USOO7235237B2

(12) United States Patent (10) Patent No.: US 7,235,237 B2 L0ScalZ0 et al. (45) Date of Patent: Jun. 26, 2007

(54) METHODS OF TREATING VASCULAR 5,968,983 A 10/1999 Kaesemeyer DISEASES CHARACTERIZED BY NITRC 5,973,011 A 10/1999 Noack et al. OXIDE INSUFFICIENCY 6,103,769 A 8, 2000 Kelm 6,117,872 A 9, 2000 Maxwell et al. (75) Inventors: Joseph Loscalzo, Dover, MA (US); 6,319,515 B1 1 1/2001 Hidaka et al. Joseph A. Vita, Hingham, MA (US); 6,458,797 B1 10/2002 Adams et al. Michael D. Loberg, Boston, MA (US); 2004, OO63719 A1 4/2004 Adams et al. Manuel Worcel, Boston, MA (US) FOREIGN PATENT DOCUMENTS (73) Assignee: NitroMed, Inc., Lexington, MA (US) EP O327263 A 8, 1989 (*) Notice: Subject to any disclaimer, the term of this EP O 968 713 A1 5, 1998 patent is extended or adjusted under 35 JP O905915.2 3, 1997 U.S.C. 154(b) by 492 days. WO WO95/26725 10, 1995 WO WO 98.21 193 5, 1998 (21) Appl. No.: 10/415,136 WO WO 99.00361 1, 1999 WO WO 99,66921 12/1999 (22) PCT Filed: May 2, 2001 WO WO 99,67231 12/1999 WO WO O1/35961 A 5, 2001 (86). PCT No.: PCT/USO1A14245 OTHER PUBLICATIONS S 371 (c)(1), (2), (4) Date: Apr. 25, 2003 Dupuis, Cardiovascular Drugs and Therapy, 8(3):501-507 (1994). Cohn et al. The New England Journal of Medicine, 325(5):303-310 (87) PCT Pub. No.: WO02/34303 (1991). Cohn et al. The New England Journal of Medicine, 314(24): 1547 PCT Pub. Date: May 2, 2002 1552 (1986). Carson et al. Circulation, Supplement I, 92(8):I31-I32, Abstract No. (65) Prior Publication Data 0145 (1995). Francis et al. Circulation, Supplement VI, 87(6):VI40-VI48 (1993). US 2004/0005306 A1 Jan. 8, 2004 Pierpont et al. Chest, 73(1): 8-13 (1978). Massie et al. The American Journal of Cardiology, 40:794-801 Related U.S. Application Data (1977). (63) Continuation-in-part of application No. 09/697.317, (Continued) filed on Oct. 27, 2000, now Pat. No. 6,635,273, and a continuation-in-part of application No. PCT/US00/ Primary Examiner Jon Weber 29582, filed on Oct. 26, 2000. Assistant Examiner Kailash C. Srivastava (74) Attorney, Agent, or Firm Wilmer Cutler Pickering (60) Provisional application No. 60 179,020, filed on Jan. Hale and Dorr LLP 31, 2000, provisional application No. 60/162,230, filed on Oct. 29, 1999. (57) ABSTRACT (51) Int. Cl. A6 IK 38/43 (2006.01) The present invention provides methods of treating or pre A6 IK 3L/25 (2006.01) venting vascular diseases caused by nitric oxide (NO) insuf AOIN 37/2 (2006.01) ficiency. The methods encompass administering a composi AOIN 33/18 (2006.01) tion comprising an antioxidant, a compound to treat (52) U.S. Cl...... 424/94.1: 514/509: 514/562 cardiovascular diseases, a nitrosated compound, a com s s 51 4676 pound that donates, transfers or relases NO, or is a NO (58) Field of Classification Search 424/94.1 synthase Substrate, or endogenously stimulates NO synthe 424/94.4, 468: 5141s. 248.458 474 509. sis, or stimulates levels of endothelium derived relaxing Is I w \s 514/562.683 factor. In the said composition, a hydralazine compound See application file for complete search history. may be an antioxidant, isosorbide mono-ordinitrate may be the compound to donate, transfer, release, or stimulate 56 References Cited endogenous9. NO SVnthesis.y The isorsorbide mavy also elevate endogenous levels of endotherlium-derived relaxing factor, U.S. PATENT DOCUMENTS or be a NO synthase Substrate and angiotensin enzyme 4.293,565 A 10, 1981 Cordes et al. inhibitor may be nitrosated compound. Disclosed in the 4.584,315 A 4, 1986 Marshall invention is also a method to treat, or prevent Renaud's 4,868,179 A 9, 1989 Cohn syndrome by administering a therapeutically effective 5,627, 191 A 5, 1997 Birch et al. amount of an antioxidant, a NO donor, a nitrosated com 5,645,839 A 7, 1997 Chobanian et al. pound and novel Sustained-release formulations (e.g. a trans 5,760,069 A 6/1998 Lukas-Laskey et al. dermal patch). 5,891.459 A 4/1999 Cooke et al. 5,902,821 A 5/1999 Lukas-Laskey et al. 17 Claims, 3 Drawing Sheets US 7,235,237 B2 Page 2

OTHER PUBLICATIONS Exner, Derek V., et al., “Lesser Response to Angiotensin-Convert ing-Enzyme Inhibitor Therapy in Black as Compared with White Kaplan et al. Annals of Internal Medicine, 84:639-645 (1976). Patients with Left Ventricular Dysfunction.” N. Engl. J. Med., vol. Bauer et al. Circulation, 84(1):35-39 (1991). 344, No. 18, May 3, 2001, pp. 1351-1357. The SOLVD Investigators, The New England Journal of Medicine, Packer, Milton, et al., “Effect of Carvedilol on Survival in Severe 327(10):685-691 (1992). Chronic Heart Failure.” N. Engl. J. Med., vol. 344, No. 22, May 31, Ziesche et al. Circulation, 87(6):VI56-VI64 (1993). 2001, pp. 1651-1658. Rector et al. Circulation, 87(6):VI71-VIT7 (1993). “A Trial of the Beta-Blocker Bucindolol in Patients with Advanced Carson et al. Journal of Cardiac Failure, 5(3):178-187 (Sep. 10, Chronic Heart Failure.” N. Engl. J. Med., vol. 344, No. 22, May 31, 1999). 2001, pp. 1659-1667. Dries et al. The New England Journal of Medicine, 340(8):609-616 Braunwald, Eugene, “Expanding Indications for Beta-Blockers in (Feb. 25, 1999). Freedman et al. Drugs, 54(Supp. 3):41-50 (1997). Heart Failure.” N. Engl. J. Med., vol. 344, No. 22, May 31, 2001, Sherman et al. Cardiologia, 42(2): 177-187 (1997). pp. 1711-1712. Biegelson et al. Coronary Artery Disease, 10-241-256 (1999). Schwartz, Robert S., “Racial Profiling in Medical Research.” N. Rudd et al. Am. J. Physiol., 277(46):H723-H739 (1999). Engl. J. Med., vol. 344, No. 18, May 3, 2001, pp. 1392-1393. Hammerman et al. Am. J. Physiol., 277(46):H1579-H1592 (1999). Levine, T. Barry, “Paradoxical Hypertension after Reversal of Heart Loscalzo et al. Transactions of the American and Climatological Failure in Patients Treated with Intensive Vasodilator Therapy.” Ass., 111:158-163 (2000). American Journal of Hypertension, Ltd., 1998; 11:1041-1047. Johnson, Clinical Pharmacy, vol. 5, pp. 536 and 541 (1986). ABSTRACTS Heart Failure 179A, JACC, Feb. 1999. Turner et al. The American Journal of Cardiology, 47:910-916 Kahn, Jonathan, “How a Drug Becomes “Ethnic'. Law, Commerce, (1981). and the Production of Racial Categories in Medicine.” Yale Journal Massie et al. Circulation, 63(3):658-664 (1981). of Heath Policy, Law, and Ethics IV: 1 (2004), pp. 1-46. Nelson etal, Journal of Cardiovascular Pharmacology, 5(4):574-579 Parker, John D., et al., “The Effect of Hydralazine on the Devel (1983). opment of Tolerance to Continuous Nitroglycerin.” The Journal of Leier et al. Circulation, 63(1): 102-109 (1981). Pharmacology and Experimental Therapeutics, vol. 280, No. 2, pp. Wilson et al. The American Journal of Medicine, 71:627-633 866-875. (1981). Kalinowski, Leszek, et al., “Race-Specific Differences in Biddle et al., J. Clin. Pharmacol., 21:343-350 (1981). Endothelial Function—Predisposition of African Americans to Vas Margorien et al. Clinical Research, 27(4): A617 (1979), incomplete. cular Diseases.” Circulation, Jun. 1, 2004, pp. 25.11-2517. Turner et al. Clinical Research, 29(2):246A (1981), abstract only. Tucker, A.T. et al. Nov. 13, 1999, “Effect of nitric-oxide-generating Hibiya et al. Japanese Circulation Journal, 45:966 (Abstract No. system on microcirculatory blood flow in skin of patients with 338) (1981), abstract only. severe Raynaud's syndrome: a randomised trial', lancet vol. 354. Franciosa et al., Circulation, 59(6):1085-1091 (1979). #13, pp. 1670-1675. Lanas et al. Rev. Med. Chile, 107:926-930 (1979), abstract only. Apr. 05, 2005. Supplementary European Search Report from Euro Usdin et al. Coeur, 10(1): 119-128 (1979), article in french only pean Patent Application No. 01932915.0. abstract considered. Jun. 21, 2004. Supplementary Partial European Search Report for Burnier et al. Hypertension, 22(3):339-347 (Sep. 1993), recieved EP OO 97 6651O. May 30, 2006. Franciosa, Joseph A., “African-American Heart Failure Trial von Lutterotti et al. American Journal of Hypertension, 4(4 Part (A-HeFT): Rationale, Design, and Methodology,” Journal of Car 2):346S-349S (1991), recd May 30, 2006. diac Failure, vol. 8, No. 3, 2002, pp. 128-135. Massie et al. Br. Heart J., 45:37.6-384 (1981), recd. May 30, 2006. Yancy, Clyde W., et al., “Race and the Response to Adrenergic Lanas et al. Rev. Med. Chile, 107:926-930 (1979). Blockage with Carvedilol in Patients with Chronic Heart Failure.” Usdin et al. Coeur, 10(1): 119-128 (1979). N. Engl. J. Med., vol. 344, No. 18, May 3, 2001, pp. 1358-1365. Parker, John D., et al., “The Effect of Hydralazine on the Devel Yancy, Clyde W. “Heart Failure in African Americans: A Cardio opment of Tolerance to Continuous Nitroglycerin.” The Journal of vascular Enigma,” Journal of Cardiac Failure, vol. 6, No. 3, 2000, Pharmacology and Experimental Therapeutics, vol. 280, No. 2, pp. pp. 183-186. 866-875, 1997. U.S. Patent Jun. 26, 2007 Sheet 1 of 3 US 7,235,237 B2

FIG. 1

Decreased Renin Release NO Insufficiency Salt/Water Retention

Increased Intracellular Increased Sensitivity to Sodium & Calcium Catecholamines and Angiotensin II

LVH a. Increased Vascular Tone

Decreased Capillary Density Myocardial Fibrosis

Hypertension Microvascular Increased LV Mass Cardiac schemia Diastolic Dysfunction

FIG. 2 -o-White hypertensives -e-Black typertensives a 15 5 O CS / S u-21 4. O

Baseline O.3 O 3.0 e 10 Methachotine Dose (glnin) U.S. Patent Jun. 26, 2007 Sheet 2 of 3 US 7,235,237 B2 FIG. 3A

-o-White normotensives -e-Black nomotensives

Baseline 0.3 1.0 3.0 10 Nitroprusside Dose (glamin)

FIG. 3B -O-White nomotensives -O-Blacknomotensives

Basettine 0.3 O 3.0 1O Methachotine Dose (g/min) U.S. Patent Jun. 26, 2007 Sheet 3 of 3 US 7,235,237 B2

FIG. 4A

-o- Salt Resistant (n=25) 1N -O - Satt Sensitive (n=6) N 9.O 15 4. S 10 u- -1

Fo- 2 Pr0.9 BaSettine 0.3 1.0 3.0 10 Methachotine Dose (g/min)

-O-Satt Resistant (n=25) To - Satt Sensitive (n=6)

