USOO6635273B1 (12) United States Patent (10) Patent No.: US 6,635,273 B1 LOScalZ0 et al. (45) Date of Patent: Oct. 21, 2003

(54) METHODS OF TREATING VASCULAR Dries et al, The New England Journal of Medicine, DISEASES CHARACTERIZED BY NITRIC 340(8):609-616 (Feb. 25, 1999). OXDE INSUFFICIENCY Freedman et al, Drugs, 54(Supp. 3):41-50 (1997). Sherman et al., Cardiologia, 42(2):177-187 (1997). (75) Inventors: Joseph Loscalzo, Dover, MA (US); Biegelson et al., Coronary Artery Disease, 10:241-256 Joseph A. Vita, Hingham, MA (US); (1999). Michael D. Loberg, Boston, MA (US); Rudd et al, Am. J. Physiol., 277(46):H732–H739 (1999). Manuel Worcel, Boston, MA (US) Hammerman et al, Am. J. Physiol., 277(46):H1579-H1592 (1999). (73) Assignees: Trustees of Boston University, Boston, LoScalzo et al., Transactions of the American and Climato MA (US); NitroMed, Inc., Bedford, logical ASS., 111:158-163 (2000). MA (US) Cohn et al, The New England Journal of Medicine, 325(5):303-310 (1991). (*) Notice: Subject to any disclaimer, the term of this Cohn et al, The New England Journal of Medicine , patent is extended or adjusted under 35 314(24): 1547–1552 (1986). U.S.C. 154(b) by 0 days. Carson et al., Circulation, Supplement I, 92(8):I31-I32, Abstract No. 0145 (1995). (21) Appl. No.: 09/697,317 (List continued on next page.) (22) Filed: Oct. 27, 2000 Primary Examiner-Jon P. Weber Related U.S. Application Data ASSistant Examiner-Kalash C. Srivastava (60) Provisional application No. 60/179,020, filed on Jan. 31, (74) Attorney, Agent, or Firm-Hale and Dorr LLP 2000, and provisional application No. 60/162,230, filed on Oct. 29, 1999. (57) ABSTRACT The present invention provides methods of treating vascular (51) Int. Cl." ...... A61L 15/16; A61K 47/00; diseases where nitric oxide insufficiency is a contributing A61 K9/20: A61 K31/00; CO7D 515/02 factor by administering a therapeutically effective amount of (52) U.S. Cl...... 424/444; 424/400; 424/439; at least one antioxidant, or a pharmaceutically acceptable 424/440, 424/443; 424/451; 424/464; 424/474; Salt thereof, and at least one of isosorbide dinitrate and 424/489; 424/944; 514/218; 514/248; 514/303; isosorbide mononitrate, and, optionally, at least one nitro 514/310; 514/364; 514/470; 54.6/118 Sated angiotensin-converting enzyme inhibitor, nitrosated (58) Field of Search ...... 424/400, 439, beta-adrenergic blocker, nitroSated calcium channel blocker, 424/444, 944, 440, 443, 451, 464, 474, nitrosated endothelin antagonist, nitrosated angiotensin II 489; 514/303,564, 218, 248, 364, 310, receptor antagonist, nitroSated renin inhibitor, and/or at least 470; 54.6/118 one compound used to treat cardiovascular diseases. The antioxidant is preferably a hydralazine compound or a (56) References Cited pharmaceutically acceptable Salt thereof. The present inven U.S. PATENT DOCUMENTS tion also provides methods of treating vascular diseases where nitric oxide insufficiency is a contributing factor by 4,868,179 A 9/1989 Cohn administering a therapeutically effective amount of at least 5,627,191. A 5/1997 Birch et al. one nitrosated angiotensin-converting enzyme inhibitor, nit 5,645,839 A 7/1997 Chobanian et al. rosated beta-adrenergic blocker, nitrosated calcium channel 5,891.459 A 4/1999 Cooke et al. 5,968.983 A 10/1999 Kaesemeyer blocker, nitrosated endothelin antagonist, nitrosated angio 5,973,011 A 10/1999 Noack et al. tensin II receptor antagonist, nitrosated renin inhibitor, and, 6,103,769 A 8/2000 Kelm optionally, at least one antioxidant and/or at least one 6,117.872 A 9/2000 Maxwell et al. compound used to treat cardiovascular diseases. The present 6,319,515 B1 11/2001 Hidaka et al. invention also provides methods of treating Raynaud's Syn drome by administering a therapeutically effective amount FOREIGN PATENT DOCUMENTS of at least one antioxidant and at least one of isosorbide WO WO95/26725 10/1995 dinitrate and isosorbide mononitrate and/or at least one WO WO 98/21193 5/1998 nitrosated angiotensin-converting enzyme inhibitor, nitro WO WO 99/00361 1/1999 Sated calcium channel blocker, nitrosated endothelin WO WO 99/66921 12/1999 antagonist, nitrosated angiotensin II receptor antagonist, WO WO 99/67231 12/1999 nitrosated renin inhibitor. The present invention also pro OTHER PUBLICATIONS vides novel transdermal patches comprising at least one antioxidant and at least one of isosorbide dinitrate and von Lutterotti et al, American Journal of Hypertension, 4 (4 isosorbide mononitrate and/or at least one nitrosated Part 2):346S-349S (1991). angiotensin-converting enzyme inhibitor, nitrosated beta Burneir et al., Hypertension, 22(3):339–347 (Sep. 1993). adrenergic blocker, nitrosated calcium channel blocker, nit Dupuis, Cardiovascular Drugs and Therapy, 8(3):501-507 rosated endothelin antagonist, nitroSated angiotensin II (1994). receptor antagonist, nitrosated renin inhibitor. Carson et al., Journal of Cardiac Failure, 5(3):178-187 (Sep. 10, 1999). 33 Claims, 3 Drawing Sheets US 6,635,273 B1 Page 2

OTHER PUBLICATIONS Carson et al., Journal of Cardiac Failure, 5(3):178-187 (Sep. Francis et al, Circulation, Supplement VI, 87(6):VI40-VI48 10, 1999). (1993). Dries et al, The New England Journal of Medicine, Pierpont et al, Chest, 73(1):8–13 (1978). 34(8):609-616 (Feb. 25, 1999). Massie et al., The American Journal of Cardiology, Freedman et al, Drugs, 54(Supp. 3):41-50 (1997). 40:794-801 (1977). Sherman et al., Cardiologia, 42(2):177-187 (1997). Kaplan et al., Annals of Internal Medicine, 84:639-645 Biegelson et al., Coronary Artery Disease, 10:241-256 (1976). (1999). Bauer et al., Circulation, 84(1):35-39 (1991). Rudd et al, Am. J. Physiol., 277(46):H732–H739 (1999). The SOLVD Investigators, The New England Journal of Hammerman et al, Am. J. Physiol., 277(46):H1579-H1592 Medicine, 327(10):685–691 (1992). (1999). Ziesche et al., Circulation, 87(6):VI56-VI64 (1993). LoScalzo et al., Transactions of the American and Climato Rector et al., Circulation, 87(6):VI71-VI77 (1993). logical ASS., 111:158-163 (2000). U.S. Patent Oct. 21, 2003 Sheet 1 of 3 US 6,635,273 B1

Fig. 1

Decreased Renin Release NO Insufficiency Salt/Water Retention

Increased Intracellular Increased Sensitivity to Sodium & Calcium a techolamines and Angiotensin II

LVH Decreased Capillary Density Increased Vascular Tone Myocardial Fibrosis

Hypertension Microvascular

Increased LV Mass Cardiac Ischemia Diastolic Dysfunction

FIG. 2 -O-White Hypertensives -o- Black Hypertensives a 15 9 9. S 10 CS O E o-1 -1 PCOOO1

Baseline O.3 1.O 3.0 10 Methacholine Dose (g/min) U.S. Patent Oct. 21, 2003 Sheet 2 of 3 US 6,635,273 B1 FIG. 3A

-o-White normotensives -o- Black nomotensives a 9.Q 15 / 9. S 10 8 N-1 5 l - O Baseline 0.3 10 3.0 1O Nitropiusside Dose (g/min)

FIG. 3B

-O-White normotensives -O-Black nomotensives

Baseline O.3 1.O 3.O 1O Methacholine Dose (g/min) U.S. Patent Oct. 21, 2003 Sheet 3 of 3 US 6,635,273 B1

FIG. 4A

-o- Salt Resistant (n=25) -e - Satt Sensitive (n-6) 15 C

Baseline 0.3 1.0 3.0 10. Methachotine Dose (g/min)

FIG. 4B

-O-Salt Resistant (n=25)

