articles Controlled Nitric Oxide Delivery Platform Based on S-Nitrosothiol Conjugated Interpolymer Complexes for Diabetic Wound Healing Yan Li and Ping I. Lee* Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario M5S 3M2, Canada Received September 20, 2009; Revised Manuscript Received December 7, 2009; Accepted December 23, 2009
Abstract: Nitric oxide (NO) is known to play a critical role in enhancing wound healing as topical NO administration has demonstrated enhanced wound healing in diabetic animal models. However, this approach has been limited by the short duration of NO release, short half-life of NO, and instability of available NO donors. To overcome these deficiencies, we have developed a new NO delivery platform based on grafting S-nitrosothiols (RSNOs), derived from endogenous glutathione (GSH) or its oligomeric derivatives, phytochelatins (PCs), onto poly(vinyl methyl etherco-maleic anhydride) (PVMMA), and their subsequent formation of interpolymer complexes with poly(vinyl pyrrolidone) (PVP). Such interpolymer complexes provide controlled release of NO for an extended duration (>10 days) and exhibit enhanced stability in the solid state over that of free RSNOs. The existence of intermolecular hydrogen bonding in such complexes and the formation of disulfide bonds following the NO release have been confirmed by FTIR and Raman. Preliminary wound healing study in a diabetic rat model demonstrates that, with a single topical application, the present controlled release NO delivery system can effectively accelerate wound closure as compared with the control (p < 0.05). The results suggest that the present NO releasing interpolymer complexes could be potentially useful for diabetic wound healing. Keywords: Nitric oxide; controlled release; interpolymer complex; S-nitrosothiols; diabetic wound healing
Introduction Nitric oxide (NO) is a ubiquitous biological mediator converted endogenously from L-arginine by nitric oxide synthases (NOS). The key physiological roles of NO in cardiovascular, nervous and immune systems have been identified and elucidated in recent years.1-3 As a result, a number of potential therapeutic applications of exogenous * To whom correspondence should be addressed. Mailing address: Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Tel: +1416-946-0606. Fax: +1-416-978-8511. E-mail: ping.lee@ utoronto.ca. (1) Barbato, J. E.; Tzeng, E. Nitric oxide and arterial disease. J. Vasc. Surg. 2004, 40, 187–193. (2) Toda, N.; Okamura, T. The pharmacology of nitric oxide in the peripheral nervous system of blood vessels. Pharmacol. ReV. 2003, 55, 271–324. 254
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NO delivery in cardiovascular diseases, respiratory diseases, cancer treatment, antimicrobial therapy, wound healing, and functional biomedical devices have been proposed and explored.4-8 Among the numerous benefits of NO, its critical (3) Taylor, E. L.; Megson, I. L.; Haslett, C.; Rossi, A. G. Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis. Cell Death Differ. 2003, 10, 418–430. (4) Groves, P. H.; Banning, A. P.; Penny, W. J.; Newby, A. C. The effects of exogenous nitric-oxide on smooth-muscle cell-proliferation following porcine carotid angioplasty. CardioVasc. Res. 1995, 30, 87–96. (5) Ricciardolo, F. L. M.; Sterk, P. J.; Gaston, B.; Folkerts, G. Nitric oxide in health and disease of the respiratory system. Physiol. ReV. 2004, 84, 731–765. (6) Wei, D. Y.; Richardson, E. L.; Zhu, K. Y.; Wang, L. W.; Le, X. D.; He, Y. J.; Huang, S. Y.; Xie, K. P. Direct demonstration of negative regulation of tumor growth and metastasis by hostinducible nitric oxide synthase. Cancer Res. 2003, 63, 3855–3859. 10.1021/mp900237f 2010 American Chemical Society
