SMA–copolymer conjugate of AHPP: A polymeric inhibitor of xanthine oxidase with potential antihypertensive effect Jun Fang a, 1 , Arun K. Iyer a,b, 1 , Takahiro Seki c , Hideaki Nakamura a , Khaled Greish a,2 , Hiroshi Maeda a, ⁎ a Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1, Kumamoto, Japan b Department of Applied Chemistry, Sojo University, Ikeda 4-22-1, Kumamoto, Japan c Innovative Collaboration Organization, Kumamoto University, Kumamoto, Japan abstract article info Article history: Received 9 September 2008 Accepted 14 January 2009 Available online 22 January 2009 Keywords: SMA AHPP Antihypertension Superoxide anion Nitric oxide In vascular system, superoxide anion (O 2 - ) generated by xanthine oxidase (XO) is known to regulate vascular tonus by reacting with, and thus consuming nitric oxide (NO), which determines vasorelaxation. We previously reported the remarkable antihypertensive effect of a potent XO inhibitor, 4-amino -6- hydroxypyrazolo[3,4-d]pyrimidine (AHPP). However, AHPP is insoluble in water, which hamper its in vivo application. Therefore, in this study we prepared a water soluble polymeric conjugate of AHPP, by using a styrene maleic acid copolymer (SMA, SMA–AHPP). SMA–AHPP showed similar inhibitory activity against XO (K i = 0.25 μM) comparable to native AHPP (K i =0.17 μM), while exhibiting good water-solubility, which now made it possible for systemic injection. In vivo experiments were carried out to examine the antihypertensive effect of SMA–AHPP using the spontaneously hypertensive rats (SHR) by i.v. injection (15, 30 mg/kg) or by oral administration (100 mg/kg) of SMA–AHPP. The results showed significantly reduced blood pressures (up to 30% reduction) of SHR rats; this antihypertensive effect continued for at least 24 h after SMA–AHPP administration. These findings strongly suggest the potential value of SMA–AHPP as an antihypertensive agent with sustained in vivo activity, which warrants further investigations. © 2009 Elsevier B.V. All rights reserved. 1. Introduction 4-Amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) is one of the most potent inhibitor of xanthine oxidase (XO), via competitively inhibiting the conversion of xanthine to uric acid catalyzed by XO [1], where superoxide (O 2 - ) is generated concomitantly. Thus O 2 - genera- tion is inhibited by AHPP. O 2 - has been well documented to play an important role in the pathogenesis of various disorders such as ischemia-reperfusion, infection and inflammatory injury [2,4]. Mean- while, it has been recently reported that O 2 - reacts with nitric oxide (NO) rapidly and hence NO was removed from the milieu [4–6]. Because XO is one of the major enzymes that produce O 2 - in vascular system [7–9], it is therefore reasonable that administration of XO inhibitor, e.g., AHPP, would prolong the biological half-life of NO and hence enhance the physiological effects of NO, especially vasodilation, by which AHPP might exhibit an antihypertentive function. NO is a well-known inorganic radical species with a variety of biological activities [10], and is produced by different isoforms of NO synthase (NOS) in many types of cells, including vascular endothelial cells, vascular smooth muscle cells, activated macrophages, neuronal cells, and glial cells. In particular, release of NO by NOS in the vascular endothelium (eNOS) modulates vascular tone, blood pressure, and tissue perfusion by activating guanylate cyclase in the smooth muscle cells under physiological conditions [10]. On the other hand, prolonged and excessive generation of NO caused by induction of inducible isoform of NOS (iNOS) under pathological conditions such as endotoxin-related septic shock, will lead to sustained vasodilation and shock [11]. NO-mediated endothelial effect is essential for maintaining normal vascular tone and functions, and deficiency of NO is greatly related with risk factors of vascular diseases, such as hypertension, hyperch- olesterolaemia, diabetes mellitus, and diseases involving smoking [12,13]. Numerous animal experiments demonstrated that NO played an important role in vascular disease pathogenesis. Targeted deletion of the eNOS gene in mice resulted in hypertension [14] and impaired vascular remodeling [15]. Whereas, increase of NO by NOS gene delivery improved endothelial function, limited neointimal prolifera- tion, and induced regression of atherosclerotic lesions [16]. The biological actions of NO are abrogated via a direct reaction with endogenous non-heme- and heme-containing proteins such as hemoglobin due to NO capturing capacity [10,17], with O 2 - [5,6], and with certain organic nitronylnitroxide radicals that scavenge NO [18]. Among these it has been indicated that increased production of O 2 - Journal of Controlled Release 135 (2009) 211–217 ⁎ Corresponding author. Tel./fax: +8196 326 4114. E-mail address: hirmaeda@ph.sojo-u.ac.jp (H. Maeda). 1 Equal contribution to this paper. 2 Present address: Dept. of Pharmaceutics, USTAR – Ghandehari Group, University of Utah, 383 Colorow Dr, Room 339, Salt Lake City, Utah 84108, USA. 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.01.006 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel