WO2010014953A1

WO2010014953A1 - Prolonged administration of a dithiol anti-oxidant protects against ventricular remodeling

Info

Publication number: WO2010014953A1
Application number: PCT/US2009/052487
Authority: WO
Inventors: Lawrence D. Horwitz; Carlin S. Long
Original assignee: The Regents Of The University Of Colorado, A Body Corporate
Priority date: 2008-08-01
Filing date: 2009-07-31
Publication date: 2010-02-04

Abstract

Use of dithiol anti-oxidants, particularly bucillamine, to protect against long- term pathological ventricular remodeling. The method of this invention is generally applicable to the reduction or prevention of pathological ventricular remodeling resulting from any injury, disease condition or infection. Administration of the dithiol anti-oxidant reduces hypertrophy, improves contractile function and/or prevents pathologic abnormal expression of myocardium specific genes. The invention further relates to methods for inhibiting or preventing the development of congestive heart failure due to pathological ventricular remodeling.

Description

PROLONGED ADMINISTRATION OF A DITHIOL ANTI-OXID ANT PROTECTS AGAINST VENTRICULAR REMODELING

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. provisional application 61/085,770, filed Aug. 1, 2008 which is incorporated in its entirety herein.

STATEMENT REGARDING GOVERNMENT RESEARCH OR DEVELOPMENT FUNDING

[0002] This application was made with United States government funding under the National Institutes of Health grant numbers HL5529, HL66399 and HL79160. The United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The pathological remodeling of the heart is a serious adverse outcome of acute myocardial infarction, despite the widespread application of reperfusion strategies. Ventricular remodeling, defined as hypertrophy of the myocytes, fibrosis of the noninfarcted myocardium, and changes in the geometry of the ventricle, presages the development of congestive heart failure (4, 29).

[0004] Ventricular remodeling and subsequent heat failure can result from various events or disorders, including among others, recent onset of acute coronary syndrome with myocardial infarction; idiopathic dilated cardiomyopathy; ischemic cardiomyopathy; postpartum or peripartum cardiomyopathy; hypertensive cardiomyopathy; alcoholic cardiomyopathy; autoimmune cardiomyopathy; cardiomyopathy due to human immunodeficiency virus; tachycardia-mediated cardiomyopathy; infectious myocarditis, including myocarditis caused by bacterial, viral, spirochetal, mycotic, rickettsial, protozoal or helminthic agents; myocarditis due to exposure to cardiotoxins, including ethanol or other alcohols, anthracyclines, cocaine or other illicit drugs, carbon monoxide, heavy metals (including lead, iron, copper), or catecholamines; myocarditis due to hypersensitivity reactions to drugs, immunizations, lithium, insect bites, snake bites, or the like; myocarditis due to systemic disorders, including collagen-vascular diseases, sarcoidosis, hypereosinophilia, Wegener's granulomatosis, thyrotoxicosis, celiac disease, or Kawasaki disease.

[0005] Ventricular remodeling is also associated with a pattern of gene expression normally present in the fetal heart, but absent in the adult heart. This abnormal pattern of gene expression generally accompanies decreased contractile function (22, 28). Changes in ventricular structure, function, and gene expression have been described in patients with heart failure (22, 35) and animal models of myocardial infarction (8, 47). Similar abnormalities in gene expression have been induced in myocardial cell cultures (2, 33).

[0006] Oxidative stress has been proposed as an important regulator of cardiac remodeling (24, 31). Cardiac ischemia-reperfusion (I/R) triggers a vigorous inflammatory response involving cytokine release, leukocyte activation, and generation of high levels of reactive oxygen species (3, 7, 19, 41). Leukocyte release of reactive oxygen species is very intense immediately after the onset of reperfusion (6), and there is evidence that anti-oxidant therapies given during the first few hours of reperfusion can reduce infarct size (12, 13, 25). However, less robust increases in oxidative activity, generated from mitochondria or other sources, may persist for weeks or months (24, 40).

[0007] A few studies in genetically altered murine models of permanent coronary artery occlusion or models of pressure overload have provided evidence that the long- term generation of oxidative stress may cause ventricular dysfunction (32, 39, 46). In addition, in vitro cell culture models support a role for oxidative signaling in the regulation of pathological remodeling at the cellular level (2, 9, 36).

[0008] A few previous studies have investigated the potential of antioxidant therapy to prevent long-term remodeling processes due to oxidative stress. Yamamoto et al. (46) reported that transgenic mice over expressing a dominant negative form of thioredoxin, an endogenous antioxidant, developed cardiac hypertrophy in the absence of exogenous stress. This hypertrophy was prevented by 4 wk of treatment with MPG. Dimethylthiourea, which has antioxidant effects but also blocks sodium/calcium exchange by an unrelated mechanism (48), improved ventricular function when given after coronary ligation in mice (15). Treatment with the antioxidant flavonoid, 7-monohydroxyethylrutoside, before ischemia partially preserved cardiac contractile responses after I/R in mice (5), although these authors did not examine cardiac function beyond 2 wk. The knockout of the myeloperoxidase gene in mice protects against the loss of cardiac function 24 days after I/R, without altering infarct size (42). Adenoviral transfer of heme oxygenase- 1 to rat myocardium prevented I/R-induced cardiac fibrosis and ventricular remodeling for up to 3 months (18).

[0009] In contrast, in a recent human clinical study, the long-term supplementation with the anti-oxidant Vitamin E was unsuccessful in preventing the development of heart failure (20). Additionally, Vitamin E has been reported to increase the risk of developing heart failure after myocardial infarction (23). Therefore, the importance of oxidative processes in remodeling or the subsequent development of heart failure and the long term benefit of anti-oxidants in treatment has not been clearly established, particularly following early reperfusion of myocardial infarctions.

[0010] The present invention relates to the use of certain dithiol anti-oxidants, such as bucillamine, for inhibiting or preventing ventricular remodeling. U.S. patent 5,670,545, relates to methods of treating ischemic disease employing bucillamine and related dithiols. U.S. patent 5,756,547 relates to a method for preserving tissue or organs for transplant which involves treatment with bucillamine and related dithiols.

SUMMARY OF THE INVENTION

[0011] The invention relates to the use of dithiol anti-oxidants, particularly bucillamine, to protect against long-term pathological ventricular remodeling. The method of this invention is generally applicable to the reduction or prevention of pathological ventricular remodeling resulting from any event, disease condition or infection and in particular is useful in pathological ventricular remodeling subsequent to ischemia-reperfusion injury. Additionally the invention relates to the use of such anti-oxidants, particularly bucillamine, to reduce hypertrophy, improve contractile function and/or prevent pathologic expression of cardiac-specific genes. The invention further relates to methods for inhibiting or preventing the development of congestive heart failure due to pathological ventricular remodeling. [0012] Redox modulation of cell signaling can exert adverse effects on ventricular shape and function that are associated with abnormal myocardial gene expression. Such redox modulation is believed to result, for example, following myocardial infarction treated with reperfusion. In the present invention, a dithiol anti-oxidant, particularly bucillamine, is administered in an amount that is effective for achieving the desired clinical benefit(s) as described herein.

[0013] In a specific embodiment, the invention relates to administration of a dithiol anti-oxidant, particularly bucillamine, for longer than one day to ameliorate or prevent pathological ventricular remodeling. The administration can be continued until risk of pathological ventricular remodeling is lowered to an acceptable level or until maximal benefit of administration is obtained.

[0014] In a specific embodiment, the invention relates to a method of administration of a dithiol anti-oxidant, particularly bucillamine, in a clinically effective amount, for a minimum of six hours, following treatment of myocardial infarction treated with reperfusion. In more specific embodiments, the administration of the dithiol anti-oxidant is continued daily for two or more days after such reperfusion treatment. In additional specific embodiments, the administration of the dithiol antioxidant is continued daily for at least one week after such reperfusion treatment. In specific embodiments, the administration of the dithiol antioxidant is continued daily for up to 1 to 2 months after such reperfusion treatment. In specific embodiments, the administration of the dithiol antioxidant is continued daily for a time ranging from 2 days to 2 months after such reperfusion treatment and more specifically for a time ranging from 1 week to 1 month after such reperfusion treatment. In a specific embodiment, the dithiol anti-oxidant is administered. In specific embodiments, the dithiol anti-oxidant is administered in oral or parenteral dosage form.

[0015] In an embodiment, the invention relates to administration of a dithiol antioxidant, particularly bucillamine, to a patient at risk of developing ventricular remodeling due to any of a variety of events, diseases or conditions, or infections. In general treatment is continued by administration of a therapeutically effective amount of the dithiol anti-oxidant until the patient is no longer at substantial risk of development of ventricular remodeling. Treatment may continue for days, months or longer. In a specific embodiment, the dithiol anti-oxidant is administered over a desired period of time, particularly over a week or more, employing a slow-release dosage form.

[0016] In a more specific embodiment, the invention provides a method for protection against ventricular remodeling following myocardial ischemia/reperfusion (I/R) injury by administration of a therapeutically effective amount of a dithiol antioxidant, particularly bucillamine. More specifically, the method is applied when cardiac reperfusion therapy, including angioplasty and administration of thrombolytic drugs, is used and in this method administration of the dithiol anti-oxidant is started during reperfusion and continued until the risk of ventricular remodeling is lowered to an acceptable level or maximal benefit is achieved. In a specific embodiment, treatment is continued for 2 weeks or more, 4 weeks or more or 6 weeks or more. In a specific embodiment, treatment is continued until pathological patterns of myocardial gene expression are no longer observed.

