Fimasartan, Anti-Hypertension Drug, Suppressed Inducible Nitric Oxide

March 2013 Note Biol. Pharm. Bull. 36(3) 467–474 (2013) 467 Fimasartan, Anti-hypertension Drug, Suppressed Inducible Nitric Oxide Synthase Expressions via Nuclear Factor-Kappa B and Activator Protein-1 Inactivation Suran Ryu,a,c Ji-Sun Shin,a,d,e Young-Wuk Cho,c,d,e Hyoung Kook Kim,f Soo Heui Paik,f Joo Han Lee,f Yong Ha Chi,f Ji Han Kim,f Je Hak Kim,f and Kyung-Tae Lee*,a,b a Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University; b Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University; c Department of Biomedical Science, College of Medical Science, Kyung Hee University; d Reactive Oxygen Species Medical Research Center, School of Medicine, Kyung Hee University; e Department of Physiology, School of Medicine, Kyung Hee University; Seoul 130–701, Republic of Korea: and f Central Research Institute, Boryung Pharm. Co., Ltd.; Ansan, Gyoenggi 425–839, Republic of Korea. Received September 28, 2012; accepted December 18, 2012 Since inhibition of angiotensin II type 1 (AT1) receptor reduces chronic inflammation associated with hypertension, we evaluated the anti-inflammatory potential and the underlying mechanism of fimasartan, a Korean Food and Drug Administration approved anti-hypertension drug, in lipopolysaccharide (LPS)- stimulated RAW264.7 macrophages. Fimasartan suppressed the expressions of inducible nitric oxide synthase (iNOS) by down-regulating its transcription, and subsequently inhibited the productions of nitric oxide (NO). In addition, fimasartan attenuated LPS-induced transcriptional and DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). These reductions were accompanied by parallel reductions in the nuclear translocation of NF-κB and AP-1. Taken together, our data suggest that fimasartan down-regulates the expression of the iNOS in macrophages via NF-κB and AP-1 inactivation. Key words fimasartan; inflammation; nitric oxide; nuclear factor-kappa B; activator protein-1 Inflammatory processes are important participants in the (LPS).15) Nitric oxide (NO), a potent pro-inflammatory media- pathophysiology of hypertension and cardiovascular disease. tor, is mainly produced by macrophages and acts as a cytotox- Hypertension patients have impaired functions of the endothe- ic agent during immune and inflammatory responses, which lium, in which inflammation plays a key role.1) Angiotensin II is generated enzymatically by inducible nitric oxide synthase is recognized as an important vascular pro-inflammatory fac- (iNOS) at inflammatory sites. iNOS is induced by pro- tor in hypertension. Angiotensin II-driven vascular inflamma- inflammatory transcription factors, such as NF-κB and AP-1 tion is considered the result of direct modulates on cytokine which are present in the cytoplasm under normal conditions release and pro-inflammatory transcription factor including and translocate to nucleus in response to the pro-inflammatory nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), stimuli.16,17) and angiotehsin II receptor blockers (ARBs) reduce the pe- Fimasartan, 2-n-butyl-5-dimethylamino-thiocarbonyl-meth- ripheral and cerebrovascular inflammation associated with this yl-6-methyl-3-{[2-(1H-tetrazole-5-yl) biphenyl-4-yl] methyl}- disease.2,3) pyrimidin-4(3H)-one, formerly known as BR-A-657, is a novel Anti-inflammatory effects of ARBs, first established in the angiotensin II receptor antagonist exhibiting potent and selec- peripheral vasculature,4) were later demonstrated in the cere- tive angiotensin II receptor type 1 (AT1) blocking activity18) bral vasculature5) and in stress-induced gastric ulcerations.6) (Fig. 1A). Fimasartan approved by Korean Food and Drug These observations suggest that ARBs may exert general anti- Administration (KFDA) for the treatment of essential hyper- inflammatory effects beyond those associated with cardiovas- tension in September 2010 exerts a direct antihypertensive cular and metabolic disease.7) Thus, apart from being a major effect on vascular smooth muscle cells and an indirect anti- vasopressor effector of the renin-angiotensin system, ARBs hypertensive effect on sodium retention, fluid retention and are probably good candidate drugs for inflammation.8) vascular tone as mediated by blockade of angiotensin II.19) Since the important role of macrophages in inflammation, It seems that the identification of the mechanisms leading many investigations about ARBs (e.g., candesartan, losartan, to inhibition of inflammation by ARBs including fimasartan telmisartan and valsartan) were performed in lipopolysac- would contribute to the development of specific therapeutic charide (LPS)-induced macrophages.9–11) In fact, there are approaches to apply in hypertension and its complications. several physiological