BaselineNitroprusside O3 (O Dose 3.0 (g/min) 1O US 7,235,237 B2 1. 2 METHODS OF TREATING VASCULAR nitrosated endothelin antagonist, nitrosated angiotensin II DISEASES CHARACTERIZED BY NITRC receptor antagonist, nitrosated renin inhibitor, and/or at least OXDE INSUFFICIENCY one compound used to treat cardiovascular diseases. The present invention also provides Sustained release formula RELATED APPLICATIONS tion comprising at least one antioxidant or a pharmaceuti cally acceptable salt thereof, and at least one nitric oxide This application is a continuation-in-part of U.S. appli donor, and, optionally, at least one nitrosated compound. cation Ser. No. 09/697,317, filed Oct. 27, 2000 now U.S. Pat. No. 6,635,273, and PCT Application No. PCT/US00/29582, BACKGROUND OF THE INVENTION filed Oct. 26, 2000, both of which claim priority to U.S. 10 Provisional Application No. 60/162,230 filed Oct. 29, 1999 The decline in cardiovascular morbidity and mortality in and U.S. Provisional Application No. 60/179,020 filed Jan. the United States over the past three decades has been the 31, 2000. result of significant advances in research on cardiovascular disease mechanisms and therapeutic strategies. The inci FIELD OF THE INVENTION 15 dence and prevalence of myocardial infarction and death from myocardial infarction, as well as that from cerebrovas The present invention provides methods of treating and/or cular accident, have decreased significantly over this period preventing vascular diseases characterized by nitric oxide largely owing to advances in prevention, early diagnosis, insufficiency by administering a therapeutically effective and treatment of these very common diseases. amount of at least one antioxidant or a pharmaceutically Analysis of outcomes by race, however, paints quite a acceptable salt thereof, and at least one compound that different picture: life expectancy and cardiovascular mor donates, transfers or releases nitric oxide, elevates endog bidity rates have improved far less for blacks than whites. enous levels of endothelium-derived relaxing factor, stimu Available data show that the likelihood of dying from lates endogenous synthesis of nitric oxide or is a substrate cardiovascular disease is far greater among black Americans for nitric oxide synthase, and, optionally, at least one nitro 25 than among white Americans. In this decade, the death rate sated angiotensin-converting enzyme inhibitor, nitrosated from cardiovascular disease for black males was 353 per beta-adrenergic blocker, nitrosated calcium channel blocker, 100,000 population, while that for white males was 244 per nitrosated endothelin antagonist, nitrosated angiotensin II 100,000; the rate for black females was 226 per 100,000; receptor antagonist, nitrosated renin inhibitor, and/or at least while that for white females was 135 per 100,000. Conso one compound used to treat cardiovascular diseases. The 30 nant with this important demographic parameter is the antioxidant may preferably be a hydralazine compound or a observation that there is a higher prevalence of several of the pharmaceutically acceptable salt thereof. The compound important risk factors for cardiovascular disease, e.g., hyper that donates, transfers or releases nitric oxide, elevates tension, Smoking, diabetes mellitus, obesity, and left ven endogenous levels of endothelium-derived relaxing factor, tricular hypertrophy, among blacks compared with whites. stimulates endogenous synthesis of nitric oxide or is a 35 In addition, outcomes of cardiovascular events are worse for substrate for nitric oxide synthase may preferably be isos blacks than whites. Following myocardial infarction, blacks orbide dinitrate and/or isosorbide mononitrate. The present have a 50% higher annual mortality rate than whites, and invention also provides methods of treating and/or prevent their five year survival is only 70%. Thus, the many ing vascular diseases characterized by nitric oxide insuffi advances in cardiovascular medicine that account for the ciency by administering a therapeutically effective amount 40 overall improvement in cardiovascular health in the general of at least one nitrosated angiotensin-converting enzyme population have failed to translate into comparable racial inhibitor, nitrosated beta-adrenergic blocker, nitrosated cal benefits. cium channel blocker, nitrosated endothelin antagonist, nit There is a need in the art for new and more effective rosated angiotensin II receptor antagonist and/or nitrosated compositions and methods for treating vascular diseases. renin inhibitor, and, optionally, at least one antioxidant 45 The present invention is directed to these, as well as other, and/or at least one compound used to treat cardiovascular important ends. diseases. The present invention also provides methods of treating and/or preventing Raynaud's syndrome by admin SUMMARY OF THE INVENTION istering a therapeutically effective amount of at least one antioxidant or a pharmaceutically acceptable salt thereof, 50 The present invention provides methods for treating and/ and at least one compound that donates, transfers or releases or preventing vascular diseases characterized by nitric oxide nitric oxide, elevates endogenous levels of endothelium insufficiency by administering to a patient a therapeutically derived relaxing factor, stimulates endogenous synthesis of effective amount of at least one antioxidant or a pharma nitric oxide or is a Substrate for nitric oxide synthase, and, ceutically acceptable salt thereof, and at least one compound optionally, at least one nitrosated angiotensin-converting 55 that donates, transfers or releases nitric oxide, elevates enzyme inhibitor, nitrosated calcium channel blocker, nit endogenous levels of endothelium-derived relaxing factor, rosated endothelin antagonist, nitrosated angiotensin II stimulates endogenous synthesis of nitric oxide or is a receptor antagonist and/or nitrosated renin inhibitor. The Substrate for nitric oxide synthase, and, optionally, at least present invention also provides novel transdermal patches one nitrosated angiotensin-converting enzyme inhibitor, nit comprising at least one antioxidant or a pharmaceutically 60 rosated beta-adrenergic blocker, nitrosated calcium channel acceptable salt thereof, and at least one compound that blocker, nitrosated endothelin antagonist, nitrosated angio donates, transfers or releases nitric oxide, elevates endog tensin II receptor antagonist, nitrosated renin inhibitor, and/ enous levels of endothelium-derived relaxing factor, stimu or at least one compound used to treat cardiovascular lates endogenous synthesis of nitric oxide or is a substrate diseases. The antioxidant may preferably be a hydralazine for nitric oxide synthase, and, optionally, at least one nitro 65 compound or a pharmaceutically acceptable salt thereof. sated angiotensin-converting enzyme inhibitor, nitrosated The compound that donates, transfers or releases nitric beta-adrenergic blocker, nitrosated calcium channel blocker, oxide, elevates endogenous levels of endothelium-derived US 7,235,237 B2 3 4 relaxing factor, stimulates endogenous synthesis of nitric Ventricular hypertrophy with inadequate capillary angiogen oxide or is a Substrate for nitric oxide synthase may pref esis, and increased matrix production with myocardial fibro erably be isosorbide dinitrate and/or isosorbide mononitrate. sis result. These intermediate phenotypes lead to the clinical The antioxidant and the nitric oxide donor and optional disorders of low-renin, salt-sensitive hypertension; dispro nitrosated compound and/or compound used to treat cardio portionate left ventricular hypertrophy and diastolic dys vascular diseases can be administered separately or as function; and microvascular myocardial ischemia. components of the same composition. FIG. 2 shows forearm blood flow responses to intra Another aspect of the present invention provides methods arterial methacholine that were assessed using venous occlu for treating and/or preventing vascular diseases character sion plethysmography in 20 white and 16 black patients with ized by nitric oxide insufficiency by administering to a 10 a clinical history of hypertension (BP>140/90). FIG. 2 patient a therapeutically effective amount of at least one shows that endothelium-derived NO action is impaired in nitrosated angiotensin-converting enzyme inhibitor, nitro the forearm microvessels of the black patients compared to sated beta-adrenergic blocker, nitrosated calcium channel the white patients. blocker, nitrosated endothelin antagonist, nitrosated angio FIGS. 3A and 3B show forearm blood flow responses to tensin II receptor antagonist and/or nitrosated renin inhibi 15 nitroprusside (FIG. 3A) and methacholine (FIG. 3B) that tor, and, optionally, at least one antioxidant and/or at least were assessed by venous occlusion plethysmography in 25 one compound used to treat cardiovascular diseases. The white and 21 black patients without hypertension. The nitrosated compound and optional antioxidant and/or com dilator response to sodium nitroprusside (FIG. 3A) was pound used to treat cardiovascular diseases can be admin significantly lower in black patients, while there was no istered separately or as components of the same composi racial difference in response to methacholine (FIG. 3B). tion. FIGS. 4A and 4B show the effect of salt-sensitivity on In another aspect, the present invention provides methods forearm microvascular function. By repeated measures for treating and/or preventing Raynaud's syndrome by ANOVA, there were trends for impaired responses to metha administering to a patient a therapeutically effective amount choline (FIG. 4A) and sodium nitroprusside (FIG. 4B) in of at least one antioxidant or a pharmaceutically acceptable 25 salt-sensitive black patients. salt thereof, and at least one compound that donates, trans fers or releases nitric oxide, elevates endogenous levels of DETAILED DESCRIPTION OF THE endothelium-derived relaxing factor, stimulates endogenous INVENTION synthesis of nitric oxide or is a substrate for nitric oxide synthase, and, optionally, at least one nitrosated angiotensin 30 As used throughout the disclosure, the following terms, converting enzyme inhibitor, nitrosated calcium channel unless otherwise indicated, shall be understood to have the blocker, nitrosated endothelin antagonist, nitrosated angio following meanings. tensin II receptor antagonist and/or nitrosated renin inhibi “Patient” refers to animals, preferably mammals, most tor. The antioxidant, nitric oxide donor, and nitrosated preferably humans, and includes males and females. compound can be administered separately or as components 35 “Therapeutically effective amount” refers to the amount of the same composition. of the compound and/or composition that is effective to In yet another aspect, the present invention provides novel achieve its intended purpose. transdermal patches comprising a therapeutically effective “Transdermal refers to the delivery of a compound by amount of at least one antioxidant at least one compound passage through the skin and into the blood stream. that donates, transfers or releases nitric oxide, elevates 40 endogenous levels of endothelium-derived relaxing factor, “Transmucosal refers to delivery of a compound by stimulates endogenous synthesis of nitric oxide or is a passage of the compound through the mucosal tissue and Substrate for nitric oxide synthase, and, optionally, at least into the blood stream. one nitrosated angiotensin-converting enzyme inhibitor, nit “Penetration enhancement” or “permeation enhance rosated beta-adrenergic blocker, nitrosated calcium channel 45 ment” refers to an increase in the permeability of the skin or blocker, nitrosated endothelin antagonist, nitrosated angio mucosal tissue to a selected pharmacologically active com tensin II receptor antagonist, nitrosated renin inhibitor, and/ pound Such that the rate at which the compound permeates or at least one compound used to treat cardiovascular through the skin or mucosal tissue is increased. diseases. “Carriers' or “vehicles' refers to carrier materials suitable In another aspect, the present invention provides Sus 50 for compound administration and include any Such material tained release formulations comprising a therapeutically known in the art such as, for example, any liquid, gel. effective amount of at least one antioxidant or a pharma solvent, liquid diluent, solubilizer, or the like, which is ceutically acceptable salt thereof, and at least one nitric non-toxic and which does not interact with any components oxide donor, and, optionally, at least one nitrosated com of the composition in a deleterious manner. pound. 55 “Sustained release' refers to the release of a therapeuti These and other aspects of the present invention are cally active compound and/or composition Such that the described in more detail herein. blood levels of the therapeutically active compound are maintained within a desirable therapeutic range over an BRIEF DESCRIPTION OF THE FIGURES extended period of time. The sustained release formulation 60 can be prepared using any conventional method known to FIG. 1 shows that nitric oxide (NO) insufficiency is one skilled in the art to obtain the desired release charac associated with increased salt and water retention and a teristics. low-renin State. Increased intracellular sodium and calcium "Nitric oxide donor or “NO donor refers to compounds in conjunction with reduced NO leads to enhanced sensi that donate, release and/or directly or indirectly transfer a tivity of vascular Smooth muscle cells and cardiomyocytes 65 nitrogen monoxide species, and/or stimulate the endogenous to the tonic and growth-stimulating properties of catechola production of nitric oxide or endothelium-derived relaxing mines and angiotensin II. Increased vascular tone, left factor (EDRF) in vivo and/or elevate endogenous levels of US 7,235,237 B2 5 6 nitric oxide or EDRF in vivo. “NO donor also includes Exemplary cycloalkyl groups include cyclopropyl, cyclobu compounds that are Substrates for nitric oxide synthase. tyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohepta, 1.3- “Nitric oxide adduct or “NO adduct refers to com dienyl, and the like. pounds and functional groups which, under physiological “Heterocyclic ring or group' refers to a saturated, unsat conditions, can donate, release and/or directly or indirectly urated, cyclic or aromatic or polycyclic hydrocarbon group transfer any of the three redox forms of nitrogen monoxide (NO", NO, NO.), such that the biological activity of the having about 2 to about 10 carbon atoms (preferably about nitrogen monoxide species is expressed at the intended site 4 to about 6 carbon atoms) where 1 to about 4 carbon atoms of action. are replaced by one or more nitrogen, oxygen and/or Sulfur "Nitric oxide releasing or "nitric oxide donating refers 10 atoms. Sulfur may be in the thio, sulfinyl or sulfonyl to methods of donating, releasing and/or directly or indi oxidation state. The heterocyclic ring or group can be fused rectly transferring any of the three redox forms of nitrogen to an aromatic hydrocarbon group. Heterocyclic groups can monoxide (NO". NO-, NO.), such that the biological activ be unsubstituted or substituted with one, two or three ity of the nitrogen monoxide species is expressed at the Substituents independently selected from alkyl, alkoxy, intended site of action. 15 amino, alkylamino, dialkylamino, arylamino, diarylamino, “Alkyl refers to a lower alkyl group, a haloalkyl group, alkylarylamino, hydroxy, OXo, thial, halo, carboxyl, car an alkenyl group, an alkynyl group, a bridged cycloalkyl boxylic ester, alkylcarboxylic acid, alkylcarboxylic ester, group, a cycloalkyl group or a heterocyclic ring, as defined aryl, arylcarboxylic acid, arylcarboxylic ester, amidyl, ester, herein. carboxamido, alkylcarboxamido, arylcarboxamido, Sulfonic acid, Sulfonic ester, Sulfonamido and nitro. Exemplary het “Lower alkyl refers to branched or straight chain acyclic erocyclic groups include pyrrolyl, 3-pyrrolinyl,4,5,6-trihy alkyl group comprising one to about ten carbon atoms dro-2H-pyranyl, pyridinyl, 1,4-dihydropyridinyl, pyrazolyl, (preferably one to about eight carbon atoms, more preferably triazolyl pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl, imi one to about six carbon atoms). Exemplary lower alkyl dazolyl, indolyl, thiophenyl, furanyl, tetrhydrofuranyl, tet groups include methyl, ethyl, n-propyl, isopropyl. n-butyl, razolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl. 25 oxazolindinyl 1,3-dioxolanyl, 2,6-dioxabicyclo[3.3.0 octa hexyl, octyl, and the like. nyl, 2-imidazonlinyl, imidazolindinyl, 2-pyrazolinyl, pyra "Haloalkyl refers to a lower alkyl group, an alkenyl Zolidinyl, isoxazolyl, isothiazolyl, 1.2.3-oxadiazolyl, 1.2.3- group, an alkynyl group, a bridged cycloalkyl group, a triazolyl, 1.3,4-thiadiazolyl, 2H-pyranyl, 4H-pyranyl. cycloalkyl group or a heterocyclic ring, as defined herein, to piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thio which is appended one or more halogens, as defined herein. 30 Exemplary haloalkyl groups include trifluoromethyl, chlo morpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1.3.5- romethyl, 2-bromobutyl, 1-bromo-2-chloro-pentyl, and the trithianyl, benzo(b)thiophenyl, benzimidazolyl, quinolinyl, like. and the like. “Alkenyl refers to a branched or straight chain C. Co “Heterocyclic compounds’ refer to mono- and polycyclic hydrocarbon (preferably a C-C hydrocarbon, more pref 35 compounds comprising at least one aryl or heterocyclic ring. erably a C-C hydrocarbon) which can comprise one or "Aryl refers to a monocyclic, bicyclic, carbocyclic or more carbon-carbon double bonds. Exemplary alkenyl heterocyclic ring system comprising one or two aromatic groups include propylenyl, buten-1-yl, isobutenyl, penten rings. Exemplary aryl groups include phenyl, pyridyl, 1-yl, 2.2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, napthyl, quinoyl, tetrahydronaphthyl, furanyl, indanyl, inde hepten-1-yl, octen-1-yl, and the like. 40 nyl, indoyl, and the like. Aryl groups (including bicylic aryl “Alkynyl refers to an unsaturated acyclic C. Cohydro groups) can be unsubstituted or Substituted with one, two or carbon (preferably a C-C hydrocarbon, more preferably a three substituents independently selected from alkyl, alkoxy, C-C hydrocarbon) which can comprise one or more car amino, alkylamino, dialkylamino, arylamino, diarylamino, bon-carbon triple bonds. Exemplary alkylnyl groups include alkylarylamino, hydroxy, carboxyl, carboxylic ester, alkyl ethynyl, propynyl, butyn-1-yl, butyn-2-yl, pentyl-1-yl, pen 45 carboxylic acid, alkylcarboxylic ester, aryl, arylcarboxylic tyl-2-yl, 3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl, hexyl acid, arylcarboxylic ester, alkylcarbonyl, arylcarbonyl, 3-yl, 3.3-dimethyl-butyn-1-yl, and the like. amidyl, ester, carboxamido, alkylcarboxamido, carbomyl. “Bridged cycloalkyl refers to two or more cycloalkyl Sulfonic acid, Sulfonic ester, Sulfonamido and nitro. Exem groups, heterocyclic groups, or a combination thereof fused plary Substituted aryl groups include tetrafluorophenyl, pen via adjacent or non-adjacent atoms. Bridged cycloalkyl 50 tafluorophenyl, Sulfonamide, alkylsulfonyl, arylsulfonyl, groups can be unsubstituted or Substituted with one, two or and the like. three substituents independently selected from alkyl, alkoxy, “Alkylaryl” refers to an alkyl group, as defined herein, to amino, alkylamino, dialkylamino, hydroxy, halo, carboxyl, which is appended an aryl group, as defined herein. Exem alkylcarboxylic acid, aryl, amidyl, ester, alkylcarboxylic plary alkylaryl groups include benzyl, phenylethyl, ester, carboxamido, alkylcarboxamido, oxo and nitro. Exem 55 hydroxybenzyl, fluorobenzyl, fluorophenylethyl, and the plary bridged cycloalkyl groups include adamantyl, decahy like. dronapthyl, quinuclidyl, 2,6-dioxabicyclo3.3.0 octane, 7-Oxabycyclo[2.2.1]heptyl, 8-azabicyclo[3.2.1]oct-2-enyl "Arylalkyl refers to an aryl radical, as defined herein, and the like. attached to an alkyl radical, as defined herein. “Cycloalkyl refers to a saturated or unsaturated cyclic 60 “Cycloalkylalkyl refers to a cycloalkyl radical, as hydrocarbon comprising from about 3 to about 10 carbon defined herein, attached to an alkyl radical, as defined atoms. Cycloalkyl groups can be unsubstituted or Substituted herein. with one, two or three substituents independently selected “Heterocyclicalkyl refers to a heterocyclic ring radical, from alkyl, alkoxy, amino, alkylamino, dialkylamino, ary as defined herein, attached to an alkyl radical, as defined lamino, diarylamino, alkylarylamino, aryl, amidyl, ester, 65 herein. hydroxy, halo, carboxyl, alkylcarboxylic acid, alkylcarboxy “Cycloalkenyl refers to an unsaturated cyclic hydrocar lic ester, carboxamido, alkylcarboxamido, oxo and nitro. bon having about 3 to about 10 carbon atoms (preferably US 7,235,237 B2 7 8 about 3 to about 8 carbon atoms, more preferably about 3 to “Dialkylamino” refers to RsoRsN—, wherein Rso and about 6 carbon atoms) comprising one or more carbon Rs are each independently an alkyl group, as defined carbon double bonds. herein. Exemplary dialkylamino groups include dimethy "Arylheterocyclic ring” refers to a bi- or tricyclic ring lamino, diethylamino, methyl propargylamino, and the like. comprised of an aryl ring, as defined herein, appended via “Diarylamino” refers to RssRN—, wherein Rss and Ro two adjacent carbon atoms of the aryl ring to a heterocyclic are each independently an aryl group, as defined herein. ring, as defined herein. Exemplary arylheterocyclic rings “Alkylarylamino” refers to RoRsN—, wherein Rs is an include dihydroindole, 1,2,3,4-tetra-hydroquinoline, and the alkyl group, as defined herein, and Rss is an aryl group, as like. defined herein. "Alkoxy' refers to RsoO—, wherein Rso is an alkyl group, 10 “Aminoalkyl refers to an amino group, an alkylamino as defined herein. Exemplary alkoxy groups include meth group, a dialkylamino group, an arylamino group, a diary oxy, ethoxy, t-butoxy, cyclopentyloxy, and the like. lamino group, an alkylarylamino group or a heterocyclic "Arylalkoxy or alkoxyaryl” refers to an alkoxy group, as ring, as defined herein, to which is appended an alkyl group, defined herein, to which is appended an aryl group, as as defined herein. defined herein. Exemplary arylalkoxy groups include ben 15 “Aminoaryl” refers to an amino group, an alkylamino Zyloxy, phenylethoxy, chlorophenylethoxy, and the like. group, a dialkylamino group, an arylamino group, a diary "Aryloxy' refers to Rs.O—, wherein Rss is an aryl group, lamino group, an alkylarylamino group or a heterocyclic as defined herein. Exemplary aryloxy groups include ring, as defined herein, to which is appended an aryl group, napthyloxy, quinolyloxy, isoquinolizinyloxy, and the like. as defined herein. “Alkoxyalkyl refers to an alkoxy group, as defined “Thio’ refers to - S -. herein, appended to an alkyl group, as defined herein. “Sulfinyl refers to S(O)-. Exemplary alkoxyalkyl groups include methoxymethyl, “Methanthial' refers to C(S)-. methoxyethyl, isopropoxymethyl, and the like. “Thial refers to =S. “Alkoxyhaloalkyl refers to an alkoxy group, as defined “Sulfonyl refers to S(O). herein, appended to a haloalkyl group, as defined herein. 25 Exemplary alkoxyhaloalkyl groups include 4-methoxy-2- "Sulfonic acid refers to —S(O)OR, wherein R, is a chlorobutyl and the like. hydrogen, an organic cation or an inorganic cation. “Cycloalkoxy” refers to RO , wherein Rs is a “Alkylsulfonic acid refers to a sulfonic acid group, as cycloalkyl group or a bridged cycloalkyl group, as defined defined herein, appended to an alkyl group, as defined herein. herein. Exemplary cycloalkoxy groups include cyclopropy 30 loxy, cyclopentyloxy, cyclohexyloxy, and the like. "Arylsulfonic acid refers to an Sulfonic acid group, as "Haloalkoxy” refers to a haloalkyl group, as defined defined herein, appended to an aryl group, as defined herein herein, to which is appended an alkoxy group, as defined "Sulfonic ester” refers to —S(O)ORss, wherein Rss is an herein. Exemplary haloalkyl groups include 1,1,1-trichloro alkyl group, an aryl group, an alkylaryl group or an aryl ethoxy, 2-bromobutoxy, and the like. 35 heterocyclic ring, as defined herein. “Hydroxy” refers to –OH. "Sulfonamido” refers to —S(O), N(Rs) (Rs.), wherein “Oxo’ refers to =O. Rs and Rs 7 are each independently a hydrogen atom, an “Oxy' refers to —OR7, wherein R, , is an organic or alkyl group, an aryl group, an alkylaryl group, or an aryl inorganic cation. heterocyclic ring, as defined herein, or Rs and Rs, taken "Organic cation” refers to a positively charged organic 40 together are a heterocyclic ring, a cycloalkyl group or a ion. Exemplary organic cations include alkyl Substituted bridged cycloalkyl group, as defined herein. ammonium cations, and the like. “Alkylsulfonamido” refers to a sulfonamido group, as “Inorganic cation” refers to a positively charged metal defined herein, appended to an alkyl group, as defined ion. Exemplary inorganic cations include Group I metal herein. cations such as for example, sodium, potassium, and the 45 "Arylsulfonamido” refers to a Sulfonamido group, as like. defined herein, appended to an aryl group, as defined herein. “Hydroxyalkyl refers to a hydroxy group, as defined “Alkylthio’ refers to Rs.S.—, wherein Rs is an alkyl herein, appended to an alkyl group, as defined herein. group, as defined herein. “Amino” refers to NH. "Arylthio’ refers to Rss S , wherein Rss is an aryl group, “Nitrate” refers to - O NO. 50 as defined herein. “Nitrite refers to - O NO. “Cycloalkylthio’ refers to RS , wherein Rs is a “Thionitrate” refers to S NO. cycloalkyl group or a bridged cycloalkyl group, as defined “Thionitrite and “nitrosothiol refer to S NO. herein. Exemplary cycloalkylthio groups include cyclopro pylthio, cyclopentylthio, cyclohexylthio, and the like. "Nitro” refers to the group - NO, and “nitrosated” refers 55 to compounds that have been substituted therewith. “Alkylsulfinyl refers to Rs S(O)—, wherein Rs is an "Nitroso’ refers to the group - NO and “nitrosylated alkyl group, as defined herein. refers to compounds that have been substituted therewith. “Alkylsulfonyl refers to Rs S(O)—, wherein Rs is “Nitrile' and “cyano” refer to CN. an alkyl group, as defined herein. "Halogen' or “halo' refers to iodine (I), bromine (Br). 60 "Arylsulfinyl' refers to Rss S(O) , wherein Rs is an chlorine (Cl), and/or fluorine (F). aryl group, as defined herein. “Alkylamino” refers to RsNH , wherein Rs is an alkyl "Arylsulfonyl refers to Rss S(O) , wherein Rss is an group, as defined herein. Exemplary alkylamino groups aryl group, as defined herein. include methylamino, ethylamino, butylamino, cyclohexy “Amidyl refers to RC(O)N(Rs.)— wherein Rs and lamino, and the like. 65 Rs7 are each independently a hydrogenatom, an alkyl group, "Arylamino” refers to RNH , wherein Rss is an aryl an aryl group, an alkylaryl group, or an arylheterocyclic group, as defined herein. ring, as defined herein. US 7,235,237 B2 9 10 “Ester refers to RC(O)O— wherein Rs is a hydrogen “Hydralazine compound” refers to a compound having atom, an alkyl group, an aryl group, an alkylaryl group, or the formula: an arylheterocyclic ring, as defined herein. “Carbamoyl refers to —O C(O)N(Rs)(Rs.), wherein R4 R3 Rs and Rs, are each independently a hydrogen atom, an a b c alkyl group, an aryl group, an alkylaryl group or an aryl R - FN FNR heterocyclic ring, as defined herein, or Rs and Rs 7 taken together are a heterocyclic ring, a cycloalkyl group or a bridged cycloalkyl group, as defined herein. 10 wherein a, b and c are independently a single or double “Carbamate” refers to Rs O C(O)N (Rs.), wherein bond; R and R are each independently a hydrogen, an Rs and Rs, are each independently a hydrogen atom, an alkyl, an ester or a heterocyclic ring, wherein alkyl, ester and alkyl group, an aryl group, an alkylaryl group or an aryl heterocyclic rind are as defined herein; R and Ra are each heterocyclic ring, as defined herein, or Rs and Rs 7 taken independently a lone pair of electrons or a hydrogen, with together are a heterocyclic ring, a cycloalkyl group or a 15 the proviso that at least one of R. R. R. and R is not a bridged cycloalkyl group, as defined herein. hydrogen. Exemplary hydralazine compounds include “Carboxyl refers to —C(O)OR, wherein Rze is a budralazine, cadralazine, dihydralazine, endralazine, hydrogen, an organic cation or an inorganic cation, as hydralazine, pildralazine, todralazine, and the like. defined herein. “Compound used to treat cardiovascular diseases’ refers to any therapeutic compound, or a pharmaceutically accept “Carbonyl refers to C(O)-. able salt thereof, used to treat any cardiovascular disease. “Alkylcarbonyl or "alkanoyl refers to Rso C(O)—, Suitable compounds include, but are not limited to, angio wherein Rso is an alkyl group, as defined herein. tensin-converting enzyme (ACE) inhibitors (such as, for "Arylcarbonyl' or “aroyl” refers to Rs. C(O)—, example, alacepril, benazepril, captopril, ceronapril, cilaza wherein Rss is an aryl group, as defined herein. 25 pril, delapril, duinapril, enalapril, enalaprilat, fosinopril, “Carboxylic ester refers to —C(O)ORss, wherein Rss is imidapril, lisinopril, losartan, moveltipril, naphthopidil, pen an alkyl group, an aryl group, an alkylaryl group or an aryl topril, perindopril, quinapril, ramipril, rentipril, spirapril, heterocyclic ring, as defined herein. temocapril, trandolapril, urapidil, Zofenopril, and the like); “Alkylcarboxylic acid and “alkylcarboxyl refer to an beta-adrenergic blockers (such as, for example, acebutolol. alkyl group, as defined herein, appended to a carboxyl 30 alprenolol, amoSulalol, arotinolol, atenolol, betaxolol. group, as defined herein. bethanidine, bevantolol, bisoprolol, bopindolol, bucumolol, “Alkylcarboxylic ester” refers to an alkyl group, as bufetolol, bufuralol, bunitrolol, bupranolol, butafilolol, cara defined herein, appended to a carboxylic ester group, as Zolol, carteolol, carvedilol, celiprolol, cetamolol, dilevalol, defined herein. epanolol, esmolol, indenolol, labetalol, mepindolol. 35 metipranolol, metoprolol, moprolol, nadolol, nadoxolol. "Arylcarboxylic acid refers to an aryl group, as defined nebivolol, nifemalol, nipradillol, oXprenolol, penbutolol, pin herein, appended to a carboxyl group, as defined herein. dolol, practolol, pronethalol, propranolol, Sotalol, Sulfinalol, "Arylcarboxylic ester and “arylcarboxyl refer to an aryl talinolol, tertatolol, tilisolol, timolol, toliprolol. xibenolol, group, as defined herein, appended to a carboxylic ester and the like); cholesterol reducers (such as, for example, group, as defined herein. 40 HMG-CoA reductase inhibitors, including, but not limited “Carboxamido” refers to —C(O)N(Rs)(Rs.), wherein to, lovastatin (MEVACORR), simvastatin (ZOCORR), Rs and Rs, are each independently a hydrogen atom, an pravastatin (PRAVACHOL(R), fluvastatin, cerivastatin alkyl group, an aryl group, an alkylaryl group or an aryl (BAYCOLOR), atorvastatin (LIPITORR), and the like: heterocyclic ring, as defined herein, or Rs and Rs, taken sequestrants, including, but not limited to, cholestyramine, together with the nitrogen to which they are attached form 45 colestipol, Sialkylaminoalkyl derivatives of cross-linked a heterocyclic ring, a cycloalkyl group or a bridged dextran, and the like: inhibitors of cholesterol absorption, cycloalkyl group, as defined herein. including, but not limited to, beta-sitosterol, acyl CoA “Alkylcarboxamido” refers to an alkyl group, as defined cholersterol acyltransferase inhibitors, melinamide, and the herein, appended to a carboxamido group, as defined herein. like); calcium channel blockers (such as, for example, 50 amlodipine, aranidipine, barnidipine, benidipine, cilnid "Arylcarboxamido” refers to an aryl group, as defined ipine, clentiazem, diltiazen, efonidipine, fantofarone, felo herein, appended to a carboxamido group, as defined herein. dipine, isradipine, lacidipine, lercanidipine, manidipine, “Urea refers to N(Rs)—C(O)N(Rs)(Rs.) wherein mibefradil, nicardipine, nifedipine, nilvadipine, nisoldipine, Rs. Rs 7, and Rs are each independently a hydrogen atom, an alkyl group, an aryl group, an alkylaryl group, or an nitrendipine, semotiadil, Veraparmil, and the like); angio 55 tensin II receptor antagonists (such as, for example, ciclosi arylheterocyclic ring, as defined herein, or Rs and Rs, taken domine, eprosartan, furosemide, irbesartan, losartan, Sarala together with the nitrogen to which they are attached form sin, Valsartan, and the like); endothelin antagonists (such as, a heterocyclic ring, as defined herein. for example, bosentan, Sulfonamide endothelin antagonists, “Phosphoryl' refers to —P(R-7)(R-7) (R), wherein Rio BQ-123, SQ 28.608, and the like); renin inhibitors (such as, is a lone pair of electrons, Sulfur or oxygen, and R7 and R7 60 for example, enalkrein, RO 42–5892. A 65317, CP80794, are each independently a covalent bond, a hydrogen, a lower ES 1005, ES 8891, SQ 34017, and the like); and mixtures alkyl, an alkoxy, an alkylamino, a hydroxy or an aryl, as thereof. defined herein. “Vascular diseases characterized by nitric oxide insuffi “Silyl refers to —Si(R)(R)(R-7s), wherein R. R. ciency’ include, but are not limited to, cardiovascular dis and Rs are each independently a covalent bond, a lower 65 eases; diseases resulting from oxidative stress; hypertension alkyl, an alkoxy, an aryl or an arylalkoxy, as defined herein. (e.g., low-renin hypertension; Salt-sensitive hypertension; “Hydrazino” refers to HN N(H)–. low-renin, Salt-sensitive hypertension; primary pulmonary US 7,235,237 B2 11 12 hypertension; thromboembolic pulmonary hypertension; The role of nitric oxide in the vascular diathesis of blacks pregnancy-induced hypertension; renovascular hyperten is illustrated by the consequences of nitric oxide insuffi sion; hypertension-dependent end-stage renal disease), heart ciency in the normal responses of the vasculature to nitric failure (e.g., microvascular cardiac ischemia), and left ven oxide. Nitric oxide insufficiency Suppresses renin release tricular hypertrophy with disproportionate microvascular from the juxtaglomerular cells, and induces a Sodium chlo ization, (i.e., inadequate vascularity) or diastolic dysfunc ride/volume sensitive increase in blood pressure. Further tion. more, nitric oxide insufficiency leads to an increased sensi “Cardiovascular diseases’ refers to any cardiovascular tivity of vascular Smooth muscle cells to vasoconstrictors, disease, including but not limited to, congestive heart fail Such as angiotensin II and catecholamines, which amplify ure, hypertension, pulmonary hypertension, myocardial and 10 cerebral infarctions, atherosclerosis, atherogenesis, throm the increase in vascular resistance. bosis, ischemic heart disease, post-angioplasty restenosis, Nitric oxide insufficiency promotes vascular smooth coronary artery diseases, renal failure, stable, unstable and muscle cell proliferation following vascular injury, and variant (Prinzmetal) angina, cardiac edema, renal insuffi Sustains Smooth muscle cell and cardiomyocyte hypertrophy ciency, nephrotic edema, hepatic edema, stroke, transient 15 in response to catecholamines and angiotensin II. Further ischemic attacks, cerebrovascular accidents, restenosis, con more, inadequate nitric oxide leads to increased production trolling blood pressure in hypertension, platelet adhesion, of extracellular matrix with consequent myocardial fibrosis. platelet aggregation, Smooth muscle cell proliferation, vas These many cardiovascular responses that result from cular complications associated with the use of medical inadequate NO in the vasculature have clear clinical corre devices, wounds associated with the use of medical devices, lates in the black population. The clinical vascular pheno pulmonary thromboembolism, cerebral thromboembolism, type of blacks that distinguishes them from whites with thrombophlebitis, thrombocytopenia, bleeding disorders, similar cardiovascular disorders is one of salt-sensitive, and the like. low-renin hypertension; left ventricular hypertrophy dispro “Diseases resulting from oxidative stress' refers to any portionate to afterload and with an inadequate angiogenic disease that involves the generation of free radicals or 25 response; and microvascular ischemia in the absence of radical compounds, such as, for example, atherogenesis, significant epicardial coronary artery disease. The net patho atheromatosis, arteriosclerosis, artherosclerosis, vascular physiological consequences of these effects are increased hypertrophy associated with hypertension, hyperlipoprotein peripheral vascular resistance with accompanying arterial aemia, normal vascular degeneration through aging, par hypertension; and an inadequately vascularized, fibrotic athyroidal reactive hyperplasia, chronic renal disease, neo 30 plastic diseases, inflammatory diseases, neurological and increase in left ventricular mass with accompanying dias acute bronchopulmonary disease, tumorigenesis, ischemia tolic dysfunction and microvascular ischemia. reperfusion syndrome, arthritis, sepsis, and the like. Given these clinical observations and the role that NO Two broad classes of cardiovascular disorders are more plays in preventing their development, the present inventors prevalent among blacks than whites and serve as areas in 35 have unexpectedly discovered that the principal cardiovas need of investigative efforts. Hypertension and left ventricu cular disorders common among blacks (such as hyperten lar hypertrophy, two related yet independent risk factors for sion, left ventricular hypertrophy, and heart failure) result coronary heart disease, are significantly more prevalent from a specific vascular diathesis that is a direct conse among blacks than whites. Blacks also have higher rates of quence of nitric oxide insufficiency. An outline of the angiographically normal coronary arteries despite a higher 40 pathogenic consequences of nitric oxide insufficiency that prevalence of risk factors for coronary atherosclerosis, and serve as the basis for these cardiovascular disorders is shown greater morbidity and mortality from coronary heart disease in FIG. 1. than whites. These paradoxical observations have led some Nitric oxide insufficiency states can be a consequence of investigators to postulate that blacks harbor a diathesis of the reduced synthesis of nitric oxide, enhanced inactivation of microvasculature that limits perfusion and serves as a stimu 45 nitric oxide, or both. Possible candidate mechanisms include lus for vascular Smooth muscle cell and cardiomyocyte alterations in the genes that code for endothelial nitric oxide hypertrophy, which, in turn, leads to hypertension and left synthase or the inducible microvascular and cardiomyocyte Ventricular hypertrophy, respectively. The underlying basis nitric oxide synthase leading to reduced expression of a for this vascular diathesis may involve the endothelium, normal gene product or appropriate expression of a less which has a limited capacity to generate vasodilator and 50 active gene product; reduction in the enzymatic activity of antiproliferative factors or an increased capacity to produce nitric oxide synthase owing to inadequate cofactor concen vasoconstrictor and proliferative factors; the vascular trations; or enhanced inactivation of nitric oxide by oxidant Smooth muscle cell, which manifests increased sensitivity to StreSS. vasoconstrictor and proliferative factors; or both, in these Data obtained by the inventors in cultured cells, animal individuals. 55 models, and human patients suggest that increased oxidant The present inventors have discovered that a major prod stress is central to the vascular diathesis of and consequent uct of the normal blood vessel that may play a role in the cardiovascular disorders common among African Ameri vascular diathesis of blacks is endothelium-derived nitric cans. Possible candidate mechanisms for the oxidant stress oxide (NO). Nitric oxide produced by the endothelial cells include enhanced production of reactive oxygen species induces vascular Smooth muscle cell relaxation, contributing 60 (ROS), decreased antioxidant defenses, or both. The inven importantly to resting vascular tone. In addition, NO inhibits tors make no a priori assumptions about the temporal or vascular Smooth muscle cell proliferation and induces apo causative relationship between oxidant stress and the vas ptosis in Smooth muscle cells, which leads to the release of cular phenotype of blacks: oxidant stress may both precede basic fibroblast growth factor and vascular endothelial cell the development of the vascular diathesis and promote its growth factor, in turn Supporting endothelial cell prolifera 65 progression once established. Recent data Suggest that tion. This sequence of cellular responses is believed to enhanced ROS production accompanies essential hyperten Sustain angiogenesis under hypoxic or ischemic conditions. Sion, atherosclerosis, thrombosis, and diabetes mellitus, and US 7,235,237 B2 13 14 appears in each case, at the very least, to be important in the In both vascular Smooth muscle cells and cardiomyocytes, progression of established disease, if not in its actual gen NO inhibits cellular proliferation and limits the proliferative CS1S. response to growth-promoting Substances (Garget al., J. Clin Endothelium-derived relaxing factor (EDRF), first Invest, 83:1774–1777 (1986)). Left ventricular hypertrophy described by Furchgott etal, Nature, 299:373-376 (1980), is 5 tends to occur in adult hearts with inadequate capillary an important mediator of vascular function. This endothelial proliferation, and this may account for the microvascular product activates guanylyl cyclase in vascular Smooth ischemia noted in patients with hypertrophy. Capillary pro muscle cells and platelets, leading to vasorelaxation and liferation is generally held to be a rare event in normal adult platelet inhibition, respectively (Loscalzo et al. Prog Car mammalian hearts. However, recent data from a hyperten diovasc Dis, 38:87–104 (1995)). The chemical nature of 10 sive rat model, in which left ventricular hypertrophy com EDRF has been studied using a variety of pharmacological monly occurs, show that treatment with a low-dose of an and analytical techniques, and is NO (Ignarro et al. Circ Res, angiotensin-converting enzyme inhibitor insufficient to pre 61:866-879 (1987); Palmer et al, Nature, 327:524–526 vent hypertension and left ventricular hypertrophy can, (1987)). nonetheless, evoke capillary angiogenesis. Compared with Nitric oxide is synthesized by one of several isoforms of 15 untreated controls, treatment with the angiotensin convert the NO synthase (NOS) family of enzymes, two of which are ing enzyme inhibitor increased myocardial capillary prolif found in the vasculature, endothelial NOS (eNOS) and eration (Unger et al. Hypertension, 20:478482 (1992)), and inducible NOS (iNOS). eNOS is synthesized by endothelial this effect was believed to be a consequence of inhibiting the cells, while iNOS is synthesized by a variety of cell types, degradation and potentiating the action of bradykinin. including vascular Smooth muscle cells, fibroblasts, and 20 Bradykinin increases myocardial blood flow by inducing (principally microvascular) endothelial cells (Balligand et release of NO from microvascular endothelial cells, and al, Am J Physiol, 268: H1293–1303 (1995)). These enzymes increased blood flow is a powerful stimulus for capillary produce NO as a result of the five-electron oxidation of proliferation (Mall et al. Bas Res Cardiol, 85:531–540 L-arginine to L-citrulline; requisite cofactors include cal (1990)). cium-calmodulin, O, FAD, FMN, tetrahydrobiopterin thi- 25 Normal metabolic processes in vascular cells are associ ols, heme, and NADPH. (Moncada et al., N Engl J Med, ated with the generation of reactive oxygen intermediates 329:2002–2012 (1993)). that must be neutralized to limit oxidative damage and The role of NO in the cardiovascular system has become cellular dysfunction. In the setting of common cardiovascu increasingly apparent over the past fifteen years (LoScalzo et lar disorders or in the presence of common risk factors for al, Prog Cardiovasc Dis, 38:87–104 (1995)). Nitric oxide 30 atherothrombotic disease, reactive oxygen species (ROS) contributes importantly to resting tone in conductance as are generated in abundance, and their rate of synthesis and well as resistance arteries (Ouyyumi et al., J. Clin Invest, flux typically exceeds the capacity of endogenous antioxi 95:1747–1755 (1995)), and plays a critical role in the dant mechanisms. Hypercholesterolemia, hyperglycemia maintenance of peripheral vascular resistance and arterial (Keaney et al. Circulation, 99:189-191 (1999)), cigarette pressure responses. Inhibition of NOS activity is associated 35 Smoking, hyperhomocysteinemia, hypertension, and frank with enhanced vascular sensitivity to vasoconstrictors, such atherosclerosis are all accompanied by an increase in plasma as norepinephrine and angiotensin II (Conrad et al. Am J and tissue ROS generation. Superoxide anion, hydrogen Physiol, 262:R1137-R1144 (1992)), and this effect appears peroxide, hydroxyl radical, peroxynitrite, and lipid peroX to be mediated, in part, by increased calcium sensitivity ides all increase in these settings. What remains unknown is (Bank et al. Hypertension, 24:322–328 (1994)). Nitric oxide 40 whether or not the increase in ROS in these disorder is a release from the cardiovascular regulatory center in the brain primary event, a secondary consequence of the underlying may also be involved in the central regulation of blood process, or both. pressure, Suggesting a role for neuronal NOS in the regula Endogenous antioxidants important for the neutralization tion of vascular tone (Cabrera et al. Biochem Biophy's Res (i.e., reduction) of ROS can be categorized into two groups: Comm, 206:77–81 (1995); Mattson et al. Hypertension, 45 Small-molecule antioxidants and antioxidant enzymes. The 28:297-303 (1996)). former group comprises molecules such as GSH, NADPH, Nitric oxide activates renin gene expression in the kidney, C-tocopherol, vitamin C, and ubiquinol-10; while the latter and is involved in the baroreceptor-mediated regulation of group comprises the Superoxide dismutases, catalase, and renin gene expression (Schricker et al., Pflug Arch, 428: glutathione peroxidases. Deficiencies in several of these 261-268 (1994)). The dependence of blood pressure on salt 50 molecular species have been shown to lead to increased intake appears to depend on NO, and NO deficiency states steady-state levels of ROS and vascular dysfunction, includ are associated with salt-sensitivity (Tolins et al. Kidney ing increased platelet activation, arterial thrombosis (Freed Internat, 46:230–236 (1994)). Selective inhibition of iNOS man et al, J Clin Invest, 97:979–987 (1996): Freedman et al. in Dahl R rats has been shown to lead to salt-sensitivity and Circulation, 98: 1481–1486 (1998)), and reduced production to the development of salt-dependent hypertension similar to 55 of platelet-derived NO (Kenet et al. Arterio Thromb Vasc Dahl S rats (Rudd et al. Am J Physiol, 277: H732-H739 Biol, 19(8): 2017–2023 (1999)), which is important for (1999)). In addition, mice deficient in iNOS (iNOS gene limiting expansion of a platelet thrombus (Freedman et al. eliminated by targeted disruption) may develop hyperten Circ Res, 84:1416–142 (1999)). sion in response to salt feeding (Rudd et al. Circulation, ROS generation accompanies the vascular dysfunction 98:1A (1998)). 60 associated with several models of atherothrombotic and Nitric oxide also affects myocardial contractility, and does hypertensive vascular diseases. Hyperhomo-cysteinemic so both by mediating muscarinic-cholinergic slowing of the mice (i.e., cyStathionine B-synthase knock-out mice) (Eber heart rate and the contractile response to beta-adrenergic hardt et al. Circulation, 98:144 (1998)), cellular glutathione stimulation (Balligand et al. Proc Nat'l Acad Sci USA, peroxidase-deficient mice (i.e., cellular glutathione peroxi 90:347–351 (1993)). This latter effect appears to be medi- 65 dase knock-out mice), and salt-induced hypertensive rats ated in vivo through the vagus nerve (Hare et al., J. Clin (i.e., salt-fed Dahl S rats) (Trolliet et al. Circulation, Invest, 95:360–366 (1995)). 98:1–725 (1998)) all manifest increased vascular ROS, and US 7,235,237 B2 15 16 this increase in ROS is accompanied by reduced NO bio derived relaxing factor, stimulates endogenous synthesis of activity through oxidative inactivation. Endothelial function nitric oxide or is a Substrate for nitric oxide synthase, and, and NO availability can be improved by improving antioxi optionally, at least one nitrosated angiotensin-converting dant status with a cysteine precursor (Vita et al., J. Clin Invest, enzyme inhibitor, nitrosated beta-adrenergic blocker, nitro 101: 1408–1414 (1998)). In addition, C-tocopherol leads to sated calcium channel blocker, nitrosated endothelin antago platelet inhibition (Freedman et al. Circulation, nist, nitrosated angiotensin II receptor antagonist, nitrosated 94:2434–2440 (1996)) as one mechanism of its atherothrom renin inhibitor, and/or at least one compound used to treat botic benefit (Stephens et al, Lancet, 347:781-786 (1996)). cardiovascular diseases. For example, the patient can be The present inventors have also discovered that salt-loading administered an antioxidant and a nitric oxide donor, or the salt-sensitive individuals (Dahl Srats) leads to an approxi 10 patient can be administered an antioxidant, a nitric oxide mate 5-fold increase in plasma F-isoprostanes (8-epi-pros donor and a nitrosated compound, or the patient can be taglandin F), and this increase precedes the development of administered an antioxidant, a nitric oxide donor, a nitro florid hypertension. These data all support the role of sated compound and a compound used to treat cardiovas oxidant stress in the genesis or evolution of vascular dys cular diseases. function and disease, and the importance of antioxidant 15 Another aspect of the present invention provides methods mechanisms in preventing this pathobiology, particularly for treating and/or preventing vascular diseases character with regard to African Americans. ized by nitric oxide insufficiency by administering to a In Support of the mechanisms illustrated above, minimum patient a therapeutically effective amount of at least one forearm vascular resistance is significantly higher among nitrosated angiotensin-converting enzyme inhibitor, nitro normotensive blacks than whites (Bassett et al. Am J Hyper sated beta-adrenergic blocker, nitrosated calcium channel tension, 5:781-786 (1992)), and forearm blood-flow blocker, nitrosated endothelin antagonist, nitrosated angio responses to isoproterenol are markedly attenuated in nor tensin II receptor antagonist and/or nitrosated renin inhibi motensive blacks, suggesting a blunted f-vasodilator tor, and, optionally, at least one antioxidant and/or at least response in these individuals (Lang et al., N Engl J Med, one compound used to treat cardiovascular diseases. 333:155–160 (1995)). Blacks tend to have greater left ven 25 The present invention also provides methods of prevent tricular mass than whites for any given level of blood ing and treating Raynaud's syndrome by administering a pressure (Koren et al. Am J Hypertension, 6:815–823 therapeutically effective amount of at least one antioxidant (1993); Chaturvedi et al. JAm Coll Cardiol, 24:1499–1505 or a pharmaceutically acceptable salt thereof, and at least (1994)). While not quantitated in any necropsy study, this one compound that donates, transfers or releases nitric response is likely to be accompanied by inadequate capillary 30 oxide, elevates endogenous levels of endothelium-derived angiogenesis which, in turn, may account for the diastolic relaxing factor, stimulates endogenous synthesis of nitric dysfunction and the microvascular ischemia observed in oxide or is a substrate for nitric oxide synthase, and, option blacks. Interestingly, blacks have been observed to have low ally, at least one nitrosated angiotensin-converting enzyme levels of urinary kallikrein (Zinner et al. Am J Epidemiol, inhibitor, nitrosated calcium channel blocker, nitrosated 104:124-132 (1976); Levy et al. J Clin Invest, 60:129–138 35 endothelin antagonist, nitrosated angiotensin II receptor (1977)), the enzyme responsible for the generation of brady antagonist and/or nitrosated renin inhibitor. For example, the kinin from high-molecular-weight kininogen. Thus, were a patient can be administered an antioxidant and a nitric oxide similar abnormality in bradykinin and bradykinin-mediated donor, or the patient can be administered an antioxidant and NO production to exist in the coronary vasculature, attenu a nitrosated compound. The antioxidant, nitric oxide donor ated blood flow responses may result that would limit 40 and nitrosated compound can be administered separately or capillary angiogenic responses and prevent the endothelial as components of the same composition. Raynaud's Syn proliferative effects of locally derived NO. drome is a condition that causes a loss of blood flow to the As discovered and described herein, African Americans fingers, toes, nose and/or ears. The affected area turns white have a unique vascular diathesis that may serve as the basis from the lack of circulation, then blue and cold, and finally for clinically important cardiovascular syndromes. For 45 numb. The affected area may also turn red, and may throb, example, differences in the outcome of left ventricular tingle or Swell. dysfunction may be a consequence of the enhanced (perhaps Another aspect of the present invention provides novel salt-dependent) increase in oxidant stress coupled with transdermal patches comprising a therapeutically effective microvascular endothelial dysfunction and an inadequately amount of at least one antioxidant and at least one compound vascularized, hypertrophied left ventricle. This constellation 50 that donates, transfers or releases nitric oxide, elevates of pathophysiological abnormalities may provide the Sub endogenous levels of endothelium-derived relaxing factor, strate for the important differences in outcome between stimulates endogenous synthesis of nitric oxide or is a blacks and whites with left ventricular dysfunction (Dreis et Substrate for nitric oxide synthase, and, optionally, at least al, N Engl J Med, 340:609-616 (1999)). In addition, these one nitrosated angiotensin-converting enzyme inhibitor, nit observations and their clinical consequences Suggest that 55 rosated beta-adrenergic blocker, nitrosated calcium channel blacks with abnormal endothelial function and nitric oxide blocker, nitrosated endothelin antagonist, nitrosated angio insufficiency states would derive direct and, perhaps, dis tensin II receptor antagonist, nitrosated renin inhibitor, and/ proportionate clinical benefit from enhancing nitric oxide in or at least one compound used to treat cardiovascular the vasculature, either by improving endothelial function, diseases providing exogenous nitric oxide donors, or both. 60 Yet another aspect of the present invention provides In view of the above, the present invention provides Sustained release formulations comprising a therapeutically methods of treating and/or preventing vascular diseases effective amount of at least one antioxidant or a pharma characterized by nitric oxide (NO) insufficiency by admin ceutically acceptable salt thereof, and at least one nitric istering a therapeutically effective amount of at least one oxide donor, and, optionally at least one nitrosated com antioxidant or a pharmaceutically acceptable salt thereof, 65 pound. and at least one compound that donates, transfers or releases In the present invention, the antioxidants include Small nitric oxide, elevates endogenous levels of endothelium molecule antioxidants and antioxidant enzymes. Antioxidant US 7,235,237 B2 17 18 refers to and includes any compound that can react and compounds include S-nitroso-polypeptides (the term quench a free radical. Suitable Small-molecule antioxidants "polypeptide' includes proteins and polyamino acids that do include, but are not limited to, hydralazine compounds, not possess an ascertained biological function, and deriva glutathione, Vitamin C, Vitamin E, cysteine, N-acetyl-cys tives thereof); S-nitrosylated amino acids (including natural teine, B-carotene, ubiquinone, ubiquinol-10, tocopherols, and synthetic amino acids and their stereoisomers and coenzyme Q, and the like. Suitable antioxidant enzymes racemic mixtures and derivatives thereof); S-nitrosylated include, but are not limited to, Superoxide dismutase, cata Sugars; S-nitrosylated, modified and unmodified, oligo lase, glutathione peroxidase, and the like. The antioxidant nucleotides (preferably of at least 5, and more preferably enzymes can be delivered by gene therapy as a viral vertor 5–200 nucleotides); straight or branched, saturated or unsat and/or a non-viral vector. Suitable antioxidants are described 10 urated, aliphatic or aromatic, Substituted or unsubstituted more fully in the literature. Such as in Goodman and Gilman, S-nitrosylated hydrocarbons; and S-nitroso heterocyclic The Pharmacological Basis of Therapeutics (9th Edition), compounds. S-nitrosothiols and methods for preparing them McGraw-Hill, 1995; and the Merck Index on CD-ROM, are described in U.S. Pat. Nos. 5,380,758 and 5,703,073; Twelfth Edition, Version 12:1, 1996; and on STN Express, WO 97/27749; WO 98/19672; and Oae et al., Org. Prep. file phar and file reg. 15 Proc. Int., 15(3):165–198 (1983), the disclosures of each of The preferred antioxidant may be a hydralazine com which are incorporated by reference herein in their entirety. pound that might preferably be administered as a pharma Another embodiment of the present invention is S-nitroso ceutically acceptable salt; more preferably as hydralazine amino acids where the nitroso group is linked to a Sulfur hydrochloride. Hydralazine hydrochloride (1-hydrazinoph group of a Sulfur-containing amino acid or derivative thalazine monohydrochloride), USP, is a white to off-white thereof. Such compounds include, for example, S-nitroso crystalline powder. It is soluble in water, slightly soluble in N-acetylcysteine, S-nitroso-captopril, S-nitroso-N-acetyl ethanol and practically insoluble in ether. Hydralazine penicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, hydrochloride is commercially available from, for example, S-nitroso-glutathione and S-nitroso-cysteinyl-glycine. Lederle Standard Products (Pearl River, N.Y.), and Par Suitable S-nitrosylated proteins include thiol-containing Pharmaceuticals Inc. (Spring Valley, N.Y.). 