Drug Metab. Pharmacokinet. 22 (1): 15–19 (2007).

Regular Article 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). Ten microliters saturated NO solution (1.9 mM) was added to 0.5 mL PBS as a standard (left panel).

Statistical analysis: Unless otherwise stated, data Fig. 4. ISDN and NTG-induced NO formation in yeast-expressed, are presented as means±SE. puriˆed human P450 isoforms. Methods are described in the legend to Results Fig. 3. ISDN or NTG (100 mM) was used in the assay. Data represent the means±SE of triplicate measurements.

Plasma levels of NOx after administration of ISDN andNCX-4016torats: Figure1showsplasmalevels of NOx after administration of ISDN (0.05 mmolWkg) or isoforms showed discrepancies between the two assay NCX-4016 (0.25 mmolWkg). Both drugs immediately systems (transfected microsomeWpuriˆed ). and continuously increased plasma NOx levels. Figure 2B shows NO generation from CYP1A2-express- NCX-4016-derived NO formation in human P450 ing microsomes, using an NO electrode. isoforms: Figures 2A and 3 show NO generation from ISDN and NTG-derived NO formation in yeast- NCX-4016 in CYP-expressing microsomes of human expressed, puriˆed P450 isoforms: NO was also gener- lymphoblast cells and yeast-expressed, puriˆed P450 ated from ISDN and NTG by some P450 isoforms isoforms, respectively. Some isoforms generated NO (Fig. 4). NO generation from ISDN or NTG was cata- fromNCX-4016similarlyinbothsystems,butother lyzed by a diŠerent isoform. 18 Yukiko MINAMIYAMA, et al.

Fig. 5. Immunohistochemistry of human CYP isoforms in the heart. Specimens were obtained at autopsy. Each section was stained with anti-CYP2J2, CYP1A2 and CYP3A4, and normal rabbit IgG. Original magniˆcation (×400)

Immunohistochemistry of human coronary artery: We reported previously that the P450 isoforms Cardiac tissue was obtained at autopsy from a male CYP1A2, CYP2E1 and CYP3A4 were present in the patient aged 20 years with lung cancer. Figure 5 shows endothelium of human cardiac blood vessels.16) typical immunohistochemistry of CYP2J2, CYP3A4 CYP2C9 and 2D6 were not present in cardiovascular and CYP1A2 observed in coronary vessel endothelium. tissues,16) whereas CYP2J2 was reported to be abundant There was also staining for CYP2E1 and CYP2A6 (data in cardiovascular tissues.17) Together, these observations not shown). No antibody binding of CYP2C9 or suggest that CYP1A2, CYP2E1, CYP3A4 and CYP2J2 CYP2D6 was observed. Smooth muscle cells were non- are responsible for NO generation from various organic speciˆcically and slightly stained. nitrates, although their potencies are diŠerent for each substrate. Discussion Tolerance is an important problem in clinical treat- This paper demonstrates that NO-aspirin is bioacti- ment with organic nitrates. Grosser et al. reported that vated through a P450-dependent pathway like other prolonged treatment with NO-aspirin also causes down- organic nitrates. Some P450 isoforms provoked bioacti- regulation of the cellular cyclic GMP response, suggest- vation of NCX-4016. The reason why diŠerent isoforms ing that tolerance occurs during therapy with NO- of P450 exerted diŠerent eŠects on NO release may have aspirin.12) We have also reported that nitrate tolerance is been due to diŠerences in substrate speciˆcity. Further- induced by P450 degradation.18) NCX-4016 is conducted more, some isoforms showed a discrepancy in their as a clinical testing drug for cardiovascular disorders ability to produce NO between the two assay systems (Phase II study). Knowledge of the pharmacokinetics used, namely, microsomes from cDNA-transfected and pharmacodynamics of these organic nitrates is human lymphoblasts and yeast-expressed, puriˆed P450 important for their long-term use for treatment of isoforms. This may be because P450 was expressed in cardiovascular diseases. lymphoblast cells with diŠerent levels of P450 reductase and cytochrome b5 cooperating with P450. The levels of Acknowledgements: We are grateful to Ms. M. these components of microsomal electron transfer diŠer Kamekawa of Okayama University Medical School, and between cell types. The system using puriˆed P450 can Ms. K. Yoshida of Osaka City University Medical be used to analyze P450-mediated NO production with School for their excellent technical assistance. constant levels of P450 reductase and cytochrome b5. NO Generation from NO-Aspirin by P450 19

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