Published on Web 12/23/2009
Controlled Nitric Oxide DeliVery Platform role in the overall wound healing process involving granulation tissue formation, epidermal migration, collagen deposition and angiogenesis has motivated the pursuit of topical administration of NO and NO donors in promoting the healing of diabetic ulcers where impaired wound healing has been linked to NO and NOS deficiencies at the wound site.9-11 Such localized and topical delivery of NO is particularly advantageous as NO is only targeted to the specific wound site without eliciting a systemic load. However, these modes of treatment have been limited by the short duration of NO release, short half-life of NO in the physiological fluid, and instability of available NO donors.9,12,13 A range of NO donors derived from two major families of NO precursors such as diazeniumdiolates (NONOates) and S-nitrosothiols have been studied extensively.14-16 In an attempt to prolong the NO release for localized and topical delivery, NONOates have been incorporated into hydrophobic polymers such as polyurethane and poly(vinyl chloride) to provide extended NO release for a duration of from several hours up to a week.17-19 However, the clinical utility of (7) Ghavari, A.; Miller, C. C.; McMullin, B.; Ghahary, A. Potential application of gaseous nitric oxide as a topical antimicrobial agent. Nitric Oxide 2006, 14, 21–29. (8) Witte, M. B.; Barbul, A. Role of nitric oxide in wound repair. Am. J. Surg. 2002, 183, 406–412. (9) Isenberg, J. S.; Ridnour, L. A.; Espey, M. G.; Wink, D. A.; Roberts, D. D. Nitric oxide in wound- healing. Microsurgery 2005, 25, 442–451. (10) Schaffer, M. R.; Tantry, U.; Efron, P. A.; Ahrendt, G. M.; Thornton, F. J.; Barbul, A. Diabetes-impaired healing and reduced wound nitric oxide synthesis: a possible pathophysiologic correlation. Surgery 1997, 121, 513–519. (11) Witte, M. B.; Kiyama, T.; Barbul, A. Nitric oxide enhances experimental wound healing in diabetes. Br. J. Surg. 2002, 89, 1594–1601. (12) Ignarro, L. J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. ReV. Pharmacol. Toxicol. 1990, 30, 535–560. (13) Wang, P. G.; Cai, T. B. Nitric Oxide Donors; Taniguchi, N., Eds.; Wiley-VCH: Weinheim, 2005. (14) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Nitric oxide donors: chemical activities and biological applications. Chem. ReV. 2002, 102, 1091–1134. (15) Napoli, C.; Ignarro, L. J. Nitric oxide-releasing drugs. Annu. ReV. Pharmacol. Toxicol. 2003, 43, 97–123. (16) Miller, M. R.; Megson, I. L. Recent development in nitric oxide donor drugs. Br. J. Pharmacol. 2008, 151, 305–321. (17) Smith, D. J.; Chakravarthy, D.; Pulfer, S.; Simmons, M. L.; Hrabie, J. A.; Citro, M. L.; Saavedra, J. E.; Davies, K. M.; Hutsell, T. C.; Mooradian, D. L.; Hanson, S. R.; Keefer, L. K. Nitric oxidereleasing polymers containing the [N(O)NO]- group. J. Med. Chem. 1996, 39, 1148–1156. (18) Batchelor, M. M.; Reoma, S. L.; Fleser, P. S.; Nuthakki, V. K.; Callahan, R. E.; Shanley, C. J.; Politis, J. K.; Elmore, J.; Merz, S. I.; Meyerhoff, M. E. More lipophilic dialkyldiamine-based diazeniumdiolates: synthesis, characterization, and application in preparing thromboresistant nitric oxide release polymeric coatings. J. Med. Chem. 2003, 46, 5153–5161. (19) Zhou, Z.; Meyerhoff, M. E. Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin. Biomaterials 2005, 26, 6506–6517.