25 proteins (where the NO group is attached to one or more Compounds contemplated for use in the present invention Sulfur groups on an amino acid or amino acid derivative (e.g., antioxidants) are used in combination with nitric oxide thereof) from various functional classes including enzymes, and compounds that release nitric oxide or otherwise Such as tissue-type plasminogen activator (TPA) and cathe directly or indirectly deliver or transfer nitric oxide to a site psin B; transport proteins, such as lipoproteins; heme pro of its activity, Such as on a cell membrane in vivo. 30 teins, such as hemoglobin and serum albumin; and biologi Nitrogen monoxide can exist in three forms: NO- (ni cally protective proteins, such as immunoglobulins, troxyl), NO. (nitric oxide) and NO" (nitrosonium). NO. is a antibodies and cytokines. Such nitrosylated proteins are highly reactive short-lived species that is potentially toxic to described in WO 93/09806, the disclosure of which is cells. This is critical because the pharmacological efficacy of incorporated by reference herein in its entirety. Examples NO depends upon the form in which it is delivered. In 35 include polynitrosylated albumin where one or more thiol or contrast to the nitric oxide radical (NO.), nitrosonium (NO") other nucleophilic centers in the protein are modified. does not react with O. or O - species, and functionalities Other examples of suitable S-nitrosothiols include: capable of transferring and/or releasing NO" and NO- are (i) HS(C(R)(R)), SNO; also resistant to decomposition in the presence of many (ii) ONS(C(R)(R)).R. and redox metals. Consequently, administration of charged NO 40 (iii) HN CH(COH)–(CH), C(O)NH-CH equivalents (positive and/or negative) does not result in the (CHSNO) C(O)NH-CH COH: generation of toxic by-products or the elimination of the wherein m is an integer from 2 to 20; R and Rare each active NO moiety. independently a hydrogen, an alkyl, a cycloalkoxy, a halo The term "nitric oxide encompasses uncharged nitric gen, a hydroxy, an hydroxyalkyl, an alkoxyalkyl, an aryl oxide (NO.) and charged nitrogen monoxide species, pref 45 heterocyclic ring, an alkylaryl, a cycloalkylalkyl, a hetero erably charged nitrogen monoxide species, such as nitroso cyclicalkyl, an alkoxy, a haloalkoxy, an amino, an nium ion (NO") and nitroxyl ion (NO-). The reactive form alkylamino, a dialkylamino, an arylamino, a diarylamino, an of nitric oxide can be provided by gaseous nitric oxide. The alkylarylamino, an alkoxyhaloalkyl, a haloalkoxy, a Sulfonic nitric oxide releasing, delivering or transferring compounds, acid, a Sulfonic ester, an alkylsulfonic acid, an arylsulfonic have the structure F NO, wherein F is a nitric oxide 50 acid, an arylalkoxy, an alkylthio, an arylthio, a cycloalky releasing, delivering or transferring moiety, include any and lthio, a cycloalkenyl, a cyano, an aminoalkyl, an aminoaryl, all Such compounds which provide nitric oxide to its an aryl, an arylalkyl, an alkylaryl, a carboxamido, a alkyl intended site of action in a form active for its intended carboxamido, an arylcarboxamido, an amidyl, a carboxyl, a purpose. The term "NO adducts' encompasses any nitrogen carbamoyl, a carbamate, an alkylcarboxylic acid, an aryl monoxide releasing, delivering or transferring compounds, 55 carboxylic acid, an alkylcarbonyl, an arylcarbonyl, an ester, including, for example, S-nitrosothiols, nitrites, nitrates, a carboxylic ester, an alkylcarboxylic ester, an arylcarboxy S-nitrothiols, Sydnonimines, 2-hydroxy-2-nitrosohydrazines lic ester, a haloalkoxy, a Sulfonamido, an alkylsulfonamido, (NONOates), (E)-alkyl-2-((E)-hydroxyimino)-5-nitro-3- an arylsulfonamido, a Sulfonic ester, a urea, a phosphoryl, a hexene amines or amides, nitroSoamines, furoxans as well as nitro, -T-Q, or (C(R)(R))-T-Q, or R, and R, taken together Substrates for the endogenous enzymes which synthesize 60 with the carbons to which they are attached form a carbonyl, nitric oxide. The “NO adducts' can be mono-nitrosylated, a methanthial, a heterocyclic ring, a cycloalkyl group or a poly-nitrosylated, mono-nitrosated and/or poly-nitrosated or bridged cycloalkyl group; Q is —NO or - NO; and T is a combination thereof at a variety of naturally susceptible or independently a covalent bond, a carbonyl, an oxygen, artificially provided binding sites for biologically active —S(O) - or —N(R)R , wherein o is an integer from 0 forms of nitrogen monoxide. 65 to 2, R is a lone pair of electrons, a hydrogen or an alkyl One group of NO adducts is the S-nitrosothiols, which are group; R, is a hydrogen, an alkyl, an aryl, an alkylcarboxylic compounds that include at least one —S NO group. These acid, an aryl carboxylic acid, an alkylcarboxylic ester, an US 7,235,237 B2 19 20 arylcarboxylic ester, an alkylcarboxamido, an arylcarboxa taerythritoltetranitrate, pentrinitrol and propatylnitrate, most mido, an alkylaryl, an alkylsulfinyl, an alkylsulfonyl, an preferred are isosorbide dinitrate and/or isosorbide mononi arylsulfinyl, an arylsulfonyl, a Sulfonamido, a carboxamido, trate. a carboxylic ester, an amino alkyl, an amino aryl, —CH2— Another group of NO adducts are N-oxo-N-nitrosoamines C(T Q)(R)(R), or -(N.O. ).M", wherein M' is an that donate, transfer or release nitric oxide and are repre organic or inorganic cation; with the proviso that when R, is sented by the formula: R'R' N(O-M'.) NO, where R' —CH, CCT-Q)(R)(R) or -(N.O. ).M"; then “ T-Q” and Rare each independently a polypeptide, an amino acid, can be a hydrogen, an alkyl group, an alkoxyalkyl group, an a Sugar, a modified or unmodified oligonucleotide, a straight aminoalkyl group, a hydroxy group or an aryl group. or branched, saturated or unsaturated, aliphatic or aromatic, In cases where R, and Rare a heterocyclic ring or R, and 10 substituted or unsubstituted hydrocarbon, or a heterocyclic R, when taken together with the carbon atoms to which they group, and M is as defined herein. are attached are a heterocyclic ring, then R, can be a Another group of NO adducts are thionitrates that donate, Substituent on any disubstituted nitrogen contained within transfer or release nitric oxide and are represented by the the radical wherein R, is as defined herein. formula: R'—(S) NO, where R' is a polypeptide, an 15 amino acid, a Sugar, a modified or unmodified oligonucle Nitrosothiols can be prepared by various methods of otide, a straight or branched, saturated or unsaturated, ali synthesis. In general, the thiol precursor is prepared first, phatic or aromatic, Substituted or unsubstituted hydrocar then converted to the S-nitrosothiol derivative by nitrosation bon, or a heterocyclic group. Preferred are those compounds of the thiol group with NaNO under acidic conditions (pH where R' is a polypeptide or hydrocarbon with a pair or pairs is about 2.5) which yields the S-nitroso derivative. Acids of thiols that are sufficiently structurally proximate, i.e., which can be used for this purpose include aqueous Sulfuric, acetic and hydrochloric acids. The thiol precursor can also vicinal, that the pair of thiols will be reduced to a disulfide. be nitrosylated by reaction with an organic nitrite Such as Compounds which form disulfide species release nitroxyl tert-butyl nitrite, or a nitrosonium salt such as nitrosonium ion (NO-) and uncharged nitric oxide (NO.). tetrafluoroborate in an inert solvent. The present invention is also directed to compounds that 25 stimulate endogenous NO or elevate levels of endogenous Another group of NO adducts for use in the present endothelium-derived relaxing factor (EDRF) in vivo or are invention, where the NO adduct is a compound that donates, Substrates for the enzyme, nitric oxide synthase. Such com transfers or releases nitric oxide, include compounds com pounds include, for example, L-arginine, L-homoarginine, prising at least one ON O - ON N— or ON.—C and N-hydroxy-L-arginine, including their nitrosated and group. The compounds that include at least one ON-O-, 30 nitrosylated analogs (e.g., nitrosated L-arginine, nitrosylated ON N— or ON C group are preferably ON O , L-arginine, nitrosated N-hydroxy-L-arginine, nitrosylated ON N or ON C-polypeptides (the term “polypeptide' N-hydroxy-L-arginine, nitrosated L-homoarginine and includes proteins and polyamino acids that do not possess an nitrosylated L-homoarginine), precursors of L-arginine and/ ascertained biological function, and derivatives thereof); or physiologically acceptable salts thereof, including, for ON O, ON N or ON C-amino acids (including natu 35 example, citrulline, ornithine, glutamine, lysine, polypep ral and synthetic amino acids and their stereoisomers and tides comprising at least one of these amino acids, inhibitors racemic mixtures); ON O—, ON N— or ON C-sugars; of the enzyme arginase (e.g., N-hydroxy-L-arginine and ON O. , ON N or ON C modified or unmodified 2(S)-amino-6-boronohexanoic acid) and the substrates for oligonucleotides (comprising at least 5 nucleotides, prefer nitric oxide synthase, cytokines, adenosin, bradykinin, cal ably 5-200 nucleotides); ON-O-, ON N- or 40 reticulin, bisacodyl, and phenolphthalein. EDRF is a vascu ON-C - Straight or branched, Saturated or unsaturated, lar relaxing factor secreted by the endothelium, and has been aliphatic or aromatic, Substituted or unsubstituted hydrocar identified as nitric oxide (NO) or a closely related derivative bons; and ON O—, ON N— or ON C-heterocyclic thereof (Palmer et al. Nature, 327:524–526 (1987); Ignarro compounds. et al. Proc. Natl. Acad. Sci. USA, 84:9265–9269 (1987)). Another group of NO adducts for use in the present 45 In the present invention the compound that donates, invention include nitrates that donate, transfer or release transfers or releases nitric oxide as a charged species, or nitric oxide, Such as compounds comprising at least one elevates levels of endogenous EDRF or nitric oxide, or is a ON O ON N.—, ON S or ON C group. substrate for nitric oxide synthase may preferably be isos Preferred among these compounds are ON O—, ON.— orbide dinitrate and/or isosorbide mononitrate, more pref N—, ON S or ON C polypeptides (the term 50 erably isosorbide dinitrate. Diluted isosorbide dinitrate (1,4, "polypeptide' includes proteins and also polyamino acids 3,6-dianhydro-D-glucitol-2,5-dinitrate), USP, is a white to that do not possess an ascertained biological function, and off-white powder that has a melting point of 70° C. and has derivatives thereof); ON O—, ON N ON S or an optical rotation of +135° (3 mg/mL, ethanol). It is freely ON-C-amino acids (including natural and synthetic soluble in organic solvents such as ethanol, ether and amino acids and their stereoisomers and racemic mixtures); 55 chloroform, but is sparingly soluble in water. Isosorbide ON O—, ON. N. , ON S or ON C-sugars; dinitrate is commercially available, for example, under the ON O ON N ON S or ON C modified trade names DILATRATER-SR (Schwarz Pharma, Milwau and unmodified oligonucleotides (comprising at least 5 kee, Wis.): ISORDIL(R) and ISORDILR TITRADOSE(R) nucleotides, preferably 5-200 nucleotides); ON O , (Wyeth Laboratories Inc., Philadelphia, Pa.); and SORBI ON N ON.—S or ON C straight or branched, 60 TRATER (Zeneca Pharmaceuticals, Wilmington, Del.). saturated or unsaturated, aliphatic or aromatic, Substituted or Isosorbide mononitrate is commercially available, for unsubstituted hydrocarbons; and ON O ON N—, example, under the trade names IMDUR(R) (A. B. Astra, ON S or ON C heterocyclic compounds. Pre Sweden); MONOKETR (Schwarz Pharma, Milwaukee, ferred examples of compounds comprising at least one Wis.); and ISMOR (Wyeth-Ayerst company, Philadelphia, ON O ON N - ON S or ON C group 65 Pa.). include isosorbide dinitrate, mononitrate, clonitrate, eryth "Nitrosated compound” refers to any compound that has rity1 tetranitrate, mannitol hexanitrate, nitroglycerin, pen been Substituted with a nitro group, i.e., —NO group. The US 7,235,237 B2 21 22 nitrosated angiotensin-converting enzyme inhibitors, nitro antagonists are described more fully in the literature, such as sated beta-adrenergic blockers, nitrosated calcium channel in Goodman and Gilman, The Pharmacological Basis of blockers, nitrosated endothelin antagonists, nitrosated Therapeutics (9th Edition), McGraw-Hill, 1995; and the angiotensin II receptor antagonists and nitrosated renin Merck Index on CD-ROM, Twelfth Edition, Version 12:1, inhibitors of the present invention include any known angio 5 1996; and on STN Express, file phar and file registry. tensin-converting enzyme inhibitors, beta-adrenergic block Suitable angiotensin II receptor antagonists, include, but ers, calcium channel blockers, endothelin antagonists, angiotensin II receptor antagonists and renin inhibitors that are not limited to, ciclosidomine, eprosartan, furosemide, have been nitrosated through one or more sites such as irbesartan, losartan, Saralasin, Valsartan, and the like. Suit oxygen (hydroxyl condensation), Sulfur (Sulfhydryl conden 10 able angiotensin II receptor antagonists are described more sation), and/or nitrogen. The nitrosated compounds of the fully in the literature, such as in Goodman and Gilman, The present invention can be prepared using conventional meth Pharmacological Basis of Therapeutics (9th Edition), ods known to one skilled in the art. For example, known McGraw-Hill, 1995; and the Merck Index on CD-ROM, methods for nitrosating compounds are described in U.S. Twelfth Edition, Version 12:1, 1996; and on STN Express, Pat. Nos. 5,380,758 and 5,703,073; WO 97/27749; WO 15 file phar and file registry. 98/19672; and Oae et al. Org. Prep. Proc. Int., 15(3): Suitable renin inhibitors, include, but are not limited to, 165–198 (1983), the disclosures of each of which are enalkrein, RO 42–5892, A 65317, CP80794, ES 1005, ES incorporated by reference herein in their entirety. WO 8891, SQ34017, and the like). Suitable renin inhibitors are 98/21193 discloses nitrosated ACE inhibitors and nitrosated described more fully in the literature, such as in Goodman beta-adrenergic blockers, the disclosure of which is incor and Gilman, The Pharmacological Basis of Therapeutics porated by reference herein in its entirety. WO 99/00361 (9th Edition), McGraw-Hill, 1995; and the Merck Index on discloses nitrate salts of ACE inhibitors, the disclosure of which is incorporated by reference herein in its entirety. CD-ROM, Twelfth Edition, Version 12:1, 1996; and on STN Suitable angiotensin-converting enzyme inhibitors, Express, file phar and file registry. include, but are not limited to, alacepril, benazepril, capto 25 In the methods of the invention, the antioxidant and at pril, ceronapril, cilaZapril, delapril, duinapril, enalapril, least one compound that donates, transfers or releases nitric enalaprilat, fosinopril, imidapril, lisinopril, losartan, movel oxide, elevates endogenous levels of endothelium-derived tipril, naphthopidil, pentopril, perindopril, quinapril, rami relaxing factor, stimulates endogenous synthesis of nitric pril, rentipril, spirapril, temocapril, trandolapril, urapidil, oxide or is a Substrate for nitric oxide synthase, and, option Zofenopril, and the like. Suitable angiotensin-converting 30 ally, at least one nitrosated compound, and/or compound enzyme inhibitors are described more fully in the literature, used to treat cardiovascular diseases can be administered as such as in Goodman and Gilman, The Pharmacological separate components or as components of the same compo Basis of Therapeutics (9th Edition), McGraw-Hill, 1995: sition. When the antioxidant and at least one nitric oxide and the Merck Index on CD-ROM, Twelfth Edition, Version donor are administered as separate components for the 12:1, 1996; and on STN Express, file phar and file registry. 35 treatment of vascular diseases characterized by nitric oxide Suitable beta-adrenergic blockers, include, but are not insufficiency or Raynaud's syndrome, they are preferably limited to, acebutolol, alprenolol, amoSulalol, arotinolol. administered to the patient at about the same time. “About atenolol, betaxolol, bethanidine, bevantolol, bisoprolol, the same time” means that within about thirty minutes of bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol. administering one compound (e.g., antioxidant or nitric bupranolol, butafilolol, carazolol, carteolol, carvedilol. 