCX-4016 is immediately converted to NO2 under methanol for the inhibition of endogenous peroxidase aerobic conditions, supernatants were used for the activity for 20 min, and normal goat serum (Dako, － － 13) analysis of NO2 and NO3 by HPLC. Part of the Kyoto; diluted to 1: 100) for the inhibition of non- study examined direct NO generation using an NO- speciˆc binding of second antibody for 30 min. The selective electrode NO-501 (Inter Medical, Nagoya, sections were treated with each polyclonal antibody Japan) as described previously.14) for human CYP2J2, CYP3A4, CYP2E1, CYP2C9, － NO3 -induced NO formation in yeast-expressed, CYP1A2, CYP2A6, or CYP2D6 (×300) for overnight puriˆed human P450 isoforms: Human P450s at 49C, and then biotin-labeled goat anti-rabbit IgG (CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, serum (Dako; diluted to 1:200) including normal serum CYP2J2 and CYP3A4) were expressed in Sac- (1:80) for 40 min, and with avidin-biotin-peroxidase charomyces cerevisiae and puriˆed as described previ- complex (Vector Laboratories, Burlingame, CA; diluted ously.15) The reaction mixture contained 0.1 mL (ˆnal to 1:100) for 40 min. The specimens were stained by volume) 0.1 M potassium phosphate buŠer (pH 7.4), 20 the bridged immunoperoxidase method (peroxidase- pmol puriˆed P450 and cytochrome b5, 0.3 U NADPH- antiperoxidaseWdiaminobenzidine) and viewed by dependent P450 reductase and 10 mg phospholipid at microscopy. Methyl green or hematoxylin was applied 379C for 3 min. The mixture was added to 1 mM for nuclear staining. Non-immune rabbit serum in place NADPH and 100 mM NCX-4016, ISDN or NTG for 15 of speciˆc antibodies was used as a negative control. min at 379C. After incubation, samples were immedi- Anti-CYP isoforms with excess fresh microsomes were ately kept on ice and 0.05 mL of 100z methanol was pre-incubated with each antibody overnight at 49C. NO Generation from NO-Aspirin by P450 17 Fig. 3. NCX-4016-derived NO formation in yeast-expressed, puriˆed human P450 isoforms. Puriˆed CYP isoforms (20 pmol) were suspended in 0.2 mL PBS (pH 7.4) and incubated with 3.3 mM G6P, 0.5 UWmL G6PDH, 1 mM NADPH, 3.3 mM MgCl2･6H2O, and 100 mM NCX-4016 for 15 min at 379C. After incubation, samples were immediately kept on ice and centrifuged at 100000 g for 60 min at 49C. Supernatants were used for the analysis of NOx by HPLC. Data represent the means±SE of triplicate measurements. Fig. 2. NCX4016-derived NO formation in human P450 isoform- expressing microsomes. (A) CYP-expressing microsomes from cDNA- transfected human lymphoblast cells (100 pmol) were suspended in 0.5 mL PBS (pH 7.4) and incubated with 3.3 mM G6P, 0.5 UWmL G6PDH, 1mM NADPH, 3.3mM MgCl2･6H2 O, and 100 mM NCX-4016 for 15 min at 379C. After incubation, samples were imme- diately kept on ice and centrifuged at 100000 g for 60 min at 49C. Supernatants were used for the analysis of NOx by HPLC. Data represent the means±SE of triplicate measurements. (B) Typical trac- ing of NO generation from CYP1A2-induced biotransformation using a NO-selective electrode. CYP1A2-expressing microsomes (1.5 mg W mL) were incubated with the buŠer, as described above, and then monitored continuously with an NO electrode (right panel).
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