-O - Satt Sensitive (n=6) g 15 9. E 10

N- 5 21 - G2-2 I- O PEO.16 Baseline O.3 1.O 3.0 10 Nitroprussiae Dose (g/min) US 6,635,273 B1 1 2 METHODS OF TREATING WASCULAR than among white Americans. In this decade, the death rate DISEASES CHARACTERIZED BY NITRIC from cardiovascular disease for black males was 353 per OXDE INSUFFICIENCY 100,000 population, while that for white males was 244 per 100,000; the rate for black females was 226 per 100,000; RELATED APPLICATIONS while that for white females was 135 per 100,000. Conso This application claims priority to U.S. Provisional Appli nant with this important demographic parameter is the cation No. 60/162,230 filed Oct. 29, 1999 and U.S. Provi observation that there is a higher prevalence of Several of the important risk factors for cardiovascular disease, e.g., sional Application No. 60/179,020 filed Jan. 31, 2000. hypertension, Smoking, diabetes mellitus, obesity, and left FIELD OF THE INVENTION Ventricular hypertrophy, among blacks compared with whites. In addition, outcomes of cardiovascular events are The present invention provides methods of treating and/or worse for blacks than whites. Following myocardial preventing vascular diseases characterized by nitric oxide infarction, blacks have a 50% higher annual mortality rate insufficiency by administering a therapeutically effective than whites, and their five year Survival is only 70%. Thus, amount of at least one antioxidant or a pharmaceutically 15 the many advances in cardiovascular medicine that account acceptable Salt thereof, and at least one of isosorbide dini for the overall improvement in cardiovascular health in the trate and isosorbide mononitrate, and, optionally, at least one general population have failed to translate into comparable nitrosated angiotensin-converting enzyme inhibitor, nitro racial benefits. Sated beta-adrenergic blocker, nitrosated calcium channel blocker, nitrosated endothelin antagonist, nitrosated angio There is a need in the art for new and more effective tensin II receptor antagonist, nitroSated renin inhibitor, and/ compositions and methods for treating vascular diseases. or at least one compound used to treat cardiovascular The present invention is directed to these, as well as other, diseases. The antioxidant is preferably a hydralazine com important ends. pound or a pharmaceutically acceptable Salt thereof. The SUMMARY OF THE INVENTION present invention also provides methods of treating and/or 25 The present invention provides methods for treating and/ preventing vascular diseases characterized by nitric oxide or preventing vascular diseases characterized by nitric oxide insufficiency by administering a therapeutically effective insufficiency by administering to a patient a therapeutically amount of at least one nitrosated angiotensin-converting effective amount of at least one antioxidant or a pharma enzyme inhibitor, nitrosated beta-adrenergic blocker, nitro ceutically acceptable Salt thereof, and at least one of isoS Sated calcium channel blocker, nitrosated endothelin orbide dinitrate and isosorbide mononitrate, and, optionally, antagonist, nitrosated angiotensin II receptor antagonist and/ at least one nitrosated angiotensin-converting enzyme or nitrosated renin inhibitor, and, optionally, at least one inhibitor, nitrosated beta-adrenergic blocker, nitrosated cal antioxidant and/or at least one compound used to treat cium channel blocker, nitrosated endothelin antagonist, nit cardiovascular diseases. The present invention also provides rosated angiotensin II receptor antagonist, nitrosated renin methods of treating and/or preventing Raynaud's Syndrome 35 inhibitor, and/or at least one compound used to treat cardio by administering a therapeutically effective amount of at vascular diseases. The antioxidant is preferably a hydrala least one antioxidant or a pharmaceutically acceptable Salt Zine compound or a pharmaceutically acceptable Salt thereof, and at least one of isosorbide dinitrate and isosor thereof. The vascular diseases characterized by nitric oxide bide mononitrate, and/or at least one nitrosated angiotensin insufficiency include, for example, hypertension (e.g., low converting enzyme inhibitor, nitroSated calcium channel 40 renin hypertension; Salt-Sensitive hypertension; low-renin, blocker, nitrosated endothelin antagonist, nitrosated angio Salt-sensitive hypertension; primary pulmonary hyperten tensin II receptor antagonist and/or nitrosated renin inhibi Sion; thromboembolic pulmonary hypertension; pregnancy tor. The present invention also provides novel transdermal induced hypertension; renovascular hypertension), heart patches comprising at least one antioxidant or a pharmaceu failure (e.g., microvascular cardiac ischemia), and left ven tically acceptable Salt thereof, and at least one of isosorbide 45 tricular hypertrophy with disproportionate dinitrate and isosorbide mononitrate, and/or at least one microvascularization, (i.e., inadequate vascularity) or dias nitrosated angiotensin-converting enzyme inhibitor, nitro tolic dysfunction. The antioxidant and the isosorbide dini Sated beta-adrenergic blocker, nitrosated calcium channel trate or isosorbide mononitrate and optional nitrosated com blocker, nitrosated endothelin antagonist, nitrosated angio pound and/or compound used to treat cardiovascular tensin II receptor antagonist and/or nitrosated renin inhibi 50 diseases can be administered Separately or as components of tor. the same composition. BACKGROUND OF THE INVENTION Another aspect of the present invention provides methods for treating and/or preventing vascular diseases character The decline in cardiovascular morbidity and mortality in ized by nitric oxide insufficiency by administering to a the United States over the past three decades has been the 55 patient a therapeutically effective amount of at least one result of Significant advances in research on cardiovascular nitrosated angiotensin-converting enzyme inhibitor, nitro disease mechanisms and therapeutic Strategies. The inci Sated beta-adrenergic blocker, nitrosated calcium channel dence and prevalence of myocardial infarction and death blocker, nitrosated endothelin antagonist, nitrosated angio from myocardial infarction, as well as that from cerebrovas tensin II receptor antagonist and/or nitrosated renin cular accident, have decreased significantly over this period 60 inhibitor, and, optionally, at least one antioxidant and/or at largely owing to advances in prevention, early diagnosis, least one compound used to treat cardiovascular diseases. and treatment of these very common diseases. The vascular diseases characterized by nitric oxide insuffi Analysis of outcomes by race, however, paints quite a ciency include, for example, hypertension (e.g., low-renin different picture: life expectancy and cardiovascular mor hypertension; Salt-Sensitive hypertension; low-renin, Salt bidity rates have improved far less for blacks than whites. 65 Sensitive hypertension; primary pulmonary hypertension; Available data show that the likelihood of dying from thromboembolic pulmonary hypertension; pregnancy cardiovascular disease is far greater among black Americans induced hypertension; renovascular hypertension), heart US 6,635,273 B1 3 4 failure (e.g., microvascular cardiac ischemia), and left ven “Patient” refers to animals, preferably mammals, most tricular hypertrophy with disproportionate preferably humans, and includes males and females. microvascularization, (i.e., inadequate vascularity) or dias “Therapeutically effective amount” refers to the amount tolic dysfunction. The nitrosated compound and optional of the compound and/or composition that is effective to antioxidant and/or compound used to treat cardiovascular diseases can be administered Separately or as components of achieve its intended purpose. the same composition. “Transdermal” refers to the delivery of a compound by In another aspect, the present invention provides methods passage through the skin and into the blood Stream. for treating and/or preventing Raynaud's Syndrome by “Transmucosal” refers to delivery of a compound by administering to a patient a therapeutically effective amount passage of the compound through the mucosal tissue and of at least one antioxidant or a pharmaceutically acceptable into the blood stream. Salt thereof, and at least one of isosorbide dinitrate and “Penetration enhancement' or “permeation enhance isosorbide mononitrate and/or at least one nitrosated ment” refers to an increase in the permeability of the skin or angiotensin-converting enzyme inhibitor, nitrosated calcium mucosal tissue to a Selected pharmacologically active com channel blocker, nitrosated endothelin antagonist, nitrosated pound Such that the rate at which the compound permeates angiotensin II receptor antagonist and/or nitrosated renin 15 inhibitor. The antioxidant, isosorbide dinitrate or isosorbide through the skin or mucosal tissue is increased. mononitrate, and nitrosated compound can be administered “Carriers' or "vehicles' refers to carrier materials Suitable Separately or as components of the Same composition. for compound administration and include any Such material In yet another aspect, the present invention provides novel known in the art Such as, for example, any liquid, gel, transdermal patches comprising a therapeutically effective Solvent, liquid diluent, Solubilizer, or the like, which is amount of at least one antioxidant at least one of isosorbide non-toxic and which does not interact with any components dinitrate and isosorbide mononitrate and/or at least one of the composition in a deleterious manner. nitrosated angiotensin-converting enzyme inhibitor, nitro “Hydralazine compound” refers to a compound having Sated beta-adrenergic blocker, nitrosated calcium channel the formula: blocker, nitrosated endothelin antagonist, nitrosated angio 25 tensin II receptor antagonist and/or nitrosated renin inhibitor, and mixtures thereof. al b C These and other aspects of the present invention are RN FN FR described in more detail herein. BRIEF DESCRIPTION OF THE FIGURES wherein a, b and c are independently a Single or double FIG. 1 shows that nitric oxide (NO) insufficiency is bond; R and R are each independently a hydrogen, an asSociated with increased Salt and water retention and a alkyl, an ester or a heterocyclic ring; R and R are each low-renin State. Increased intracellular Sodium and calcium independently a lone pair of electrons or a hydrogen, with in conjunction with reduced NO leads to enhanced Sensi the proviso that at least one R, R2, R and R is not a tivity of vascular Smooth muscle cells and cardiomyocytes 35 hydrogen. Exemplary hydralazine compounds include to the tonic and growth-stimulating properties of catechola budralazine, cadralazine, dihydralazine, endralazine, mines and angiotensin II. Increased vascular tone, left hydralazine, pildralazine, todralazine, and the like. Ventricular hypertrophy with inadequate capillary "Antioxidant” refers to a compound that can react and angiogenesis, and increased matrix production with myo quench a free radical. cardial fibrosis result. These intermediate phenotypes lead to 40 “Alkyl” refers to a lower alkyl group, a haloalkyl group, the clinical disorders of low-renin, Salt-Sensitive hyperten a hydroxyalkyl group, an alkenyl group, an alkynyl group, Sion; disproportionate left ventricular hypertrophy and dias a bridged cycloalkyl group, a cycloalkyl group or a hetero tolic dysfunction; and microvascular myocardial ischemia. cyclic ring, as defined herein. FIG. 2 shows forearm blood flow responses to intra “Lower alkyl” refers to branched or straight chain acyclic arterial methacholine that were assessed using venous occlu 45 alkyl group comprising one to about ten carbon atoms Sion plethysmography in 20 white and 16 black patients with (preferably one to about eight carbon atoms, more preferably a clinical history of hypertension (BP>140/90). FIG. 2 one to about six carbon atoms). Exemplary lower alkyl shows that endothelium-derived NO action is impaired in groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, the forearm microVessels of the black patients compared to isobutyl, Sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl, the white patients. 50 hexyl, octyl, and the like. FIGS. 3A and 3B show forearm blood flow responses to “Haloalkyl” refers to a lower alkyl group, an alkenyl nitroprusside (FIG. 3A) and methacholine (FIG. 3B) that group, an alkynyl group, a bridged cycloalkyl group, a were assessed by Venous occlusion plethysmography in 25 white and 21 black patients without hypertension. The cycloalkyl group or a heterocyclic ring, as defined herein, to dilator response to sodium nitroprusside (FIG. 3A) was which is appended one or more halogens, as defined herein. Significantly lower in black patients, while there was no 55 Exemplary haloalkyl groups include trifluoromethyl, racial difference in response to methacholine (FIG. 3B). chloromethyl, 2-bromobutyl, 1-bromo-2-chloro-pentyl, and FIGS. 4A and 4B show the effect of salt-sensitivity on the like. forearm microvascular function. By repeated measures “Alkenyl refers to a branched or Straight chain C-Co ANOVA, there were trends for impaired responses to metha hydrocarbon (preferably a C-C hydrocarbon, more pref choline (FIG. 4A) and sodium nitroprusside (FIG. 4B) in 60 erably a C-C hydrocarbon) which can comprise one or Salt-sensitive black patients. more carbon-carbon double bonds. Exemplary alkenyl groups include propylenyl, buten-1-yl, isobutenyl, penten DETAILED DESCRIPTION OF THE 1-yl, 2.2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, INVENTION hepten-1-yl, octen-1-yl, and like. AS used throughout the disclosure, the following terms, 65 “Alkynyl' refers to an unsaturated acyclic C-Cohydro unless otherwise indicated, shall be understood to have the carbon (preferably a C-C hydrocarbon, more preferably a following meanings. C-C hydrocarbon) which can comprise one or more US 6,635,273 B1 S 6 carbon-carbon triple bonds. Exemplary alkynyl groups arylcarbonyl, amn idyl, e Ster, carbo X amido, include ethynyl, propynyl, butyn-1-yl, butyn-2-yl, pentyl-1- alkylcarboxamido, carbomyl, Sulfonic acid, Sulfonic ester, yl, pentyl-2-yl, 3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl, Sulfonamido and nitro. Exemplary Substituted aryl groups hexyl-3-yl, 3,3-dimethyl-butyn-1-yl, and the like. include tetrafluorophenyl, pentafluorophenyl, Sulfonamide, “Bridged cycloalkyl” refers to two or more cycloalkyl 5 alkylsulfonyl, arylsulfonyl, and the like. groups, heterocyclic groups, or a combination thereof fused “Hydroxy” refers to -OH. via adjacent or non-adjacent atoms. Bridged cycloalkyl “Hydroxyalkyl” refers to a hydroxy group, as defined groups can be unsubstituted or Substituted with one, two or herein, appended to an alkyl group, as defined herein. three Substituents independently Selected from alkyl, alkoxy, “Alkylcarbonyl” refers to Rs-C(O), wherein R is amino, alkylamino, dialkylamino, hydroxy, halo, carboxyl, an alkyl group, as defined herein. alkylcarboxylic acid, aryl, amidyl, ester, alkylcarboxylic “Arylcarbonyl” refers to Rss-C(O), wherein Rss is an ester, carboxamido, alkylcarboxamido, OXO and nitro. Exem aryl group, as defined herein. plary bridged cycloalkyl groups include adamantyl, “Ester” refers to RC(O)O- wherein Rs is a hydrogen decahydronapthyl, quinuclidyl, 2,6-dioxabicyclo3.3.0 atom, an alkyl group, an aryl group, an alkylaryl group, or octane, 7-Oxabycyclo2.2.1]heptyl, 8-azabicyclo3.2.1]oct 15 an arylheterocyclic ring, as defined herein. “Alkylaryl' 2-enyl, and the like. refers to an alkyl group, as defined herein, to which is “Cycloalkyl” refers to a Saturated or unsaturated cyclic appended an aryl group, as defined herein. Exemplary hydrocarbon comprising from about 3 to about 8 carbon alkylaryl groups include benzyl, phenylethyl, atoms. Cycloalkyl groups can be unsubstituted or Substituted hydroxybenzyl, fluorobenzyl, fluorophenylethyl, and the with one, two or three Substituents independently Selected like. from alkyl, alkoxy, amino, alkylamino, dialkylamino, "Arylheterocyclic ring” refers to a bi- or tricyclic ring arylamino, diarylamino, alkylarylamino, aryl, amidyl, ester, comprised of an aryl ring, as defined herein, appended via hydroxy, halo, carboxyl, alkylcarboxylic acid, alkylcarboxy two adjacent carbon atoms of the aryl ring to a heterocyclic lic ester, carboxamido, alkylcarboxamido, OXO and nitro. ring, as defined herein. Exemplary arylheterocyclic rings Exemplary cycloalkyl groups include cyclopropyl, 25 include dihydroindole, 1,2,3,4-tetra-hydroquinoline, and the cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, like. cyclohepta, 1,3-dienyl, and the like. “Hydrazino” refers to HN-N(H)-. “Heterocyclic ring or group' refers to a Saturated or “Compound used to treat cardiovascular diseases” refers unsaturated cyclic hydrocarbon group having about 2 to to any therapeutic compound, or a pharmaceutically accept about 10 carbon atoms (preferably about 4 to about 6 carbon able Salt thereof, used to treat any cardiovascular disease. atoms) where 1 to about 4 carbon atoms are replaced by one Suitable compounds include, but are not limited to, or more nitrogen, oxygen and/or Sulfur atoms. Sulfur maybe angiotensin-converting enzyme (ACE) inhibitors (Such as, in the thio, Sulfinyl or Sulfonyl oxidation State. The hetero for example, alacepril, benazepril, captopril, ceranapril, cyclic ring or group can be fused to an aromatic hydrocarbon cilaZapril, delapril, duinapril, enalapril, enalaprilat, group. Heterocyclic groups can be unsubstituted or Substi 35 foSinopril, imidapril, lisinopril, moveltipril, pentopril, tuted with one, two or three Substituents independently perindopril, quinapril, ramipril, rentipril, Spirapril, Selected from alkyl, alkoxy, amino, alkylamino, temocapril, trandolapril, Zofenopril, and the like), beta dialkylamino, arylamino, diarylamino, alkylarylamino, adrenergic blockers (such as, for example, amoSulalol, hydroxy, OXO, thial, halo, carboxyl, carboxylic ester, alkyl atenolol, betaxolol, bethanidine, bevantolol, bisoprolol, carboxylic acid, alkylcarboxylic ester, aryl, arylcarboxylic 40 bopindolol, bufuralol, bunitrolol, bupranolol, butafilolol, acid, arylcarboxylic ester, alkylcarbonyl, arylcarbonyl, carazolol, carteolol, carvedilol, celiprolol, cetamolol, amidyl, ester, carboxamido, alkylcarboxamido, dilevalol, epanolol, indenolol, mepindolol, metipranolol, arylcarboxamido, hydrazino, Sulfonic acid, Sulfonic ester, metoprolol, moprolol, nadolol, nebivolol, nipradillol, Sulfonamido and nitro. Exemplary heterocyclic groups penbutolol, pindolol, pronethalol, propranolol, Sotalol, include pyrrolyl, 3-pyrrolinyl, 4,5,6-trihydro-2H-pyranyl, 45 Sulfinalol, talinolol, tertatolol, tilisolol, timolol, toliprolol, pyridinyl, 1,4-dihydropyridinyl, pyrazolyl, triazolyl, and the like); cholesterol reducers (Such as, for example, pyrimidinyl, pyridaZinyl, oxazolyl, thiazolyl, imidazolyl, HMG-CoA reductase inhibitors, including, but not limited indolyl, thiophenyl, furanyl, tetrhydrofuranyl, tetrazolyl, to, lovastatin (MEVACOR(R), simvastatin (ZOCOR(R), 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, oxazolindinyl 1,3- pravastatin (PRAVACHOLE), fluvastatin, cerivastatin dioxolanyl, 2-imidazonlinyl, imidazolindinyl, 2-pyrazolinyl, 50 (BAYCOLOR), atorvastatin (LIPITOR(R), and the like; pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, Sequestrants, including, but not limited to, cholestyramine, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4H-pyranyl, colestipol, Sialkylaminoalkyl derivatives of croSS-linked piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, dextran, and the like; inhibitors of cholesterol absorption, thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl, 1.3, including, but not limited to, beta-sitosterol, acyl CoA 5-trithianyl, benzo(b)thiophenyl, benzimidazolyl, 55 cholersterol acyltransferase inhibitors, melinamide, and the quinolinyl, and the like. like), calcium channel blockers (such as, for example, "Aryl refers to a monocyclic, bicyclic, carbocyclic or amlodipine, aranidipine, barnidipine, benidipine, heterocyclic ring System comprising one or two aromatic cilnidipine, clentiaZem, diltiazen, efonidipine, fantofarone, rings. Exemplary aryl groups include phenyl, pyridyl, felodipine, isradipine, lacidipine, lercanidipine, manidipine, napthyl, quinoyl, tetrahydronaphthyl, furanyl, indanyl, 60 mibefradil, nicardipine, nifedipine, nilvadipine, nisoldipine, indenyl, indoyl, and the like. Aryl groups (including bicylic nitrendipine, Semotiadil, Veraparmil, and the like), angio aryl groups) can be unsubstituted or Substituted with one, tensin II receptor antagonists (such as, for example, two or three Substituents independently Selected from alkyl, ciclosidomine, eprosartan, furosemide, irbesartan, losartan, alkoxy, amino, alkylamino, dialkylamino, arylamino, Saralasin, Valsartan, and the like); endothelin antagonists diarylamino, alkylarylamino, hydroxy, carboxyl, carboxylic 65 (Such as, for example, bosentan, Sulfonamide endothelin ester, alkylcarboxylic acid, alkylcarboxylic ester, aryl, aryl antagonists, BQ-123, SQ28608, and the like); renin inhibi carboxylic acid, arylcarboxylic ester, alkylcarbonyl, tors (such as, for example, enalkrein, RO 42-5892, A65317, US 6,635,273 B1 7 8 CP80794, ES 1005, ES 8891, SQ34017, and the like); and portionate to afterload and with an inadequate angiogenic mixtures thereof. response; and microvascular ischemia in the absence of “Cardiovascular diseases” refers to any cardiovascular Significant epicardial coronary artery disease. The net patho disease, including but not limited to, congestive heart physiological consequences of these effects are increased failure, hypertension, pulmonary hypertension, myocardial peripheral vascular resistance with accompanying arterial and cerebral infarctions, atherosclerosis, atherogenesis, hypertension; and an inadequately vascularized, fibrotic thrombosis, ischemic heart disease, post-angioplasty increase in left ventricular mass with accompanying dias restenosis, coronary artery diseases, renal failure, Stable, tolic dysfunction and microvascular ischemia. unstable and variant (Prinzmetal) angina, cardiac edema, Given these clinical observations and the role that NO renal insufficiency, nephrotic edema, hepatic edema, Stroke, playS in preventing their development, the present inventors and the like. have unexpectedly discovered that the principal cardiovas Two broad classes of cardiovascular disorders are more cular disorders common among blacks (such as prevalent among blacks than whites and Serve as areas in hypertension, left ventricular hypertrophy, and heart failure) need of investigative efforts. Hypertension and left ventricu result from a specific vascular diathesis that is a direct lar hypertrophy, two related yet independent risk factors for 15 consequence of nitric oxide insufficiency. An outline of the coronary heart disease, are significantly more prevalent pathogenic consequences of NO insufficiency that Serve as among blacks than whites. BlackS also have higher rates of the basis for these cardiovascular disorders is shown in FIG. angiographically normal coronary arteries despite a higher 1. prevalence of risk factors for coronary atherosclerosis, and NO insufficiency States can be a consequence of reduced greater morbidity and mortality from coronary heart disease synthesis of NO, enhanced inactivation of NO, or both. than whites. These paradoxical observations have led Some PoSSible candidate mechanisms include alterations in the investigators to postulate that blackSharbor a diathesis of the genes that code for endothelial NO synthase or the inducible microvasculature that limits perfusion and Serves as a Stimu microvascular and cardiomyocyte NO Synthase leading to lus for vascular Smooth muscle cell and cardiomyocyte reduced expression of a normal gene product or appropriate hypertrophy, which, in turn, leads to hypertension and left 25 expression of a leSS active gene product, reduction in the Ventricular hypertrophy, respectively. The underlying basis enzymatic activity of NO Synthase owing to inadequate for this vascular diathesis may involve the endothelium, cofactor concentrations, or enhanced inactivation of NO by which has a limited capacity to generate vasodilator and oxidant StreSS. antiproliferative factors or an increased capacity to produce Data obtained by the inventors in cultured cells, animal vasoconstrictor and proliferative factors, the vascular models, and human patients Suggest that increased oxidant Smooth muscle cell, which manifests increased Sensitivity to StreSS is central to the vascular diathesis of and consequent vasoconstrictor and proliferative factors, or both, in these cardiovascular disorders common among African Ameri individuals. cans. PoSSible candidate mechanisms for the oxidant StreSS The present inventors have discovered that a major prod include enhanced production of reactive oxygen Species uct of the normal blood vessel that may play a role in the 35 (ROS), decreased antioxidant defenses, or both. The inven vascular diathesis of blacks is endothelium-derived nitric tors make no a priori assumptions about the temporal or oxide (NO). Nitric oxide produced by the endothelial cells causative relationship between oxidant StreSS and the vas induces vascular Smooth muscle cell relaxation, contributing cular phenotype of blackS: Oxidant StreSS may both precede importantly to resting vascular tone. In addition, NO inhibits the development of the vascular diathesis and promote its vascular Smooth muscle cell proliferation and induces apo 40 progression once established. Recent data Suggest that ptosis in Smooth muscle cells, which leads to the release of enhanced ROS production accompanies essential basic fibroblast growth factor and vascular endothelial cell hypertension, atherosclerosis, thrombosis, and diabetes growth factor, in turn Supporting endothelial cell prolifera mellitus, and appears in each case, at the very least, to be tion. This sequence of cellular responses is believed to important in the progression of established disease, if not in Sustain angiogenesis under hypoxic or ischemic conditions. 45 its actual genesis. The role of NO in the vascular diathesis of blacks is Endothelium-derived relaxing factor (EDRF), first illustrated by the consequences of NO insufficiency in the described by Furchgott et al, Nature, 299:373-376 (1980), is normal responses of the vasculature to NO. Nitric oxide an important mediator of vascular function. This endothelial insufficiency Suppresses renin release from the juxtaglom product activates guanylyl cyclase in vascular Smooth erular cells, and induces a Sodium chloride/volume Sensitive 50 muscle cells and platelets, leading to vasorelaxation and increase in blood pressure. Furthermore, NO insufficiency platelet inhibition, respectively (LoScalzo et al., Prog Car leads to an increased Sensitivity of vascular Smooth muscle diovasc Dis, 38:87–104 (1995)). The chemical nature of cells to vasoconstrictors, Such as angiotensin II and EDRF has been Studied using a variety of pharmacological catecholamines, which amplify the increase in Vascular and analytical techniques, and is NO (Ignarro et al., Circ Res, resistance. 55 61:866–879 (1987); Palmer et al, Nature, 327:524–526 Nitric oxide insufficiency promotes vascular Smooth (1987)). muscle cell proliferation following vascular injury, and Nitric oxide is synthesized by one of several isoforms of Sustains Smooth muscle cell and cardiomyocyte hypertrophy the NO synthase (NOS) family of enzymes, two of which are in response to catecholamines and angiotensin II. found in the vasculature, endothelial NOS (eNOS) and Furthermore, inadequate NO leads to increased production 60 inducible NOS (iNOS). eNOS is synthesized by endothelial of extracellular matrix with consequent myocardial fibrosis. cells, while iNOS is synthesized by a variety of cell types, These many cardiovascular responses that result from including vascular Smooth muscle cells, fibroblasts, and inadequate NO in the vasculature have clear clinical corre (principally microvascular) endothelial cells (Balligand et lates in the black population. The clinical vascular pheno al, Am J Physiol, 268: H1293–1303 (1995)). These enzymes type of blacks that distinguishes them from whites with 65 produce NO as a result of the five-electron oxidation of Similar cardiovascular disorders is one of Salt-sensitive, L-arginine to L-citrulline, requisite cofactors include low-renin hypertension; left ventricular hypertrophy dispro calcium-calmodulin, O, FAD, FMN, tetrahydrobiopterin US 6,635,273 B1 9 10 thiols, heme, and NADPH. (Moncada et al, N Engl J Med, that must be neutralized to limit oxidative damage and 329:2002–2012 (1993)). cellular dysfunction. In the Setting of common cardiovascu The role of NO in the cardiovascular system has become lar disorders or in the presence of common risk factors for increasingly apparent over the past fifteen years (LoScalzo et atherothrombotic disease, reactive oxygen species (ROS) al, Prog Cardiovasc Dis, 38:87–104 (1995)). Nitric oxide are generated in abundance, and their rate of Synthesis and contributes importantly to resting tone in conductance as flux typically exceeds the capacity of endogenous antioxi well as resistance arteries (Ouyyumi et al., J. Clin Invest, dant mechanisms. Hypercholesterolemia, hyperglycemia 95:1747–1755 (1995)), and plays a critical role in the (Keaney et al, Circulation, 99:189-191 (1999)), cigarette maintenance of peripheral vascular resistance and arterial Smoking, hyperhomocysteinemia, hypertension, and frank pressure responses. Inhibition of NOS activity is associated 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-R 1144 (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 whether or not the increase in ROS in these disorder is a release from the cardiovascular regulatory center in the brain 15 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, 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, a-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, glutathione peroxidases. Deficiencies in Several of these 428:261-268 (1994)). The dependence of blood pressure on molecular species have been shown to lead to increased salt intake appears to depend on NO, and NO deficiency 25 steady-state levels of ROS and vascular dysfunction, includ States are associated with Salt-sensitivity (Tolins et al., Kid ing increased platelet activation, arterial thrombosis ney Internat, 46:230–236 (1994)). Selective inhibition of (Freedman et al., J. Clin Invest, 97.979–987 (1996); Freed iNOS in Dahl R rats has been shown to lead to Salt man et al, Circulation, 98: 1481-1486 (1998)), and reduced Sensitivity and to the development of Salt-dependent hyper production of platelet-derived NO (Kenet et al, Arterio tension similar to Dahl S rats (Rudd et al, Am J Physiol, Thromb Vasc Biol, 19(8): 2017-2023 (1999)), which is 277:H732-H739 (1999)). In addition, mice deficient in important for limiting expansion of a platelet thrombus iNOS (iNOS gene eliminated by targeted disruption) may (Freedman et al., Circ Res, 84:1416-142 (1999)). develop hypertension in response to Salt feeding (Rudd etal, ROS generation accompanies the vascular dysfunction Circulation, 98:1A (1998)). associated with several models of atherothrombotic and Nitric oxide also affects myocardial contractility, and does 35 hypertensive vascular diseases. Hyperhomo-cysteinemic So both by mediating muscarinic-cholinergic Slowing of the mice (i.e., cyStathionine f-synthase knock-out mice) heart rate and the contractile response to beta-adrenergic (Eberhardt et al., Circulation, 98:144 (1998)), cellular glu stimulation (Balligand et al, Proc Nat'l Acad Sci USA, tathione peroxidase-deficient mice (i.e., cellular glutathione 90:347–351 (1993)). This latter effect appears to be medi peroxidase knock-out mice), and Salt-induced hypertensive ated in Vivo through the vagus nerve (Hare et al., J. Clin 40 rats (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 In both vascular Smooth muscle cells and cardiomyocytes, this increase in ROS is accompanied by reduced NO bio NO inhibits cellular proliferation and limits the proliferative activity through oxidative inactivation. Endothelial function response to growth-promoting Substances (Garget al., J. Clin and NO availability can be improved by improving antioxi Invest, 83:1774–1777 (1986)). Left ventricular hypertrophy 45 dant status with a cysteine precursor (Vita et al., J. Clin Invest, tends to occur in adult hearts with inadequate capillary 101: 1408-1414 (1998)). In addition, C-tocopherol leads to proliferation, and this may account for the microvascular plate let inhibition (Freedman et al., Circulation, ischemia noted in patients with hypertrophy. Capillary pro 94:2434-2440 (1996)) as one mechanism of its atherothrom liferation is generally held to be a rare event in normal adult botic benefit (Stephens et al, Lancet, 347:781-786 (1996)). mammalian hearts. However, recent data from a hyperten 50 The present inventors have also discovered that Salt-loading Sive rat model, in which left Ventricular hypertrophy com Salt-sensitive individuals (Dahl S rats) leads to an approxi monly occurs, show that treatment with a low-dose of an mate 5-fold increase in plasma F-isoprostanes (8-epi angiotensin-converting enzyme inhibitor insufficient to pre prostaglandin F), and this increase precedes the develop vent hypertension and left Ventricular hypertrophy can, ment of florid hypertension. These data all Support the role nonetheless, evoke capillary angiogenesis. Compared with 55 of oxidant StreSS in the genesis or evolution of vascular untreated controls, treatment with the angiotensin convert dysfunction and disease, and the importance of antioxidant ing enzyme inhibitor increased myocardial capillary prolif mechanisms in preventing this pathobiology, particularly eration (Unger et al, Hypertension, 20:478-482 (1992)), and with regard to African Americans. this effect was believed to be a consequence of inhibiting the In Support of the mechanisms illustrated above, minimum degradation and potentiating the action of bradykinin. 60 forearm Vascular resistance is significantly higher among Bradykinin increases myocardial blood flow by inducing normotensive blacks than whites (Bassett et al, Am J release of NO from microvascular endothelial cells, and Hypertension, 5:781-786 (1992)), and forearm blood-flow increased blood flow is a powerful stimulus for capillary responses to isoproterenol are markedly attenuated in nor proliferation (Mall et al, Bas Res Cardiol, 85:531-540 motensive blacks, Suggesting a blunted B-vasodilator (1990)). 65 response in these individuals (Lang et al, N Engl J Med, Normal metabolic processes in vascular cells are associ 333:155–160 (1995)). Blacks tend to have greater left ven ated with the generation of reactive oxygen intermediates tricular mass than whites for any given level of blood US 6,635,273 B1 11 12 pressure (Koren et al, Am J Hypertension, 6:815-823 tensin II receptor antagonist and/or nitrosated renin (1993); Chaturvedi et al, J Am Coll Cardiol, 10 inhibitor, and, optionally, at least one antioxidant and/or at 24: 1499–1505 (1994)). While not quantitated in any least one compound used to treat cardiovascular diseases. necropsy Study, this response is likely to be accompanied by The present invention also provides methods of prevent inadequate capillary angiogenesis which, in turn, may ing and treating Raynaud's Syndrome by administering a account for the diastolic dysfunction and the microvascular therapeutically effective amount of at least one antioxidant ischemia observed in blacks. Interestingly, blacks have been or a pharmaceutically acceptable Salt thereof, and at least observed to have low levels of urinary kallikrein (Zinner et one of isosorbide dinitrate and isosorbide mononitrate, and, al, Am J Epidemiol, 104:124-132 (1976); Levy et al., J Clin optionally, at least one nitrosated angiotensin-converting Invest, 60:129-138 (1977)), the enzyme responsible for the enzyme inhibitor, nitrosated calcium channel blocker, nit generation of bradykinin from high-molecular-weight kini rosated endothelin antagonist, nitroSated angiotensin II nogen. Thus, were a similar abnormality in bradykinin and receptor antagonist and/or nitrosated renin inhibitor. For bradykinin-mediated NO production to exist in the coronary example, the patient can be administered an antioxidant vasculature, attenuated blood flow responses may result that band isosorbide dinitrate, or the patient can be administered would limit capillary angiogenic responses and prevent the 15 an antioxidant and isosorbide mononitrate, or the patient can endothelial proliferative effects of locally derived NO. be administered an antioxidant, isosorbide dinitrate and AS discovered and described herein, African Americans isosorbide mononitrate. The antioxidant and isosorbide dini have a unique vascular diathesis that may serve as the basis trate or isosorbide mononitrate can be administered Sepa for clinically important cardiovascular Syndromes. For rately or as components of the same composition. Raynaud's example, differences in the outcome of left ventricular Syndrome is a condition that causes a loSS of blood flow to dysfunction may be a consequence of the enhanced (perhaps the fingers, toes, nose and/or ears. The affected area turns Salt-dependent) increase in oxidant stress coupled with white from the lack of circulation, then blue and cold, and microvascular endothelial dysfunction and an inadequately finally numb. The affected area may also turn red, and may vascularized, hypertrophied left ventricle. This constellation throb, tingle or Swell. of pathophysiological abnormalities may provide the Sub 25 Another aspect of the present invention provides novel Strate for the important differences in outcome between transdermal patches comprising a therapeutically effective blacks and whites with left ventricular dysfunction (Dreis et amount of at least one antioxidant and at least one of al, N Engl J Med, 340:609-616 (1999)). In addition, these isosorbide dinitrate and isosorbide mononitrate and/or at observations and their clinical consequences Suggest that least one nitrosated angiotensin-converting enzyme blacks with abnormal endothelial function and nitric oxide inhibitor, nitrosated beta-adrenergic blocker, nitrosated cal insufficiency States would derive direct and, perhaps, dis cium channel blocker, nitrosated endothelin antagonist, nit proportionate clinical benefit from enhancing nitric oxide in rosated angiotensin II receptor antagonist, nitrosated renin the vasculature, either by improving endothelial function, inhibitor, and mixtures thereof. providing exogenous nitric oxide donors, or both. In the present invention, the antioxidants include Small In view of the above, the present invention provides 35 molecule antioxidants and antioxidant enzymes. Suitable methods of treating and/or preventing vascular diseases Small-molecule antioxidants include, but are not limited to, characterized by nitric oxide (NO) insufficiency by admin hydralazine compounds, glutathione, Vitamin C, Vitamin E, istering a therapeutically effective amount of at least one cysteine, N-acetyl-cy Steine, B-caroteine, ubiquinone, antioxidant or a pharmaceutically acceptable Salt thereof, ubiquinol-10, tocopherols, coenzyme Q, and the like. Suit and at least one of iSOSorbide dinitrate and isosorbide 40 able antioxidant enzymes include, but are not limited to, mononitrate, and, optionally, at least one nitrosated Superoxide dismutase, catalase, glutathione peroxidase, and angiotensin-converting enzyme inhibitor, nitroSated beta the like. Suitable antioxidants are described more fully in the adrenergic blocker, nitrosated calcium channel blocker, nit literature, Such as in Goodman and Gilman, The Pharma rosated endothelin antagonist, nitrosated angiotensin II cological Basis of Therapeutics (9th Edition), McGraw-Hill, receptor antagonist, nitroSated renin inhibitor, and/or at least 45 1995; and the Merck Index on CD-ROM, Twelfth Edition, one compound used to treat cardiovascular diseases. For Version 12:1, 1996; and on STN Express, file phar and file example, the patient can be administered an antioxidant and reg. isosorbide dinitrate, or the patient can be administered an The preferred antioxidant is a hydralazine compound that antioxidant and isosorbide mononitrate, or the patient can be is preferably administered in the form of a pharmaceutically administered an antioxidant, isosorbide dinitrate and isos 50 acceptable Salt, more preferably in the form of hydralazine orbide mononitrate. The vascular diseases characterized by hydrochloride. Hydralazine hydrochloride is commercially NO insufficiency include, for example, hypertension (e.g., available from, for example, Lederle Standard Products low-renin hypertension; Salt-Sensitive hypertension; low (Pearl River, N.Y.), and Par Pharmaceuticals Inc. (Spring renin, Salt-Sensitive hypertension; primary pulmonary Valley, N.Y.). hypertension; thromboembolic pulmonary hypertension; 55 Isosorbide dinitrate is commercially available, for pregnancy-induced hypertension; renovascular example, under the trade names DILATRATE(R-SR hypertension), heart failure (e.g., microvascular cardiac (Schwarz Pharma, Milwaukee, Wis.); ISORDIL(R) and ischemia), and left ventricular hypertrophy with dispropor ISORDILR TITRADOSE(R) (Wyeth Laboratories Inc., tionate microvascularization, (i.e., inadequate vascularity) Philadelphia, Pa.); and or diastolic dysfunction. 60 SORBITRATE(E) (Zeneca Pharmaceuticals, Wilmington, Another aspect of the present invention provides methods Del.). Isosorbide mononitrate is commercially available, for for treating and/or preventing vascular diseases character example, under the trade names IMDUROR (A. B. Astra, ized by nitric oxide insufficiency by administering to a Sweden); MONOKET(R) (Schwarz Pharma, Milwaukee, patient a therapeutically effective amount of at least one Wis.); and ISMO(E) (Wyeth-Ayerst company, Philadelphia, nitrosated angiotensin-converting enzyme inhibitor, nitro 65 Pa.). Sated beta-adrenergic blocker, nitrosated calcium channel The nitrosated angiotensin-converting enzyme inhibitors, blocker, nitrosated endothelin antagonist, nitrosated angio nitrosated beta-adrenergic blockers, nitrosated calcium US 6,635,273 B1 13 14 channel blockers, nitrosated endothelin antagonists, nitro Suitable angiotensin II receptor antagonists, include, but Sated angiotensin II receptor antagonists and nitroSated renin are not limited to, ciclosidomine, eprosartan, furosemide, inhibitors of the present invention include any known irbesartan, losartan, Saralasin, Valsartan, and the like. Suit angiotensin-converting enzyme inhibitors, beta-adrenergic able angiotensin II receptor antagonists are described more blockers, calcium channel blockers, endothelin antagonists, fully in the literature, Such as in Goodman and Gilman, The angiotensin II receptor antagonists and renin inhibitors that Pharmacological Basis of Therapeutics (9th Edition), have been nitroSated through one or more Sites Such as McGraw-Hill, 1995; and the Merck Index on CD-ROM, oxygen (hydroxyl condensation), Sulfur (Sulfiydryl Twelfth Edition, Version 12:1, 1996; and on STN Express, condensation) and/or nitrogen. The nitrosated compounds of file phar and file registry. the present invention can be prepared using conventional Suitable renin inhibitors, include, but are not limited to, methods known to one skilled in the art. For example, enalkrein, RO 42–5892, A 65317, CP80794, ES 1005, ES known methods for nitroSating compounds are described in 8891, SQ34017, and the like). Suitable renin inhibitors are U.S. Pat. Nos. 5,380,758 and 5,703,073; WO 97/27749; WO described more fully in the literature, Suchas in Goodman 98/19672; and Oae et al., Org. Prep. Proc. Int., 15(3) and Gilman, The Pharmacological Basis of Therapeutics :165-198 (1983), the disclosures of each of which are 15 (9th Edition), McGraw-Hill, 1995; and the Merck Index on incorporated by reference herein in their entirety. WO CD-ROM, Twelfth Edition, Version 12:1, 1996; and on STN 98/21193 discloses nitrosated ACE inhibitors and nitrosated Express, file phar and file registry. beta-adrenergic blockers, the disclosure of which is incor In the methods of the invention, the antioxidant and at porated by reference herein in its entirety. WO 99/00361 least one of iSOSorbide dinitrate and isosorbide mononitrate, discloses nitrate Salts of ACE inhibitors, the disclosure of and, optionally, at least one nitrosated compound, and/or which is incorporated by reference herein in its entirety. compound used to treat cardiovascular diseases can be Suitable angiotensin-converting enzyme inhibitors, administered as Separate components or as components of include, but are not limited to, alacepril, benazepril, the same composition. When the antioxidant and at least one captopril, ceranapril, cilaZapril, delapril, duinapril, enalapril, of isosorbide dinitrate and isosorbide mononitrate are enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, 25 administered as Separate components for the treatment of pentopril, perindopril, quinapril, ramipril, rentipril, Spirapril, vascular diseases characterized by NO insufficiency or temocapril, trandolapril, Zofenopril, and the like. Raynaud's Syndrome, they are preferably administered to Suitable angiotensin-converting enzyme inhibitors are the patient at about the same time. “About the same time” described more fully in the literature, Such as in Goodman means that within about thirty minutes of administering one and Gilman, The Pharmacological Basis of Therapeutics compound (e.g., antioxidant or isosorbide dinitrate or isos (9th Edition), McGraw-Hill, 1995; and the Merck Index on orbide mononitrate) to the patient, the other compound (e.g., CD-ROM, Twelfth Edition, Version 12:1, 1996; and on STN isosorbide dinitrate or isosorbide mononitrate or Express, file phar and file registry. antioxidant) is administered to the patient." About the same Suitable beta-adrenergic blockers, include, but are not time' also includes Simultaneous administration of the com linited to, amoSulalol, atenolol, betaxolol, bethanidine, 35 pounds. bevantolol, bisoprolol, bopindolol, bufuralol, bunitrolol, In addition to the administration of the combination of the bupranolol, butafilolol, carazolol, carteolol, carvedilol, antioxidant and isosorbide dinitrate or isosorbide mononi celiprolol, cetamolol, dile Valol, epanolol, indenolol, trate for the treatment of Vascular diseases characterized by mepindolol, metipranolol, metoprolol, moprolol, nadolol, NO insufficiency, the patients can receive digitalis Such as nebivolol, nipradillol, penbutolol, pindolol, pronethalol, 40 digoxin and/or diuretics and/or at least one nitrosated propranolol, Sotalol, Sulfinalol, talinolol, tertatolol, tilisolol, angiotensin-converting enzyme inhibitor, nitrosated beta timolol, toliprolol, and the like. Suitable beta-adrenergic adrenergic blocker, nitrosated calcium channel blocker, nit blockers are described more fully in the literature, Such as in rosated endothelin antagonist, angiotensin II receptor Goodman and Gilman, The Pharmacological Basis of Thera antagonist, nitrosated renin inhibitor, and/or at least one peutics (9th Edition), McGraw-Hill, 1995; and the Merck 45 compound used to treat cardiovascular diseases. Index on CD-ROM, Twelfth Edition, Version 12:1, 1996; The digoxin is preferably administered orally to achieve and on STN Express, file phar and file registry. a steady State blood Serum concentration of at least about 0.7 Suitable calcium channel blockers, include, but are not nanograms per ml to about 2.0 nanograms per ml. The limited to, amlodipine, aranidipine, barnidipine, benidipine, diuretic is administered, preferably orally, to manage edema. cilnidipine, clentiaZem, diltiazen, efonidipine, fantofarone, 50 Suitable diuretics include, but are not limited to, thiazides felodipine, isradipine, lacidipine, lercanidipine, manidipine, (Such as, for example, chlorothiazide, hydrochlorothiazide); mibefradil, nicardipine, nifedipine, nilvadipine, nisoldipine, ethacrynic acid, furosemide, Spironalactone, triamterene or nitrendipine, Semotiadil, Veraparmil, and the like. Suitable mixtures thereof. Depending on the diuretic used, potassium calcium channel blockers are described more fully in the may also be administered to the patient in order to optimize literature, Such as in Goodman and Gilman, The Pharma 55 the fluid balance while avoiding hypokalemic alkalosis. The cological Basis of Therapeutics (9th Edition), McGraw-Hill, administration of potassium can be in the form of potassium 1995; and the Merck Index on CD-ROM, Twelfth Edition, chloride or by the daily ingestion of foods with high potas Version 12:1, 1996; and on STN Express, file phar and file Sium content Such as, for example, bananas, orange juice, registry. and the like. The method of administration of these com Suitable endothelin antagonists, include, but are not lim 60 pounds is described in further detail in U.S. Pat. No. ited to, boSentan, Sulfonamide endothelin antagonists, 4,868,179, the disclosure of which is incorporated by ref BO-123, SO 28608, and the like. Suitable endothelin erence herein in its entirety. antagonists are described more fully in the literature, Such as The compounds and compositions of the invention can be in Goodman and Gilman, The Pharmacological Basis of administered by any available and effective delivery system Therapeutics (9th Edition), McGraw-Hill, 1995; and the 65 including, but not limited to, orally, bucally, parenterally, by Merck Index on CD-ROM, Twelfth Edition, Version 12:1, inhalation spray, topically (including transdermally), or rec 1996; and on STN Express, file phar and file registry. tally in dosage unit formulations containing conventional US 6,635,273 B1 15 16 nontoxic pharmaceutically acceptable carriers, adjuvants, and perfuming agents. Suppositories for rectal administra and vehicles as desired. The preferred methods of adminis tion of the compounds or compositions can be prepared by tration are by oral administration or topical application mixing the drug with a Suitable nonirritating excipient Such (transdermal application). as cocoa butter and polyethylene glycols which are Solid at Topical administration can also involve the use of trans room temperature but liquid at rectal temperature, Such that dermal administration Such as transdermal patches or ion they will melt in the rectum and release the drug. tophoresis devices. Dosage forms for topical administration The term parenteral includes Subcutaneous injections, of the compounds and compositions can include creams, intravenous, intramuscular, intrasternal injection, or infusion Sprays, lotions, gels, ointments, and the like. In Such dosage techniques. Injectable preparations, for example, Sterile forms, the compositions of the invention can be mixed to injectable aqueous or oleaginous Suspensions can be formu form white, Smooth, homogeneous, opaque cream or lotion lated according to the known art using Suitable dispersing with, for example, benzyl alcohol 1% or 2% (wt/wt) as a agents, wetting agents and/or Suspending agents. The Sterile preservative, emulsifying wax, glycerin, isopropyl injectable preparation can also be a sterile injectable Solution palmitate, lactic acid, purified water and Sorbitol Solution. In or Suspension in a nontoxic parenterally acceptable diluent addition, the compositions can contain polyethylene glycol 15 or Solvent, for example, as a Solution in 1,3-butanediol. 