40 oxide donor) to the patient, the other compound (e.g., nitric celiprolol, cetamolol, dilevalol, epanolol, esmolol, inde oxide donor or antioxidant) is administered to the patient. nolol, labetalol, mepindolol, metipranolol, metoprolol. "About the same time' also includes simultaneous admin moprolol, nadolol, nadoxolol, nebivolol, nifenalol, istration of the compounds. nipradillol, oXprenolol, penbutolol, pindolol, practolol. In addition to the administration of the combination of the pronethalol, propranolol, Sotalol, Sulfinalol, talinolol, terta 45 antioxidant and nitric oxide donor for the treatment of tolol, tilisolol, timolol, toliprolol, Xibenolol, and the like. vascular diseases characterized by nitric oxide insufficiency, Suitable beta-adrenergic blockers are described more fully the patients can receive digitalis Such as digoxin and/or in the literature, Such as in Goodman and Gilman, The diuretics and/or at least one nitrosated angiotensin-convert Pharmacological Basis of Therapeutics (9th Edition), ing enzyme inhibitor, nitrosated beta-adrenergic blocker, McGraw-Hill, 1995; and the Merck Index on CD-ROM, 50 nitrosated calcium channel blocker, nitrosated endothelin Twelfth Edition, Version 12:1, 1996; and on STN Express, antagonist, angiotensin II receptor antagonist, nitrosated file phar and file registry. renin inhibitor, and/or at least one compound used to treat Suitable calcium channel blockers, include, but are not cardiovascular diseases. limited to, amlodipine, aranidipine, barnidipine, benidipine, The digoxin is preferably administered orally to achieve cilnidipine, clentiazem, diltiazen, efonidipine, fantofarone, 55 a steady state blood serum concentration of at least about 0.7 felodipine, isradipine, lacidipine, lercanidipine, manidipine, nanograms per ml to about 2.0 nanograms per ml. The mibefradil, nicardipine, nifedipine, nilvadipine, nisoldipine, diuretic is administered, preferably orally, to manage edema. nitrendipine, semotiadil, Veraparmil, and the like. Suitable Suitable diuretics include, but are not limited to, thiazides calcium channel blockers are described more fully in the (such as, for example, chlorothiazide, hydrochlorothiazide); literature, such as in Goodman and Gilman, The Pharma 60 ethacrynic acid, furosemide, spironalactone, triamterene or cological Basis of Therapeutics (9th Edition), McGraw-Hill, mixtures thereof. Depending on the diuretic used, potassium 1995; and the Merck Index on CD-ROM, Twelfth Edition, may also be administered to the patient in order to optimize Version 12:1, 1996; and on STN Express, file phar and file the fluid balance while avoiding hypokalemic alkalosis. The registry. administration of potassium can be potassium chloride or by Suitable endothelin antagonists, include, but are not lim 65 the daily ingestion of foods with high potassium content ited to, bosentan, Sulfonamide endothelin antagonists, Such as, for example, bananas, orange juice, and the like. BQ-123, SQ 28.608, and the like. Suitable endothelin The method of administration of these compounds is US 7,235,237 B2 23 24 described in further detail in U.S. Pat. No. 4,868,179, the starch, amylopectin, or cellulose derivatives of gelatin. Tab disclosure of which is incorporated by reference herein in its lets and pills can be prepared with enteric coatings. entirety. Liquid dosage forms for oral administration can include The compounds and compositions of the invention can be pharmaceutically acceptable emulsions, Solutions, Suspen administered by any available and effective delivery system sions, syrups, and elixirs containing inert diluents com including, but not limited to, orally, bucally, parenterally, by monly used in the art, Such as water. Such compositions can inhalation spray, topically (including transdermally), or rec also comprise adjuvants, such as wetting agents, emulsifying tally in dosage unit formulations containing conventional and Suspending agents, and Sweetening, flavoring, and per nontoxic pharmaceutically acceptable carriers, adjuvants, fuming agents. and vehicles as desired. The preferred methods of adminis 10 Suppositories for rectal administration of the compounds tration are by oral administration or topical application or compositions can be prepared by mixing the drug with a (transdermal application). Suitable nonirritating excipient such as cocoa butter and Topical administration can also involve the use of trans polyethylene glycols which are solid at room temperature dermal administration Such as transdermal patches or ion but liquid at rectal temperature, such that they will melt in tophoresis devices. Dosage forms for topical administration 15 the rectum and release the drug. of the compounds and compositions can include creams, The term parenteral includes Subcutaneous injections, sprays, lotions, gels, ointments, and the like. In such dosage intravenous, intramuscular, intrasternal injection, or infusion forms, the compositions of the invention can be mixed to techniques. Injectable preparations, for example, sterile form white, Smooth, homogeneous, opaque cream or lotion injectable aqueous or oleaginous Suspensions can be formu with, for example, benzyl alcohol 1% or 2% (wit/wt) as a lated according to the known art using Suitable dispersing preservative, emulsifying wax, glycerin, isopropyl palmi agents, wetting agents and/or Suspending agents. The sterile tate, lactic acid, purified water and Sorbitol Solution. In injectable preparation can also be a sterile injectable solution addition, the compositions can contain polyethylene glycol or Suspension in a nontoxic parenterally acceptable diluent 400. They can be mixed to form ointments with, for or solvent, for example, as a solution in 1,3-butanediol. example, benzyl alcohol 2% (wit/wt) as preservative, white 25 Among the acceptable vehicles and solvents that can be used petrolatum, emulsifying wax, and tenox II (butylated are water, Ringer's solution, and isotonic sodium chloride hydroxyanisole, propyl gallate, citric acid, propylene gly solution. Sterile fixed oils are also conventionally used as a col). Woven pads or rolls of bandaging material, e.g., gauze, Solvent or Suspending medium. can be impregnated with the compositions in solution, The compounds and compositions of the invention will lotion, cream, ointment or other Such form can also be used 30 typically be administered in a pharmaceutical composition for topical application. comprising one or more carriers or excipients. Examples of The compositions can also be applied topically using a suitable carriers include, for example, water, silicone, transdermal system, such as one of an acrylic-based polymer waxes, petroleum jelly, polyethylene glycols, propylene adhesive with a resinous crosslinking agent impregnated glycols, liposomes, Sugars, salt solutions, alcohol, vegetable with the composition and laminated to an impermeable 35 oils, gelatins, lactose, amylose, magnesium Stearate, talc, backing. In a particular embodiment, the compositions of the Surfactants, silicic acids, viscous paraffins, perfume oils, present invention are administered as a transdermal patch, fatty acid monoglycerides and diglycerides, petroethral fatty more particularly as a Sustained-release transdermal patch. acid esters, hydroxymethylcelluloses, polyvinyl-pyrroli The transdermal patches of the present invention can include dones, and the like. The pharmaceutical preparations can be any conventional form such as, for example, adhesive 40 sterilized and if desired, mixed with auxiliary agents, e.g., matrix, polymeric matrix, reservoir patch, matrix or mono lubricants, preservatives, stabilizers, wetting agents, emul lithic-type laminated structure, and are generally comprised sifiers, salts for influencing osmotic pressure, buffers, col of one or more backing layers, adhesives, penetration orings, flavorings and/or aromatic Substances and the like enhancers, an optional rate controlling membrane and a which do not deleteriously react with the active compounds. release liner which is removed to expose the adhesives prior 45 For topical application, the compositions can also include to application. Polymeric matrix patches also comprise a one or more permeation enhancers including, for example, polymeric-matrix forming material. Suitable transdermal dimethylsulfoxide (DMSO), dimethyl formamide (DMF), patches are described in more detail in, for example, U.S. N,N-dimethylacetamide (DMA), decylmethylsulfoxide Pat. Nos. 5,262,165, 5,948,433, 6,010,715 and 6,071,531, (C1OMSO), polyethylene glycol monolaurate (PEGML), the disclosure of each of which are incorporated herein in 50 glyceral monolaurate, lecithin, 1-substituted azacyclohep their entirety. tan-2-ones, particularly 1-N-dodecylcyclazacycoheptan-2- Solid dosage forms for oral administration can include ones (available under the trademark AZONE from Nelson capsules, Sustained-release capsules, tablets, Sustained Research & Development Co., Irvine Calif.), alcohols and release tablets, chewable tablets, sublingual tablets, effer the like. For parenteral application, particularly suitable Vescent tablets, pills, powders, granules and gels. In Such 55 vehicles consist of solutions, preferably oily or aqueous Solid dosage forms, the active compounds can be admixed Solutions, as well as Suspensions, emulsions, or implants. with at least one inert diluent, such as Sucrose, lactose or Aqueous Suspensions can contain Substances which increase starch. Such dosage forms can also comprise, as is normal the viscosity of the Suspension and include, for example, practice, additional Substances other than inert diluents, e.g., sodium carboxymethyl cellulose, sorbitol and/or dextran. lubricating agents. Such as magnesium Stearate. In the case 60 Optionally, the Suspension can contain stabilizers. The com of capsules, tablets, effervescent tablets, and pills, the dos positions, if desired, can also contain minor amounts of age forms can also comprise buffering agents. Soft gelatin wetting agents, emulsifying agents and/or pH buffering capsules can be prepared to contain a mixture of the active agents. compound or composition and vegetable oil. Hard gelatin The composition, if desired, can also contain minor capsules can contain granules of the active compound in 65 amounts of wetting agents, emulsifying agents and/or pH combination with a solid, pulverulent carrier, such as lac buffering agents. The composition can be a liquid solution, tose, Saccharose, Sorbitol, mannitol, potato starch, corn Suspension, emulsion, tablet, pill, capsule, Sustained release US 7,235,237 B2 25 26 formulation, or powder. The composition can be formulated Sustain release formulation comprises hydralazine hydro as a Suppository, with traditional binders and carriers such as chloride and isosorbide dinitrate and/or isosorbide mononi triglycerides. Oral formulations can include standard carri trate. ers such as pharmaceutical grades of mannitol, lactose, While individual needs may vary, determination of opti starch, magnesium Stearate, sodium saccharine, cellulose, mal ranges for effective amounts of the compounds and/or magnesium carbonate, and the like. compositions is within the skill of the art and can be Various delivery systems are known and can be used to determined by standard clinical techniques, including refer administer the compounds or compositions of the present ence to Goodman and Gilman, supra: The Physicians Desk invention, including, for example, encapsulation in lipo Reference, Medical Economics Company, Inc., Oradell, Somes, microbubbles, emulsions, microparticles, microcap 10 N.J., 1995; and DrugFacts and Comparisons, Inc., St. Louis, Sules, nanoparticles, and the like. The required dosage can Mo., 1993. Generally, the dosage required to provide an be administered as a single unit or in a Sustained release effective amount of the compounds and compositions, which form. can be adjusted by one of ordinary skill in the art, will vary The bioavailabilty of the compositions can be enhanced depending on the age, health, physical condition, sex, diet, by micronization of the formulations using conventional 15 weight, extent of the dysfunction of the recipient, frequency techniques such as grinding, milling, spray drying and the of treatment and the nature and scope of the dysfunction or like in the presence of Suitable excipients or agents such as disease, medical condition of the patient, the route of phospholipids or Surfactants. administration, pharmacological considerations such as the Sustained release dosage forms of the invention may activity, efficacy, pharmacokinetic and toxicology profiles of comprise microparticles and/or nanoparticles having a thera the particular compound used, whether a drug delivery peutic agent dispersed therein or may comprise the thera system is used, and whether the compound is administered peutic agent in pure, preferably crystalline, Solid form. For as part of a drug combination. Sustained release administration, microparticle dosage forms The compounds and compositions of the present inven comprising pure, preferably crystalline, therapeutic agents tion can be formulated as pharmaceutically acceptable salts. are preferred. The therapeutic dosage forms of this aspect of 25 Pharmaceutically acceptable salts include, for example, the present invention may be of any configuration Suitable alkali metal salts and addition salts of free acids or free bases. The nature of the salt is not critical, provided that it for Sustained release. is pharmaceutically-acceptable. Suitable pharmaceutically Nanoparticle Sustained release therapeutic dosage forms acceptable acid addition salts may be prepared from an are preferably biodegradable and, optionally, bind to the 30 inorganic acid or from an organic acid. Examples of Such vascular Smooth muscle cells and enter those cells, primarily inorganic acids include, but are not limited to, hydrochloric, by endocytosis. The biodegradation of the nanoparticles hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phos occurs over time (e.g., 30 to 120 days; or 10 to 21 days) in phoric acid and the like. Appropriate organic acids include, prelysosomic vesicles and lysosomes. Preferred larger but are not limited to, aliphatic, cycloaliphatic, aromatic, microparticle therapeutic dosage forms of the present inven 35 heterocyclic, carboxylic and Sulfonic classes of organic tion release the therapeutic agents for Subsequent target cell acids, such as, for example, formic, acetic, propionic, suc uptake with only a few of the Smaller microparticles entering cinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascor the cell by phagocytosis. A practitioner in the art will bic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, appreciate that the precise mechanism by which a target cell benzoic, anthranilic, mesylic, Salicylic, p-hydroxybenzoic, assimilates and metabolizes a dosage form of the present 40 phenylacetic, mandelic, embonic (pamoic), methane invention depends on the morphology, physiology and meta Sulfonic, ethanesulfonic, benzenesulfonic, pantothenic, tolu bolic processes of those cells. The size of the particle enesulfonic, 2-hydroxyethanesuifonic, Sulfanilic, Stearic, Sustained release therapeutic dosage forms is also important algenic, B-hydroxybutyric, cyclohexylaminosulfonic, galac with respect to the mode of cellular assimilation. For taric and galacturonic acid and the like. Suitable pharma example, the Smaller nanoparticles can flow with the inter 45 ceutically-acceptable base addition salts include, but are not stitial fluid between cells and penetrate the infused tissue. limited to, metallic salts made from aluminum, calcium, The larger microparticles tend to be more easily trapped lithium, magnesium, potassium, Sodium and Zinc or organic interstitially in the infused primary tissue, and thus are salts made from primary, secondary and tertiary amines, useful to deliver anti-proliferative therapeutic agents. cyclic amines, N,N'-dibenzylethylenediamine, chlorop Particular Sustained release dosage forms of the present 50 rocaine, choline, diethanolamine, ethylenediamine, meglu invention comprise biodegradable microparticles or nano mine (N-methylglucamine) and procaine and the like. All of particles. More particularly, biodegradable microparticles or