[0017] The invention also relates to the use of dithiol antioxidants, particularly those of formula 1 and more particularly bucillamine, for the manufacture of a medicament for inhibition, reduction or prevention of ventricular remodeling. In specific embodiments, the medicament is an oral dosage form or a parenteral dosage form. In another specific embodiment, the medicament is a slow- or sustained-release dosage form, particularly for oral or parenteral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a graph of infarct sizes in bucillamine-treated and control mice. Infarct size as a percentage of the left ventricle was measured after 48 h of reperfusion as described in The Examples. Individual values for each mouse are represented by squares (saline control) and triangles (Bucillamine), and the mean value for each group is designated by a horizontal line.

[0019] Figs. 2A and B are graphs showing that bucillamine protects against cardiac hypertrophy following ischemia/reperfusion (I/R) injury. Fig. 2A is a graph of mouse body mass as a function of indicated treatment. Each mouse was weighed before surgery, weekly during the treatment interval, and just before death. Group averages are shown for the weights just before surgery (presurgery) and at the end of the experiment interval [28-day (28d) postsurgery]. Fig. 2B is a graph of normalized cardiac mass (n, number of mice/group) as a function of indicated treatment. Twenty- eight days after surgical induction of I/R injury, hearts were excised and trimmed of atria and major blood vessels. The ventricles were blotted dry before weighing. Ventricular weights were normalized to body weights for each mouse (heart weight- to-body weight ratio, in mg/g).

[0020] Figs. 3A and B illustrate representative echocardiographic M-mode tracings. Echocardiograms were obtained as detailed in The Examples. M-mode echoes representative for each experimental treatment group, Fig. 3A saline/control and Fig. 3B bucillamine/ control are shown.

[0021] Fig. 4 is a graph showing that bucillamine protects against the loss of cardiac contractile function measured as the change in fractional shortening (FS) following I/R injury. Fractional shortening (FS) was measured as described in Table 1. The change in FS over the experimental interval in each individual animal was calculated as (FSpre + FS28d)/FSpre xlOO%, where FSpre is FS before surgery and FS28d is FS 28 days after surgery). The individual values were averaged to determine the means ± SE for each experimental group as indicated.

[0022] Figs. 5A-F are graphs showing that bucillamine attenuates the expression of the pathological fetal gene program following I/R injury. Ventricular tissue was analyzed by ribonuclease protection assay for expression of pathological marker gene mRNAs as described in The Examples. Individual values for each mRNA species were normalized to the mean sham value (saline or bucillamine, as appropriate) for the same mRNA. Each figure A-F indicated the individual mRNA species measured, as follows: β-myosin heavy chain (β-MHC; Fig. 5A); atrial natriuretic peptide (ANP; Fig. 5B); skeletal α-actin (Fig. 5C); α-MHC (Fig. 5D); α-MHC-to-β-MHC ratio (Fig. 5E); and sarco(endo)plasmic reticulum Ca²⁺ ATPase 2a (SERCA2a; Fig. 5F). DETAILED DESCRIPTION OF THE INVENTION

[0023] Bucillamine [iV-(2-mercapto-2-methylpropionyl)-L-cysteine] is a member of a group of low molecular weight, cysteine-derived thiol donors that include N- acetylcysteine (NAC) and iV-2-mercaptopropionyl glycine (MPG). These compounds readily enter cells through the cysteine transport pathway and exert their antioxidant effect by maintaining the endogenous glutaredoxin and thioredoxin systems in a reduced state by transfer of thiol groups (1, 43). Bucillamine, in contrast to NAC and MPG, contains two donatable thiol groups, making it a considerably more potent antioxidant than NAC or MPG, which each contain only one thiol group (11, 13, 43). Bucillamine has been proven effective in counteracting oxidative stress (1, 11, 13, 26, 38, 43).

[0024] Dithiol antioxidants include those of formula 1 :

or a pharmaceutically acceptable salt thereof where:

[0025] Rl and R2, independently of one another, are alkyl groups, particularly

[0026] lower alkyl groups having 1-6 or 1-3 carbon atoms;

[0027] R3 is carboxylic acid, carboxylate or salt thereof or an ester(-CO-OR_E) or amide (-CO-N(R_N)₂ of the carboxylate;

[0028] R4 or R5, independently of one another, are selected from the group consisting of a hydrogen, an alkyl group, an alkanoyl group, a phenyl-alkylgroup or a phenylcarbonyl group and the phenyl ring in the phenyl-alkyl and phenylcarbonyl groups can be unsubstituted or substituted by at least one group selected from halogen, hydroxyl, alkyl or alkoxy;

[0029] A is an alkylene group having 1-6 or 1-3 carbon atoms, such as a -CH₂- group; and