400. They can be mixed to form ointments with, for Among the acceptable vehicles and Solvents that can be used example, benzyl alcohol 2% (wit/wt) as preservative, white are water, Ringer's Solution, and isotonic Sodium chloride petrolatum, emulsifying wax, and tenox II (butylated Solution. Sterile fixed oils are also conventionally used as a hydroxyanisole, propyl gallate, citric acid, propylene Solvent or Suspending medium. glycol). Woven pads or rolls of bandaging material, e.g., The compounds and compositions of the invention will gauze, can be impregnated with the compositions in typically be administered in a pharmaceutical composition Solution, lotion, cream, ointment or other Such form can also comprising one or more carriers or excipients. be used for topical application. Examples of Suitable carriers include, for example, water, The compositions can also be applied topically using a Silicone, waxes, petroleum jelly, polyethylene glycols, pro transdermal System, Such as one of an acrylic-based polymer 25 pylene glycols, lipoSomes, Sugars, Salt Solutions, alcohol, adhesive with a resinous crosslinking agent impregnated Vegetable oils, gelatins, lactose, amylose, magnesium with the composition and laminated to an impermeable Stearate, talc, Surfactants, Silicic acids, Viscous paraffins, backing. In a preferred embodiment, the compositions of the perfume oils, fatty acid monoglycerides and diglycerides, present invention are administered in the form of a trans petroethral fatty acid esters, hydroxymethylcelluloses, dermal patch, more preferably in the form of a Sustained polyvinyl-pyrrollidones, and the like. The pharmaceutical release transdermal patch. The transdermal patches of the preparations can be Sterilized and if desired, mixed with present invention can include any conventional form Such auxiliary agents, e.g., lubricants, preservatives, Stabilizers, as, for example, adhesive matrix, polymeric matrix, reser Wetting agents, emulsifiers, Salts for influencing OSmotic voir patch, matrix or monolithic-type laminated Structure, preSSure, buffers, colorings, flavorings and/or aromatic Sub and are generally comprised of one or more backing layers, 35 stances and the like which do not deleteriously react with the adhesives, penetration enhancers, an optional rate control active compounds. For topical application, the compositions ling membrane and a release liner which is removed to can also include one or more permeation enhancers expose the adhesives prior to application. Polymeric matrix including, for example, dimethylsulfoxide (DMSO), dim patches also comprise a polymeric-matrix forming material. ethyl formamide (DMF), N,N-dimethylacetamide (DMA), Suitable transdermal patches are described in more detail in, 40 decylmethylsulfoxide (C10MSO), polyethylene glycol for example, U. S. Pat. Nos. 5,262,165, 5.948,433, 6,010, monolaurate (PEGML), glyceral monolaurate, lecithin, 715 and 6,071,531, the disclosure of each of which are 1-Substituted azacycloheptan-2-ones, particularly 1-N- incorporated herein in their entirety. dodecylcyclazacycoheptan-2-ones (available under the Solid dosage forms for oral administration can include trademark AZONE from Nelson Research & Development capsules, Sustained-release capsules, tablets, chewable 45 Co., Irvine Calif.), alcohols and the like. For parenteral tablets, Sublingual tablets, effervescent tablets, pills, application, particularly Suitable vehicles consist of powders, granules and gels. In Such Solid dosage forms, the Solutions, preferably oily or aqueous Solutions, as well as active compounds can be admixed with at least one inert Suspensions, emulsions, or implants. Aqueous Suspensions diluent, Such as Sucrose, lactose or Starch. Such dosage can contain Substances which increase the Viscosity of the forms can also comprise, as is normal practice, additional 50 Suspension and include, for example, Sodium carboxymethyl Substances other than inert diluents, e.g., lubricating agents, cellulose, Sorbitol and/or dextran. Optionally, the Suspension Such as magnesium Stearate. In the case of capsules, tablets, can contain Stabilizers. The compositions, if desired, can effervescent tablets, and pills, the dosage forms can also also contain minor amounts of wetting agents, emulsifying comprise buffering agents. Soft gelatin capsules can be agents and/or pH buffering agents. prepared to contain a mixture of the active compound or 55 Various delivery Systems are known and can be used to composition and vegetable oil. Hard gelatin capsules can administer the compounds or compositions of the present contain granules of the active compound in combination invention, including, for example, encapsulation in with a Solid, pulverulent carrier, Such as lactose, Saccharose, liposomes, microbubbles, emulsions, microparticles, micro Sorbitol, mannitol, potato Starch, corn Starch, amylopectin, capsules and the like. The required dosage can be adminis or cellulose derivatives of gelatin. Tablets and pills can be 60 tered as a Single unit or in a Sustained release form. prepared with enteric coatings. While individual needs may vary, determination of opti Liquid dosage forms for oral administration can include mal ranges for effective amounts of the compounds and/or pharmaceutically cceptable emulsions, Solutions, compositions is within the skill of the art and can be Suspensions, Syrups, and elixirs containing inert diluents determined by Standard clinical techniques, including refer commonly used in the art, Such as water. Such compositions 65 ence to Goodman and Gilman, Supra; The Physician's Desk can also comprise adjuvants, Such as wetting agents, emul Reference, Medical Economics Company, Inc., Oradell, Sifying and Suspending agents, and Sweetening, flavoring, N.J., 1995; and DrugFacts and Comparisons, Inc., St. Louis, US 6,635,273 B1 17 18 Mo., 1993. Generally, the dosage required to provide an EXAMPLES effective amount of the compounds and compositions, which can be adjusted by one of ordinary skill in the art, will vary The following examples are for purposes of illustration depending on the age, health, physical condition, Sex, diet, only, and are not intended to limit the Scope of the Specifi weight, extent of the dysfunction of the recipient, frequency cation or claims. of treatment and the nature and Scope of the dysfunction or disease, medical condition of the patient, the route of Example 1 administration, pharmacological considerations Such as the activity, efficacy, pharmacokinetic and toxicology profiles of As described herein, NO deficiency is a central patho the particular compound used, whether a drug delivery physiologic mechanism for the black vascular diathesis. To System is used, and whether the compound is administered 1O examine this issue in forearm microVessels, the vasodilator as part of a drug combination. responses to intra-arterial infusions of methacholine, Sodium The compounds and compositions of the present inven nitroprusside, and Verapamil were examined using venous tion can be formulated as pharmaceutically acceptable Salts. occlusion plethysmography in 36 white and black hyperten Pharmaceutically acceptable Salts include, for example, Sive patients. These patients had no other coronary factors, alkali metal Salts and addition Salts of free acids or free 15 Such as Smoking, diabetes mellitus, or hypercholesterolemia, bases. The nature of the salt is not critical, provided that it and the two groups were matched in terms of age, gender, is pharmaceutically-acceptable. Suitable pharmaceutically lipid levels, blood pressure, and anti-hypertensive treatment. acceptable acid addition Salts may be prepared from an Similar to previous reports (Lang et al, N Engl J Med, inorganic acid or from an organic acid. Examples of Such 333:155–160 (1995); Panza et al, N Engl J Med, 323:22–27 inorganic acids include, but are not limited to, hydrochloric, (1990)), the dilator response to methacholine, but not hydrobromic, hydroiodic, nitric, carbonic, Sulfuric and phos nitroprusside, was Significantly reduced in these hyperten phoric acid and the like. Appropriate organic acids include, Sive patients compared to age-matched normotensive con but are not limited to, aliphatic, cycloaliphatic, aromatic, trols (Sherman et al, Circulation, 98:1-376 (1998)). Regard heterocyclic, carboxylic and Sulfonic classes of organic ing racial differences, as shown in FIG. 2, vasodilation in acids, Such as, for example, formic, acetic, propionic, 25 Succinic, glycolic, gluconic, lactic, malic, tartaric, citric, response to methacholine was markedly worse in the black ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, hypertensive patients compared to white hypertensive glutamic, benzoic, anthranilic, meSylic, Salicylic, patients. There were no racial differences in the responses to p-hydroxybenzoic, phenylacetic, mandelic, embonic Sodium nitroprusside or Verapamil (data not shown), Sug (p amo ic), me thane Sulfonic, ethane Sulfonic, gesting that. this impairment of NO action in black hyper benzene Sulfonic, panto the nic, toluene Sulfonic, tensives is at the endothelial level and that dysfunction of 2-hydroxyethaneSuifonic, Sulfanilic, Stearic, algenic, vascular Smooth muscle does not account for the impaired B-hydroxybutyric, cyclohexylaminoSulfonic, galactaric and response. These findings have important implications for the galacturonic acid and the like. Suitable pharmaceutically pathogenesis of hypertension and myocardial ischemia. acceptable base addition Salts include, but are not limited to, metallic Salts made from aluminum, calcium, lithium, 35 Example 2 magnesium, potassium, Sodium and Zinc or organic Salts made from primary, Secondary and tertiary amines, cyclic To investigate the issue of whether NO deficiency is a amines, N,N'-dibenzylethylenediamine, chloroprocaine, primary or Secondary phenomenon in hypertensive black choline, diethanolamine, ethylenediamine, meglumine patients, 46 normotensive white and black patients who (N-methylglucamine) and procaine and the like. All of these 40 were matched for age and gender were compared. AS shown Salts may be prepared by conventional means from the in FIG. 3B, the vasodilator responses to methacholine were corresponding compound by reacting, for example, the not significantly different in black and white normotensive appropriate acid or base with the compound. patients. However, the response to Sodium nitroprusside In preferred embodiments, the hydralazine is adminis (FIG.3A) was significantly lower in the black normotensive tered in an amount of about 30 milligrams per day to about 45 patients. This finding shows that there is an impairment in 300 milligrams per day; the isosorbide dinitrate is adminis the vasodilator response exogenous Source of NO in black tered in an amount of about 20 milligrams per day to about patients even prior to the development of hypertension, 200 milligrams per day; and the isosorbide mononitrate is anobservation that would be consistent with a primary rather administered in an amount of about 10 milligrams per day to than Secondary role in the black vascular diathesis. These about 120 milligrams per day. The preferred amounts of 50 findings are consistent with recently published results by hydralazine and/or isosorbide dinitrate or isosorbide mono other investigators (Lang et al, N Engl J Med, 333:155-160 nitrate can be administered as a single dose once a day; in (1995); Cardillo et al., Hypertension 31:1235–1239 (1998)). multiple doses Several times throughout the day; or in a Example 3 Sustained-release formulation. The present invention also provides pharmaceutical kits 55 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, Physiol, 277:H732–H739 (1999)). To explore the relation nitrosated compounds, compounds used to treat cardiovas 60 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 a form prescribed by a governmental agency regulating the deprivation using an established inpatient protocol