articles NONOates has been limited by the potential toxicity of the parent compound and byproduct of NONOate metabolism, particularly the possibility of forming carcinogenic secondary nitrosamines.20,21 Although C-based NONOates have been proposed to avoid the formation of carcinogenic nitrosoamines,22 not all such NONOates can release NO spontaneously,23 nor can they be readily adaptable to wound healing applications.24 On the other hand, S-nitrosothiols (with generic structure, RSNO) represent an important and a more desirable class of NO donors because they occur endogenously as Snitrosoglutathione (GSNO) or as RSNOs of sulfur-containing peptides and proteins.15,25 However, the stability of these RSNOs is often less than desirable as the S-NO bond is both thermally and photolytically labile, and susceptible to homolytic cleavage catalyzed by metal ions leading to the rapid release of NO with the corresponding formation of disulfides,26 thus limiting their suitability for localized and topical delivery of NO. In order to improve the stability and prolong the effective duration of RSNOs, a number of approaches have been pursued. One strategy involves the modification of molecular structure of low-molecular weight RSNOs to increase their lipophilicity,27 or to impart pHcontrolled NO release properties.28 These synthetic RSNOs and their decomposition products often involve chemical moieties of unknown pharmacological and toxicological nature thereby limiting their acceptability for in vivo applications. Another approach involves conjugating NO to larger peptides and proteins such as bovine serum albumin (BSA) or PEG-conjugated BSA through the cysteine residues.29,30 In addition to rapid NO release, another major drawback of these NO donors is their low level of achievable NO loading due to the limited cysteine content of BSA. (20) Kro¨ncke, K. D.; Suschek, C. V. Adulterated effect of nitric oxidegenerating donors. J. InVest. Dermatol. 2008, 128, 258–260. (21) Bauer, J. A.; Rao, W.; Smith, D. J. Evaluation of linear polyethyleneimine/nitric oxide adduct on wound repair: therapy versus toxicity. Wound Rep. Reg. 1998, 6, 569–577. (22) Arnold, E. V.; Doletski, B. G.; Raulli, R. E. Nitric oxide-releasing molecules. International Patent Application No. PCT/US05/ 000174. (23) http://home.ncifcrf.gov/lcc/nitricoxide/NOdonors.asp. (24) http://www.amuletpharma.com/techplatforms.html. (25) Richardson, G.; Benjamin, N. Potential therapeutic uses for S-nitrosothiols. Clin. Sci. 2002, 102, 99–105. (26) Williams, D. L. H. The mechanism of nitric oxide formation from S-nitrosothiols (thionitrites). Chem. Commun. 1996, 10, 1085– 1091. (27) Roy, B.; dı¨Hardemare, A. D.; Fontecave, M. New thionitrites: synthesis, stability, and nitric oxide generation. J. Org. Chem. 1994, 59, 7019–7026. (28) Lu, D.; Nadas, J.; Zhang, G.; Johnson, W.; Zweier, J. L.; Cardounel, A. J.; Villamena, F. A.; Wang, P. G. 4-Aryl-1,3,2oxathiazolylium-5-olates as pH-controlled NO-donors: the next generation of S-nitrosothiols. J. Am. Chem. Soc. 2007, 129, 5503– 5514. (29) Katsumi, H.; Nishikawa, M.; Ma, S. F.; Yamashita, F.; Hashida, M. Physicochemical, tissue distribution, and vasodilation characteristics of nitrosated serum albumin: delivery of nitric oxide in ViVo. J. Pharm. Sci. 2004, 93, 2343–2352. VOL. 7, NO. 1 MOLECULAR PHARMACEUTICS 255
articles Alternatively, synthetic polymers physically blended with RSNO have also been evaluated as NO delivery vehicles. In this regard, de Oliveira and co-workers incorporated NO donors such as GSNO and/or S-nitroso-N-acetylcysteine (SNAC) into films and gels based on water-soluble polymers such as poly(vinyl alcohol), poly(vinyl pyrrolidone), or Pluoronic F127 hydrogel, and evaluated such physical blends for topical NO delivery.31-33 Their animal results suggest that daily repeated application of GSNO-blended hydrogels during the early phases of cutaneous wound repair may accelerate wound closure and re-epithelialization.34 However, these physically blended NO donor systems still inherit the same drawbacks of free RSNOs such as rapid NO release and inactivation upon contact with physiological fluids due to thermal or metal ion catalyzed degradation. As a result, the delivery of NO from these existing RSNO based systems cannot be maintained for any extended duration, generally not more than several hours, thus requiring frequent reapplication from freshly prepared systems. In order to achieve a meaningful extension of NO release duration, the macromolecular prodrug approach with Snitrosothiol groups covalently attached to the polymer backbone appears to be a viable strategy for enhancing the stability of RSNOs, as well as in controlling the release duration of NO. In this regard, Bohl and West synthesized S-nitrosocysteine (CysNO)-derived PEG-hydrogels which exhibited reduced platelet adhesion and smooth muscle cell proliferation in cell culture.35 Despite this seemingly encouraging result, most of the NO release occurred in the first 4 h and the achievable level of NO loading in these hydrogels remained very low (