[0030] R_E and R_N, independently of one another, can be alkyl, or alkylaryl, particularly alkyl phenyl groups. [0031] In a specific embodiment, Rl and R2 are C1-C3 alkyl groups, particularly methyl groups. In a specific embodiment, R3 is a carboxylic acid (-COOH), carboxylate (-COO^') or a carboxylate salt (-COO^"X⁺, where X ⁺ is a pharmaceutically acceptable cation). In a specific embodiment, R4 and R5 are hydrogens. In a specific embodiment, m is 0, 1 or 2. In a specific embodiment, Rl and R2 are methyl groups, R4 and R5 are hydrogens and m is 0 or 1. In a specific embodiment, Rl and R2 are methyl groups, R4 and R5 are hydrogens, m is 0 or 1 and R3 is a carboxylic acid (- COOH), carboxylate (-COO^") or a carboxylate salt (-COO^"X⁺, where X ⁺ is a pharmaceutically acceptable cation).

[0032] For all R groups of formula 1 that contain alky, alkenyl, alkanoyl, alkoxy. alkylenedioxy or alkylamino groups. Preferred groups are those that contain from 1 to about 6 carbon atoms (i.e.* lower alkyl) or those having 1-3 carbon atoms.

[0033] In embodiments, the methods of the invention employ one or more compounds of formula 1. In specific embodiments, the methods of the invention employ bucillamine. In other specific embodiments, the methods of the invention employ:

[0034] Certain dithiols of formula 1 are described in U.S. patent 5,292,926.

[0035] Ventricular remodeling also called cardiac remodeling as used herein refers to pathological changes in size, shape and function of the heart which ultimately lead to heart failure (Cohn, JN; Ferrari, R; Sharpe, N (2000) Cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. Behalf of an International Forum on Cardiac Remodeling, J. Am. Coll. Cardiol, 35(3):569-582). Ventricular remodeling results from injury to the heart from a specific injury event, e.g., acute myocardial infarction, or various disease conditions which stress the heart (e.g., increase pressure or volume overload), such as chronic hypertension, congenital heart disease with intracardiac shunting, or valvular heart disease. Ventricular remodeling is associated with histopathological and structural changes in the ventricular myocardium that lead to progressive decline in ventricular performance. Ventricular remodeling is associated with an abnormal pattern of gene expression normally present in the fetal heart, but absent in the adult heart. This abnormal pattern of gene expression generally accompanies decreased contractile function (22, 28).

[0036] Ventricular remodeling and subsequent heat failure can result from various events or disorders, including among others, myocardial infarction; cardiomyopathy or myocarditis. More specifically, ventricular remodeling can result from idiopathic dilated cardiomyopathy; ischemic cardiomyopathy; postpartum or peripartum cardiomyopathy; hypertensive cardiomyopathy; alcoholic cardiomyopathy; autoimmune cardiomyopathy; cardiomyopathy due to human immunodeficiency virus; tachycardia-mediated cardiomyopathy; infectious myocarditis, including myocarditis caused by bacterial, viral, spirochetal, mycotic, rickettsial, protozoal or helminthic agents; myocarditis due to exposure to cardiotoxins, including ethanol or other alcohols, anthracyclines, cocaine or other illicit drugs, carbon monoxide, heavy metals (including lead, iron, copper), or catecholamines; myocarditis due to hypersensitivity reactions to drugs, immunizations, lithium, insect bites, snake bites, or the like; myocarditis due to systemic disorders, including collagen- vascular diseases, sarcoidosis, hypereosinophilia, Wegener's granulomatosis, thyrotoxicosis, celiac disease, or Kawasaki disease. The methods of this invention are useful in the treatment, reduction and prevention of ventricular remodeling from any of such causes and are particularly useful for treatment, reduction or prevention of ventricular remodeling which results from myocardial infarction followed by reperfusion therapy.

[0037] Methods of this invention administer a clinically effective amount of a dithiol anti-oxidant, particularly a dithiol anti-oxidant of formula 1 or a salt, ester or amide thereof to a patient at risk of ventricular remodeling until the risk of ventricular remodeling has been lowered to an acceptable level or until maximal benefit has been achieved. Administration of the dithiol anti-oxidant is typically continued for at least two days. Administration of the dithiol anti-oxidant in the present method for at a minimum of six hours and typically significantly longer (weeks or months) is distinguished from treatment with anti-oxidants for ischemic diseases or reperfusion treatment in which administration is typically over several hours. See US patent 5,670,545. More specifically, administration of the dithiol anti-oxidant for reducing or preventing ventricular remodeling occurs for a period of weeks or months to achieve desired benefit. It will be appreciated in the art, that the administration of dithiol anti-oxidant may have some level of toxicity and as such the dosage and length of time of administration will be balanced to achieve maximal benefit with minimal toxic side effects.

[0038] In a specific application to the reduction or prevention of ventricular remodeling after reperfusion therapy, dithiol antioxidants are administered for a significantly longer time than they would be for treatment of reperfusion injury.