these salts may be prepared by conventional means from the nanoparticles are formed of a polymer containing matrix that corresponding compound by reacting, for example, the biodegrades by random, nonenzymatic, hydrolytic Scission appropriate acid or base with the compound. ing to release therapeutic agent, thereby forming pores 55 In particular embodiments, the hydralazine hydrochloride within the particulate structure. can be administered in an amount of about 30 milligrams per In a particular embodiment, the compositions of the day to about 400 milligrams per day; the isosorbide dinitrate present invention are orally administered as a Sustained can be administered in an amount of about 5 milligrams per release tablet or a Sustained release capsule. For example, day to about 200 milligrams per day; and the isosorbide the Sustained release formulations can comprise a therapeu 60 mononitrate can be administered in an amount of about 5 tically effective amount of at least one antioxidant or a milligrams per day to about 120 milligrams per day. In a pharmaceutically acceptable salt thereof, and at least one more particular embodiment, the hydralazine hydrochloride nitric oxide donor, or the Sustained release formulations can can be administered in an amount of about 50 milligrams per comprise a therapeutically effective amount of at least one day to about 300 milligrams per day; the isosorbide dinitrate antioxidant or a pharmaceutically acceptable salt thereof, 65 can be administered in an amount of about 20 milligrams per and at least one nitric oxide donor, and, optionally at least day to about 160 milligrams per day; and the isosorbide one nitrosated compound. In a particular embodiment the mononitrate can be administered in an amount of about 15 US 7,235,237 B2 27 28 milligrams per day to about 100 milligrams per day. In an Example 2 even more particular embodiment, the hydralazine hydro chloride can be administered in an amount of about 75 To investigate the issue of whether NO deficiency is a milligrams one to four times per day; the isosorbide dinitrate primary or secondary phenomenon in hypertensive black can be administered in an amount of about 40 milligrams 5 patients, 46 normotensive white and black patients who one to four time per day; and the isosorbide mononitrate can were matched for age and gender were compared. As shown be administered in an amount of about 20 milligrams one to in FIG. 3B, the vasodilator responses to methacholine were four times per day. The particular amounts of hydralazine not significantly different in black and white normotensive and/or isosorbide dinitrate or isosorbide mononitrate can be patients. However, the response to Sodium nitroprusside administered as a single dose once a day; or in multiple 10 (FIG. 3A) was significantly lower in the black normotensive patients. This finding shows that there is an impairment in doses several times throughout the day; or as a Sustained the vasodilator response exogenous source of NO in black release oral formulation; or as a transdermal Sustained patients even prior to the development of hypertension, an release patch. observation that would be consistent with a primary rather The dose of nitric oxide donor in the composition will be than secondary role in the black vascular diathesis. These dependent on the specific nitric oxide donor compound and 15 findings are consistent with recently published results by the mode of administration. For example, when L-arginine other investigators (Lang et al, N Engl J Med, 333:155–160 is the orally administered nitric oxide donor, it can be (1995); Cardillo et al., Hypertension 31:1235–1239 (1998)). administered in an amount of about 3 grams to about 15 grams to provide a plasma level in the range of about 0.2 Example 3 mM to about 30 mM. The present invention also provides pharmaceutical kits Black patients have a preponderance of salt-sensitive comprising one or more containers filled with one or more hypertension, and the data described herein show that NO of the ingredients of the pharmaceutical compounds and/or deficiency is a pathogenic mechanism of salt-sensitive compositions. Such kits can also include, for example, other hypertension in experimental models (Rudd et al. Am J compounds and/or compositions (e.g., diuretics, digoxin, 25 Physiol, 277: H732–H739 (1999)). To explore the relation nitrosated compounds, compounds used to treat cardiovas between salt-sensitivity and endothelium-derived NO action cular diseases and the like), a device(s) for administering the in black patients prior to the development of hypertension, compounds and/or compositions, and written instructions in the blood pressure response to salt-loading and salt-depri a form prescribed by a governmental agency regulating the Vation using an established inpatient protocol (Weinberger et manufacture, use or sale of pharmaceuticals or biological 30 al, Hypertension, 8: II-127-II-134 (1986)) in a group of normotensive black patients was assessed. Briefly, blood products, which instructions can also reflects approval by pressure was continuously monitored non-invasively during the agency of manufacture, use or sale for human adminis a salt load (Sodium 458 mEq over 24 hours) and during a tration. period of Salt depletion (furosemide treatment and sodium intake 10 mEq over 24 hours). Patients were considered to EXAMPLES 35 be salt-sensitive if mean blood pressure was at least 10 mm Hg higher during the salt loading period (Weinberger et al. The following examples are for purposes of illustration Hypertension, 8:II-127-II-134 (1986)). As shown in FIGS. only, and are not intended to limit the scope of the specifi 4A-B, there were trends for impaired vasodilator responses cation or claims. to both methacholine (FIG. 4A) and sodium nitroprusside 40 (FIG. 4B). The vasodilator responses to Verapamil were Example 1 equivalent in Salt-sensitive and salt-resistant patients (data not shown). These data show impaired NO action in the As described herein, NO deficiency is a central patho microvasculature of salt sensitive individuals prior to the physiologic mechanism for the black vascular diathesis. To development of hypertension. examine this issue in forearm microVessels, the vasodilator 45 responses to intra-arterial infusions of methacholine, sodium Example 4 nitroprusside, and Verapamil were examined using venous occlusion plethysmography in 36 white and black hyperten The effects of hypertension and race on conduit vessel sive patients. These patients had no other coronary factors, function were examined using a well-established brachial Such as Smoking, diabetes mellitus, or hypercholesterolemia, 50 ultrasound technique (Vita et al. Lanzer & Lipton, Eds. and the two groups were matched in terms of age, gender, Diagnostics of Vascular Diseases. Principles and Technol lipid levels, blood pressure, and anti-hypertensive treatment. ogy. Berlin: Springer-Verlag, pp.249-259 (1996)) in 370 Similar to previous reports (Lang et al., N Engl J Med, patients (178 black, 192 white). As shown in Table 1, there 333:155–160 (1995); Panza et al, N Engl J Med, 323:22–27 were no significant differences in flow-mediated dilation or (1990)), the dilator response to methacholine, but not nitro nitroglycerin-mediated dilation according to race. However, prusside, was significantly reduced in these hypertensive 55 hypertension was associated with a highly significant reduc patients compared to age-matched normotensive controls tion in both flow-mediated dilation and nitroglycerin-medi (Sherman et al. Circulation, 98:1–376 (1998)). Regarding ated dilation. Further, systolic blood pressure was inversely racial differences, as shown in FIG. 2, vasodilation in correlated with flow-mediated dilation (r—-0.30, P-0.001) response to methacholine was markedly worse in the black and nitroglycerin-mediated dilation (r=-0.33, P-0.001). hypertensive patients compared to white hypertensive 60 Diastolic pressure correlated to a similar extent with vascu patients. There were no racial differences in the responses to lar function. By multiple linear regression analysis, Vessel Sodium nitroprusside or Verapamil (data not shown), Sug size and systolic blood pressure were the only independent gesting that this impairment of NO action in black hyper predictors of flow-mediated dilation in this sizable group of tensives is at the endothelial level and that dysfunction of patients. Thus, both black and white patients with hyperten vascular Smooth muscle does not account for the impaired 65 sion demonstrate a significant impairment of NO action in response. These findings have important implications for the conduit arteries (Gokce et al. Circulation, 99(25) 3234–3240 pathogenesis of hypertension and myocardial ischemia. (1999)). US 7,235,237 B2 29 30 S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, TABLE 1. S-nitroso-cysteine, S-nitroso-glutathione and S-nitroso-cys teinyl-glycine. Conduit Vasonotor Function. By Race 4. The method of claim 1, wherein the at least one White Patients Black Patients P compound that donates, transfers, or releases nitric oxide, or induces the production of endogenous nitric oxide, or endot FMD (%) helium-derive relaxing factors, or is a Substrate for nitric Normotensive 11.5 - 5.9 13.3 7.2 NS oxide synthase is selected from the group consisting of Hypertensive 9.SS.3* 8.8 6.2* NS L-arginine, L-homoarginine, N-hydroxy-L-arginine, nitro NTG-response (%) 10 sated L-arginine, nitrosylated L-arginine, nitrosated N-hy droxy-L-arginine, nitrosylated N-hydroxy-L-arginine, cit Normotensive 18.1 - 8.1 20.6 - 8.4 NS Hypertensive 14.6 + 6.2* 16.4 7.1* NS rulline, omithine, glutamine; and lysine. 5. The method of claim 1, wherein the at least one Data are meant SD. antioxidant enzyme and the at least one compound that *p < 0.001 compared to race matched normotensives. 15 donates, transfers, or releases nitric oxide, or induces the Each of the above examples demonstrate that NO action production of endogenous nitric oxides, or endothelium is impaired in the microvasculature of black hypertensive derived relaxing factor, or is a substrate for nitric oxide patients to a greater extent than in white hypertensive synthase, or a pharmaceutically acceptable salt thereof are patients. There is a suggestion that this abnormality may administered orally, or parenterally. precede the development of hypertension, particularly in 6. The method of claim 5, wherein the at least one salt-sensitive individuals, consistent with a pathogenic role. antioxidant enzyme and the at least one compound that In conduit vessels, a marked impairment of NO action that donates, transfers, or releases nitric oxide, or induces the may contribute to ischemic heart disease and stroke has been production of endogenous nitric oxide, or endothelium demonstrated. This abnormality appears to be independent derived relaxing factor, or is a substrate for nitric oxide of race, a finding that is consistent with possibility that the 25 synthase, or a pharmaceutically acceptable salt thereof are impairment is a consequence of blood pressure elevation of administered orally. any cause. Since African Americans have a greater incidence 7. The method of claim 6, wherein the at least one of hypertension and a preponderance of salt-sensitive hyper antioxidant enzyme and the at least one compound that tension, these findings lend further Support to the hypothesis donates, transfers, or releases nitric oxide, or induces the that nitric oxide insufficiency contributes to the pathogenesis 30 production of endogenous nitric oxide, or endothelium of ischemic heart disease in this population. derived relaxing factor, or is a substrate for nitric oxide The disclosure of each patent, patent application and synthase, or a pharmaceutically acceptable salt thereof are publication cited herein is hereby incorporated by reference administered orally as a Solid dose, a liquid dose or a herein in its entirety. Suspension. 35 8. The method of claim 7, wherein the solid dose is a Although the invention has been set forth in detail, one tablet or capsule. skilled in the art will appreciate that numerous changes and 9. The method of claim 7, wherein the solid dose is a modifications can be made to the invention without depart Sustained release capsule or a Sustained release tablet. ing from the spirit and scope thereof. 10. The method of claim 8, wherein the tablet or capsule 40 is a Sublingual tablet or a Sublingual capsule. What is claimed is: 11. The method of claim 8, wherein the tablet or capsule 1. A method to treat a vascular disease resulting from is a chewable tablet or a chewable capsule. nitric oxide insufficiency in a patient comprising adminis 12. The method of claim 1, further comprising adminis tering to the patient a therapeutically effective amount of at tering a therapeutically effective edema managing amount of least one antioxidant enzyme, and at least one compound 45 a diuretic compound. that donates, transfers, or releases nitric oxide, or induces the 13. The method of claim 12, wherein the diuretic com production of endogenous nitric oxide, or endothelium pound is a thiazide, ethacrynic acid, a furosemide, a spira derived relaxing factor, or is a substrate for nitric oxide nolactone, a triamterene, or a mixture thereof. synthase, or a pharmaceutically acceptable Salt thereof, 14. The method of claim 12, further comprising admin wherein the antioxidant enzyme is Superoxide dismutase, 50 istering a therapeutically effective amount of potassium. catalase, glutathione peroxidase, or a mixture thereof and the 15. The method of claim 14, wherein the potassium is vascular disease resulting from nitric oxide insufficiency is administered as potassium chloride or by the daily ingestion a disease resulting from oxidative stress and wherein the at of foods with high potassium content. least one antioxidant enzyme, and the at least one compound 16. The method of claim 1, further comprising adminis that donates, transfers, or releases nitric oxide, or induces the 55 tering at least one nitrosated angiotensin-converting enzyme production of endogenous nitric oxide, or endothelium inhibitor, nitrosated beta-adrenergic blocker, nitrosated cal derived relaxing factor, or is a substrate for nitric oxide cium channel blocker, nitrosated endothelin antagonist, nit synthase are administered as components of the same com rosated angiotensin II receptor antagonist, nitrosated renin position, or are administered as separate components at inhibitor, or a mixture thereof. about the same time. 60 17. The method of claim 1, wherein the at least one 2. The method of claim 1, further comprising administer compound that donates, transfers, or releases nitric oxide, or ing a pharmaceutically acceptable carrier. induces the production of endogenous nitric oxide or endot 3. The method of claim 1, wherein the said at least one helium-derived relaxing factor or is a substrate for nitric compound that donates, transfers, or releases nitric oxide, or oxide synthase is isosorbide dinitrate and/or isosorbide induces the production of endogenous nitric oxide, or endot 65 mononitrate. helium-derived relaxing factor, or is a substrate for nitric oxide synthase is selected from the group consisting of