roles of angiotensin II type 1 (AT1) re- Therefore, it is necessary to investigate whether fimasartan ceptor in macrophages, such as positive regulation of peroxide has the anti-inflammatory potential, likewise other ARBs. production12) and shift M1/M2 polarization balance.13) How- In the present study, we investigated the anti-inflammatory ever, in macrophages AT1a receptor expression level is report- effects of the fimasartan in LPS-stimulated RAW264.7 macro- edly low and AT1b receptor is not detected at all14) and the phages. most important feature of macrophages is that macrophages are widely distributed in the body and provide an immedi- Materials and Methods ate defense against foreign agents, such as lipopolysaccharide Materials The fimasartan used for this study was syn- The authors declare no conflict of interest. thesized at Boryung Pharm. Co. (Seoul, Republic of Korea) * To whom correspondence should be addressed. e-mail: [email protected] © 2013 The Pharmaceutical Society of Japan 468 Vol. 36, No. 3 Fig. 1. effects of Fimasartan on the LPS-Induced NO Production and iNOS Expressions in RAW264.7 Macrophages (A) Chemical structure of fimasartan. (B) Following pretreatment with fimasartan (62.5, 125, or 250 µM) for 1 h, cells were treated with LPS (1 µg/mL) for 24 h (con- trols were not treated with LPS or fimasartan). L-NIL (20 µM) was used as a positive control. (C) Lysates were prepared from control, 24 h LPS (1 µg/mL) alone treated or LPS plus fimasartan (62.5, 125, or 250 µM) treated cells. Total cellular proteins (30 µg) were resolved by SDS-PAGe, transferred to PVDF membranes, and detected with specific antibodies. (D) Total RNA was prepared for qRT-PCR analysis of iNOS from cells stimulated with LPS (1 µg/mL) with/without fimasartan (62.5, 125, or 250 µM) for 4 h. The mRNA expressions of iNOS were determined as described in Materials and Methods. (e) Cells were transfected with a pGL3-iNOS promoter (−1592/+185) vector and the phRL-TK vector as an internal control. The level of luciferase activities were determined as described in Materials and Methods. Data are presented as the means±S.D. of three independent experiments. # p<0.05 vs. control cells; * p<0.05 ** p<0.01, *** p<0.001 vs. LPS-stimulated cells. ANOVA and Dunnett’s post-hoc test were used to determine the significances of differences. and its chemical structure was determined by spectral data transcriptase were purchased from Promega (Madison, WI, as described previously.18) The identity of this compound U.S.A.). SYBR green ex Taq was obtained from TaKaRa was confirmed by LC-MS and was found to be >98% pure. (Shiga, Japan). iNOS, β-actin oligonucleotide primers were Dulbecco’s modified eagle’s medium (DMeM), fetal bovine purchased from Bioneer (Seoul, Korea). 3-(4,5-Dimethylthi- serum, penicillin, and streptomycin were obtained from Life azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenyl- Technologies Inc. (Grand Island, NY, U.S.A.). iNOS, p65, methylsulfonylfluoride (PMSF), DL-dithiothreitol (DTT), c-Jun, c-Fos, poly(ADP ribose) polymerase (PARP), β-actin NS-398, LPS (Escherichia coli, serotype 0111:B4), and all monoclonal antibodies, and peroxidase-conjugated secondary other chemicals were purchased from Sigma Chemical Co. (St. antibody were purchased from Santa Cruz Biotechnology, Louis, MO, U.S.A.). Inc. (Santa Cruz, CA, U.S.A.). Random oligonucleotide prim- Cell Culture and Sample Treatment The RAW264.7 ers and Moloney Murine Leukemia Virus (M-MLV) reverse macrophage cell line was obtained from the Korea Cell Line March 2013 469 Bank (Seoul, Korea). These cells were grown at 37°C in Nuclear Extraction and Electrophoretic Mobility Shift DMeM supplemented with 10% fetal bovine serum (FBS), Assay (EMSA) RAW264.7 macrophages were plated in penicillin (100 units/mL) and streptomycin sulfate (100 µg/mL) 100-mm dishes (106 cells/mL), and treated with fimasartan in a humidified atmosphere 5% CO2. Cells were incubated (62.5, 125, or 250 µM), stimulated with LPS for 1 h, washed with fimasartan at concentration of 62.5, 125 or 250 µM, and once with PBS, scraped into 1 mL of cold PBS, and pelleted then stimulated with LPS 1 µg/mL for the indicated time. by centrifugation. Nuclear extracts were prepared as described Nitrite Determination RAW264.7 macrophages were previously.20) Nuclear extracts (5 µg) were mixed with double- plated at 5×105 cells/well in 24 well-plates and then incubated stranded NF-κB and AP-1 oligonucleotide, 5′-AGT TGA GGG with or without LPS (1 µg/mL) in the absence or presence of GAC TTT CCC AGG C-3′ and 5′-CGC TTG ATG ACT CAG various concentrations (62.5, 125, or 250 µM) of fimasartan for CCG GAA-3′, respectively, and end-labeled by biotin. DNA- 24 h. The nitrite accumulated

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