[0039] Treatment methods of this invention comprise the step of administering a clinically effective amount of one or more compounds of this invention, in particular bucillamine, or a salt, ester, solvate or prodrug thereof to an individual to reduce or prevent ventricular remodeling. The term "clinically effective amount," as used herein, refers to the amount of the compound, that, when administered to the individual is effective to at least partially reduce or prevent the condition or symptom that is recited, e.g., ventricular remodeling, or to at least partially ameliorate a symptom of such condition. Symptoms associated with ventricular remodeling include among others, abnormal expression of myocardial genes, histopathologic and structural changes in the ventricular myocardium and changes in shape, size, structure or function of the heart. Amelioration of ventricular remodeling can be assessed, for example, as a decrease in expression level of one or more genes the expression of which is associated with heart-recognized abnormal pattern of myocardial gene expression described herein. Amelioration of ventricular remodeling can also be assessed, by reversion of the abnormal pattern of myocardial gene expression to a normal non-pathologic pattern of myocardial gene expression. Alternatively, amelioration of ventricular remodeling can be assessed by decreases in histopathological and structural changes in the ventricular myocardium. Amelioration of ventricular remodeling may also be assessed by inhibition or retardation of the development of histopathological and structural changes in the ventricular myocardium. Amelioration of ventricular remodeling may also be assessed by inhibition or retardation of the development of detrimental changes to the size, shape or function of the heart.

[0040] In an embodiment, administration of a dithiol anti-oxidant is continued until the risk of ventricular remodeling is lowered to an acceptable level. For purposes herein "lowering to an acceptable level" relates to minimizing the rate of increase in appearance or severity of symptoms of ventricular remodeling or to the decrease in extent or disappearance of symptoms. In one embodiment, "lowering to an acceptable level" refers to decrease in abnormal myocardial gene expression levels and preferably to no observable abnormal myocardial gene expression levels.

[0041] As noted above, ventricular remodeling can be caused by a variety of events and disease conditions. A person at risk of ventricular remodeling is a person who is diagnosed to have or is suspected to have experienced any such event (i.e., myocardial infarction) or to have any such disease condition that is understood in the art to potentially lead to ventricular remodeling (cardiomyopathy, myocarditis). The methods of the invention can be applied to those at risk of developing ventricular remodeling as well as those who show symptoms, such as abnormal myocardial gene expression, of ventricular remodeling.

[0042] As is understood in the art, the clinically effective amount of a given compound will depend at least in part upon, the mode of administration, any carrier or vehicle (e.g., solution, emulsion, etc.) employed, the extent of damage and the specific individual to whom the compound is to be administered (age, weight, condition, sex, etc.). The dosage requirements needed to achieve the "clinically effective amount" vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Based on the results obtained in standard pharmacological test procedures, projected daily dosages of active compound can be determined as is understood in the art.

[0043] Compounds of this invention can be employed in unit dosage form, e.g. as tablets or capsules. In such form, the active compound or more typically a pharmaceutical composition containing the active compound is sub-divided in unit dose containing appropriate quantities of the active compound; the unit dosage forms can be packaged compositions, for example, packaged powders, vials, ampules, pre- filled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.

[0044] The dosage can vary within wide limits and as is understood in the art will have to be adjusted to the individual requirements in each particular case. With respect to the preferred administration route is oral or parenteral administration. The invention encompasses, slow release or controlled release dosage forms.

[0045] Any suitable form of administration can be employed in the method herein. The compounds of this invention can, for example, be administered in oral dosage forms including tablets, capsules, pills, powders, granules, solutions, elixirs, tinctures, suspensions, syrups and emulsions. Oral dosage forms may include sustained release or timed release formulations. The compounds of this invention may also be administered topically. Parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. Administration by various means uses appropriate dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0046] The clinically active compounds of the invention can be administered alone, but generally will be administered with a pharmaceutical carrier selected upon the basis of the chosen route of administration and standard pharmaceutical practice.

[0047] Administration includes any form of administration that is known in the art and is intended to encompass administration in any appropriate dosage form and further is intended to encompass administration of a compound, alone or in a pharmaceutically acceptable carrier.

[0048] Pharmaceutical compositions and medicaments of this invention comprise one or more compounds in combination with a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and medicaments are prepared in accordance with acceptable pharmaceutical procedures, such as, for example, those described in Remingtons Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference in its entirety.

[0049] Pharmaceutically acceptable carriers are those carriers that are compatible with the other ingredients in the formulation and are biologically acceptable. Carriers can be solid or liquid. Solid carriers can include one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders, tablet-disintegrating agents, or encapsulating materials. Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water (of appropriate purity, e.g., pyrogen- free, sterile, etc.), an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid carrier can contain other suitable pharmaceutical additives such as, for example, solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Compositions for oral administration can be in either liquid or solid form.