manufacture, use or Sale of pharmaceuticals or biological (Weinberger et al, Hypertension, 8:11-127-II-134 (1986)) in products, which instructions can also reflects approval by 65 a group of normotensive black patients was assessed. the agency of manufacture, use or Sale for human adminis Briefly, blood pressure was continuously monitored non tration. invasively during a salt load (sodium 458 mEq over 24 US 6,635,273 B1 19 20 hours) and during a period of Salt depletion (furosemide The disclosure of each patent, patent application and treatment and Sodium intake 10 mEq over 24 hours). publication cited herein is hereby incorporated by reference Patients were considered to be salt-sensitive if mean blood herein in its entirety. preSSure was at least 10 mm Hg higher during the Salt loading period (Weinberger et al, Hypertension, 8:II-127-II Although the invention has been Set forth in detail, one 134 (1986)). As shown in FIGS. 4A-B, there were trends for skilled in the art will appreciate that numerous changes and impaired vasodilator responses to both methacholine (FIG. modifications can be made to the invention without depart 4A) and sodium nitroprusside (FIG. 4B). The vasodilator ing from the Spirit and Scope thereof. responses to Verapamil were equivalent in Salt-Sensitive and What is claimed is: 1. A method of treating a vascular disease characterized Salt-resistant patients (data not shown). These data show 1O impaired NO action in the microvasculature of Salt Sensitive by nitric oxide insufficiency in a patient in need thereof individuals prior to the development of hypertension. comprising administering to the patient a therapeutically effective amount of at least one antioxidant, or a pharma Example 4 ceutically acceptable Salt thereof, and at least one of isos The effects of hypertension and race on conduit vessel orbide dinitrate and isosorbide mononitrate, wherein the function were examined using a well-established brachial 15 vascular disease characterized by nitric oxide insufficiency ultrasound technique (Vita et al, Lanzer & Lipton, Eds., is low-renin hypertension; Salt-Sensitive hypnsion; or low Diagnostics of Vascular Diseases. Principles and Technol renin, Salt-sensitive hypertension. ogy. Berlin:Springer-Verlag, pp.249-259 (1996)) in 370 2. The method of claim 1, further comprising administer patients (178 black, 192 white). As shown in Table 1, there ing a pharmaceutically acceptable carrier. were no significant differences in flow-mediated dilation or 3. The method of claim 1, wherein the antioxidant is a nitroglycerin-mediated dilation according to race. However, Small-molecule antioxidant, or a pharmaceutically accept hypertension was associated with a highly significant reduc able Salt thereof, or an antioxidant enzyme. tion in both flow-mediated dilation and nitroglycerin 4. The method of claim 3, wherein the Small-molecule mediated dilation. Further, Systolic blood pressure was antioxidant is a hydralazine compound of formula I, inversely correlated with flow-mediated dilation (r=-0.30, 25 glutathione, Vitamin C, Vitamin E, cysteine, N-acetyl P<0.001) and nitroglycerin-mediated dilation (r=-0.33, cysteine, B-carotene, ubiquinone, ubiquinol-10, tocopherol, P<0.001). Diastolic pressure correlated to a similar extent coenzyme Q, or mixture thereof, wherein the hydralazine with vascular function. By multiple linear regression compound of formula (I) is: analysis, vessel Size and Systolic blood pressure were the only independent predictors of flow-mediated dilation in this (I) Sizable group of patients. R4 R3 al b C Thus, both black and white patients with hypertension RN - N - R. demonstrate a significant impairment of NO action in con duit arteries (Gokce et al., Circulation, 99(25) 3234-3240 35 wherein a, b and c are independently a Single or double (1999)). bond; R and R are each independently a hydrogen, an alkyl, an ester or a heterocyclic ring; R and R are each TABLE 1. independently a lone pair of electrons or a hydrogen; with Conduit Vasomotor Function. By Race the proviso that at least one of R, R2, R and R4 is not a 40 hydrogen. White Patients Black Patients P 5. The method of claim 3, wherein the antioxidant enzyme is a Superoxide dismutase, a catalase, a glutathione FMD (%) peroxidase, or a mixture thereof. Normotensive 11.5 + 5.9 13.3 7.2 NS 6. The method of claim 4, wherein the hydralazine com Hypertensive 9.5 - 5.3* 8.8 6.2 NS 45 pound is budralazine, cadralazine, dihydralazine, NTG-response (%) endralazine, hydralazine, pildralazine or todralazine or a Normotensive 18.1 - 8.1 20.6 - 8.4 NS pharmaceutically,acceptable Salt thereof. Hypertensive 14.6 6.2 16.4 7.1 NS 7. The method of claime 6, wherein the hydralazine compound is hydralazine hydrochloride. Data are meant SD. *p < 0.001 compared to race matched normotensives. 50 8. The method of claim 7, wherein the hydralazine hydro chloride is administered in an amount of about 30 milli Each of the above examples demonstrate that NO action grams per day to about 300 milligrams per day. is impaired in the microvasculature of black hypertensive 9. The method of claim 1, wherein the isosorbide dinitrate patients to a greater extent than in white hypertensive is administered in an amount of about 20 milligrams per day patients. There is a Suggestion that this abnormality may 55 to about 200 milligrams per day. precede the development of hypertension, particularly in 10. The method of claim 1, wherein the isosorbide mono Salt-sensitive individuals, consistent with a pathogenic role. nitrate is administered in an amount of about 10 milligrams In conduit vessels, a marked impairment of NO action that per day to about 120 milligrams per day. may contribute to ischemic heart disease and Stroke has been 11. The method of claim 1, wherein the at least one demonstrated. This abnormality appears to be independent 60 antioxidant and the at least one of isosorbide dinitrate and of race, a finding that is consistent with possibility that the isosorbide mononitrate are administered orally. impairment is a consequence of blood preSSure elevation of 12. The method of claim 11, wherein the at least one any cause. Since African Americans have a greater incidence antioxidant and the at least one of isosorbide dinitrate and of hypertension and a preponderance of Salt-Sensitive isosorbide mononitrate are orally administered in the form hypertension, these findings lend further Support to the 65 of a Solid dose. hypothesis that NO insufficiency contributes to the patho 13. The method of claim 12, wherein the Solid dose is in genesis of ischemic heart disease in this population. the form of a tablet or capsule. US 6,635,273 B1 21 22 14. The method offlaim 13, wherein the capsule is in the 26. The method of claim 25, wherein the diuretic com form of a Sustained release capsule. pound is a thiazide, ethacrynic acid, a furosemide, a 15. The method of claim 13, wherein the tablet is in the Spiranolactone, a triamterene, or a mixture thereof. form of a sublingual tablet. 27. The method of claim 25, further comprising admin 16. The method of claim 13, wherein the tablet is in the istering a therapeutically effective amount of potassium. form of a chewable tablet. 17. The method of claim 1, wherein the at least one 28. The method of claim 27, wherein the potassium is antioxidant and the at least one of isosorbide dinitrate and administered in the form of potassium chloride or by the isosorbide mononitrate are administered as components of daily ingestion of foods with high potassium content. the same composition. 1O 29. The method of claim 1, further comprising adminis 18. The method of claim 1, wherein the at least one tering at least one nitrosated angiotensin-converting enzyme antioxidant and the at least one of isosorbide dinitrate and inhibitor, nitrosated beta-adrenergic blocker, nitrosated cal isosorbide mononitrate are administered to the patient as cium channel blocker, nitrosated endothelin antagonist, nit Separate components. rosated angiotensin II receptor antagonist, nitrosated renin 19. The method of claim 18, wherein the at least one 15 inhibitor, or a mixture thereof. antioxidant and the at least one isosorbide dinitrate and isosorbide mononitrate are administered to the patient as 30. The method of claim 1, further comprising adminis Separate components at about the Same time. tering at least one compound used to treat cardiovascular 20. The method of claim 1, wherein the at least one diseases, or a pharamaceutically acceptable Salt thereof. antioxidant and the at least one of isosorbide dinitrate and 31. The method of claim 30, wherein the at least one isosorbide mononitrate are administered transdermally. compound used to treat cardiovascular diseases is an 21. The method of claim 20, wherein the transdermal angiotensin-converting enzyme inhibitor, a beta-adrenergic application is in the form of a Sustained-release patch. blocker, a cholesterol reducer, a calcium channel blocker, an 22. The method of claim 1, further comprising adminis angiotensin II receptor antagonist, an endothelin antagonist, tering a digitalis. 25 a renin inhibitor, or a mixture thereof. 23. The method of claim 22, wherein the digitalis is 32. The method of claim 1, comprising administering to digoxin. the patient a therapeutically effective amount of hydralazine 24. The method of claim 23, wherein the digoxin is administered in an amount to achieve a blood Serum con hydrochloride and isosorbide dinitrate. centration of at least about 0.7 nanograms per milliliter to 33. The method of claim 1, comprising administering to about 2.0 nanograms per milliliter. the patient a therapeutically effective amount of hydralazine 25. The method of claim 1 further comprising adminis hydrochloride and isosorbide. tering a therapeutically effective edema managing amount of a diuretic compound. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,635,273 B1 Page 1 of 1 DATED : October 21, 2003 INVENTOR(S) : Loscalzo et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 10 Line 32, delete “142 and insert -- 1421 -- Column 13 Line 8, delete “(sulffiydryl” and insert -- sulfhydryl -- Line 35, delete “linited' and insert -- limited -- Column 15 Line 63, delete “cceptable” and insert -- acceptable -- Column 18 Line 29, delete “that.' and insert -- that -- Line 48, delete “anobservation' and insert -- an observation -- Column 20 Line 16, delete “hypnsion' and insert -- hypertension -- Column 22 Line 32, after “isOSorbide’ insert -- mononitrate --

Signed and Sealed this Sixteenth Day of March, 2004 WDJ

JON W. DUDAS Acting Director of the United States Patent and Trademark Office