[0050] The present invention provides methods of treating disorders, diseases conditions and symptoms in a mammal and particularly in a human, by administering to an individual in need of treatment or prophylaxis, a therapeutically effective amount of a compound of this invention to the mammal in need thereof. The result of treatment can be partially or completely alleviating, inhibiting, preventing, ameliorating and/or relieving the disorder, condition or one or more symptoms thereof. Administration includes any form of administration that is known in the art to be effective for a given type of disease or disorder, is intended to encompass administration in any appropriate dosage form and further is intended to encompass administration of a compound, pharmaceutically acceptable salt, solvate or ester thereof alone or in a pharmaceutically acceptable carrier thereof or administration of a prodrug derivative or analog of a compound of this invention which will form an equivalent amount of the active compound or substance within the body. An individual in need of treatment or prophylaxis includes those who have been diagnosed to have a given disorder or condition and to those who are suspected, for example, as a consequence of the display of certain symptoms, or as a consequence of certain treatments as having such disorders or conditions or the potential for developing such disorders or conditions..

[0051] The term "pharmaceutically acceptable salts" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, preferably hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, N-acetylcystein and the like.

[0052] In addition these salts may be prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts and the like. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins and the like. Compounds of formula I can also be present in the form of zwitterions.

[0053] The invention expressly includes pharmaceutically usable solvates of compounds of the invention, particularly bucillamine. The compounds of the invention can be solvated, e.g. hydrated. The solvation can occur in the course of the manufacturing process or can take place, e.g. as a consequence of hygroscopic properties of an initially anhydrous compound (hydration).

[0054] In certain embodiments, the present invention is directed to prodrugs of dithiol antioxidants, particularly bucillamine. The term "prodrug," as used herein, means a compound that is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of Formula 1. Various forms of prodrugs are known in the art such as those discussed in, for example, Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al. (ed.), "Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991), Bundgaard, et al., Journal of Drug Delivery Reviews, 8:1-38(1992), Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (19SS); and Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975), each of which is hereby incorporated by reference in its entirety.

[0055] Pharmaceutically acceptable carriers are those carriers that are compatible with the other ingredients in the formulation and are biologically acceptable. Carriers can be solid or liquid.

[0056] In specific embodiments, the invention provides a method for inhibiting, reducing or preventing ventricular remodeling in a patient at risk of ventricular remodeling by administering to such patient a clinically effective amount of a dithiol anti-oxidant, particularly a dithiol anti -oxidant of formula 1 or a pharmaceutically acceptable salt thereof (above). Administration is continued to the patient at risk until the risk of ventricular remodeling is lowered to an acceptable level or until maximal benefit of administration is achieved. Administration can be continued for at least two days. Administration can be continued for at least one week. Administration can be continued for at least one month.

[0057] In a more specific embodiment, ventricular remodeling is the result of myocardial infarction, cardiomyopathy or myocarditis. In another specific embodiment, the risk of ventricular remodeling is associated with acute myocardial infarction treated with reperfusion and the dithiol anti-oxidant is administered during reperfusion and is continued for a minimum of six hours following treatment reperfusion. Treatment can be continued for two or more days after such reperfusion treatment. Treatment can be continued for at least one week after such reperfusion treatment. Treatment can be continued for at least one month after such reperfusion treatment. [0058] In another embodiment, the method of this invention is applied in the absence of reperfusion treatment or therapy. More specifically, the method of this invention can be applied even in the absence of reperfusion treatment or therapy.

[0059] The method of claim 1 wherein reperfusion treatment is not employed prior to administration of the dithiol anti-oxidant.

[0060] In another specific embodiment, administration is continued until abnormal myocardial gene expression is no longer observed. In another embodiment, in which reperfusion treatment has occurred, administration begins during reperfusion and continues until it is determined that symptoms of ventricular remodeling have not occurred or if they have occurred that a minimization of such symptoms has been achieved. For example, administration can continue until it is ascertained that abnormal myocardial gene expression or histopathological and structural changes in the ventricular myocardium that could result from reperfusion, myocardial infarction, cardiomyopathy, or myocarditis have not appeared.

[0061] In specific embodiments, administration is oral or parenteral. In other embodiments, administration employs a slow- or sustained release dosage form, which may be administered by any appropriate route including oral or parenteral administration.

[0062] The invention provides for the use of a dithiol anti-oxidant of formula 1 for the manufacture of a medicament for the reduction, inhibition or prevention of ventricular remodeling. The invention also provides for the use of a dithiol antioxidant of formula 1 for the manufacture of a medicament for the reduction, inhibition or prevention of ventricular remodeling subsequent to myocardial infarction and reperfusion treatment. The invention additionally provides for the use of a dithiol anti-oxidant of formula 1 for the manufacture of a medicament for reduction, inhibition or prevention of heart failure subsequent to ventricular remodeling. More specifically in such uses in the dithiol anti-oxidant of formula 1 , R3 is carboxylate (- COO-) or a carboxylate salt (-COO^"X⁺, where X ⁺ is a pharmaceutically acceptable cation). More specifically in such uses in the dithiol anti-oxidant of formula 1, Rl and R2 are C1-C3 alkyl groups. More specifically in such uses in the dithiol anti- oxidant of formula 1, R4 and R5 are hydrogens. More specifically in such uses in the dithiol anti -oxidant of formula 1, m is 0 or 1. More specifically in such uses in the dithiol anti-oxidant of formula 1, Rl and R2 are methyl groups. More specifically in such uses the dithiol anti-oxidant is bucillamine. More specifically in such uses the dithiol antioxidant is the compound of formula 1 where m is 1, Rl and R2 are methyl groups, R4 and R5 are hydrogens and R3 is carboxylate or a salt thereof. More specifically in such uses the dithiol anti-oxidant is

CH,

[0063] In specific embodiments, the medicament is a pharmaceutically acceptable composition comprising one or more compounds of formula 1 herein or salts, esters or amides thereof. The medicament can further comprise a pharmaceutically acceptably carrier. The medicament can be prepared for any suitable method of administration in any suitable dosage form. Oral and parenteral dosage forms are preferred. The medicament can be a slow- or sustained-release formulation as is known in the art.

[0064] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers of the group members, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. A number of specific groups of variable definitions have been described herein. It is intended that all combinations and subcombinations of the specific groups of variable definitions are individually included in this disclosure. Compounds described herein may exist in one or more isomeric forms, e.g., structural or optical isomers. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer (e.g., cis/trans isomers, R/S enantiomers) of the compound described individual or in any combination. Additionally, unless otherwise specified, all isotopic variants of compounds disclosed herein are intended to be encompassed by the disclosure. For example, it will be understood that any one or more hydrogens in a molecule disclosed can be replaced with deuterium or tritium. Isotopic variants of a molecule are generally useful as standards in assays for the molecule and in chemical and biological research related to the molecule or its use. Isotopic variants, including those carrying radioisotopes, may also be useful in diagnostic assays and in therapeutics. Methods for making such isotopic variants are known in the art. Specific names of compounds are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same compounds differently.

[0065] Molecules disclosed herein may contain one or more ionizable groups [groups from which a proton can be removed (e.g., -COOH) or added (e.g., amines) or which can be quaternized (e.g., amines)]. All possible ionic forms of such molecules and salts thereof are intended to be included individually in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art can select from among a wide variety of available counterions those that are appropriate for preparation of salts of this invention for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt may result in increased or decreased solubility of that salt.

[0066] Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.

[0067] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.

[0068] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. The broad term comprising is intended to encompass the narrower consisting essentially of and the even narrower consisting of. Thus, in any recitation herein of a phrase "comprising one or more claim element" (e.g., "comprising A and B), the phrase is intended to encompass the narrower, for example, "consisting essentially of A and B" and "consisting of A and B." Thus, the broader word "comprising" is intended to provide specific support in each use herein for either "consisting essentially of or "consisting of." The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0069] One of ordinary skill in the art will appreciate that starting materials, catalysts, reagents, synthetic methods, purification methods, analytical methods, and assay methods, other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by examples, preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0070] All references cited herein are hereby incorporated by reference to the extent that there is no inconsistency with the disclosure of this specification. Some references provided herein are incorporated by reference to provide details concerning sources of starting materials; alternative starting materials, reagents, methods of synthesis, purification methods, and methods of analysis; as well as additional uses of the invention.

The Examples

Materials and Methods

[0071] Experimental animals. All procedures were conducted in conformance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the University of Colorado at Denver and Health Sciences Center Institutional AnimalCare and Use Committee. C57B1/6J mice (11 to 12 wk old) were purchased from Jackson Laboratory (Bar Harbor, ME) and allowed to acclimate for 1 wk before any experimental intervention.

[0072] Surgical generation of myocardial I/R in mice. Mice were anesthetized by an injection of 2% 2,2,2-tribromoethanol (0.66 mg/g ip; Aldrich Chemical, St. Louis, MO). The mice were then orally intubated with a 20-gauge angiocath and mechanically ventilated with 90% 02-10% room air at a tidal volume of 0.4 ml and a rate of 120 breaths/min (model CIV-101, Columbus Instruments, Columbus,OH). The heart was accessed via a parasternal thoracotomy at the fourth intercostal space and a 7-0 silk suture passed under the left anterior descending coronary artery (LAD) at the point where it emerged from under the left atrial flap. Myocardial ischemia was achieved by occluding the LAD against a 22-gauge J-shaped stainless steel probe and verified by visually noting the regional akinesis and blanching of the left ventricle. The chest was closed in layers, with the long end of the probe remaining outside the chest wall, allowing the animal to be removed from the ventilator. After 30 min of ischemia, reperfusion was initiated by carefully pulling the probe out from under the ligature and then removing it from the chest cavity.

[0073] Other investigators have shown that a 30-min ischemic interval in wild-type mice results in infarction of -50% of the area at risk (45). Following the surgical procedure, the mouse was allowed to recover on a warmed surface, with supplemental oxygen delivered through a nose cone. Sham-operated animals underwent all procedures described, except the actual occlusion of the LAD. I/R was verified by three-lead electrocardiograms, which were obtained preoperatively, at the end of the ischemic interval and immediately after the initiation of reperfusion. Mice fully recovered from the surgical procedure were returned to standard animal housing conditions. Postsurgical pain was controlled with buprenorphine injections (2 μg/g sc, bid) for the first 48 hr following surgery and acetaminophen (2 mg/ml, ad libitum in the drinking water) for 7 days.

[0074] Bucillamine treatment. Powdered bucillamine (>99% purity) was obtained from Keystone Biomedical (Los Angeles, CA). Stock solutions of bucillamine (5 mg/ml) were made in normal saline, pH adjusted to ~7.4 with equimolar NaOH, and filter sterilized. Within 5 min of reperfusion being initiated, an intravenous bolus of bucillamine (10 μg/g) was administered via tail-vein injection. The mice were subsequently treated with daily injections of bucillamine (10 μg/g sc), rotating the injection sites. Control mice received saline injections.

[0075] Echocardiography. Cardiac function was assessed in the University of Colorado at Denver Small Animal Hemodynamic Core Facility by two-dimensional transthoracic echocardiography (echo). The mice were sedated with intraperitoneal injections of fentanyl (34 ng/g) plus droperidol (1.7 μg/g) to maintain heart rates consistently above 550 beats/min. Echoes were obtained with an HP Sonos 5500 echocardiograph machine using a 15-MHz linear array intraoperative probe (Philips Ultrasound, Andover, MA). Parasternal short-axis views, long-axis views, and M- modes (at the level of the short axis) were routinely obtained. Echo images were obtained on the mice 4-6 days before surgical intervention (baseline) and then 4 wk following surgery just before death. All analyses were performed off-line by an individual blinded as to treatment status.

[0076] Infarct size. Region at risk and infarct size were determined in mice that underwent I/R surgery as described in Surgical generation of myocardial I/R in mice, except that a slip knot was tied in the suture used to occlude the LAD. Forty-eight hours after reperfusion began, the mice were anesthetized and heparinized, and the hearts were excised. The slip knot was then pulled taut to reocclude the LAD. The aorta was cannulated and the heart perfused with 10 ml of cardioplegia solution containing (in mM) 140.0 NaCl, 15.0 KCl, 1.0 MgSO₄, 1.0 Na₂HPO₄, 11.0 glucose, 15.07V,jV-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 10.0 EGTA, and 30.0 2,3-butanedione monoxime and 0.10% BSA and 10 U/ml heparin. Regions of the heart still receiving blood flow during LAD occlusion were identified by perfusion with 5 ml of 2% Evans blue. After the heart was removed from the cannula, the atria and right ventricle were trimmed away before transversely slicing the left ventricle into four sections. Infarcted myocardium was identified by incubating the heart slices with 1% triphenyltetrazolium chloride at 37⁰C for 15 min. Each slice was weighed and then imaged with a Nikon SMZ800 stereoscope equipped with a Cool-Snap CCD camera. Perfused (dark blue), nonperfused but noninfarcted (brick red), and infarcted (white) myocardial regions were quantitated by planimetry using ImagePro software software. The region at risk and infarct size were determined using the following equations: Weight of region of interest = (Al xWtl) + (Λ2χWt2) + (A3 χWt3) + (A4 xWt4), where A is area of the region of interest determined by planimetry from each of the four heart sections and Wt is the weight of each section. Region of interest as percentage of the left ventricle = (Wt of region of interest/Wt of left ventricle) x

100%.

[0077] Tissue harvest. At the end of the 28-day experimental interval, the mice were weighed before heart excision. The excised hearts were rinsed in cardioplegia solution containing (in mM) 140.0 NaCl, 5.4 KCl, 1.0 MgSO₄, 1.0 Na₂HPO₄, 11.0 glucose, 15.0 BES, 1.0 EGTA, and 30.0 2,3-butanedione monoxime and 0.1% BSA (pH 7.4), and the atria and major vessels were removed. The combined ventricles were blotted dry, weighed, and stored in RNALater (Ambion, Austin, TX) at - 20°C. The lung and liver were also excised, trimmed of vascular tissue, blotted dry, and weighed.

[0078] Ribonuclease protection assay. Tissue RNA was purified using TRIzol reagent (Invitrogen, Carlsbad, CA). Blinded analysis of myocardia mRNAs was performed by ribonuclease protection assay using a cassette of mouse cardiac riboprobes as described previously (14). Briefly, 10 μg aliquots of RNA were hybridized with [³²P]-labeled anti-sense probes for 1) cardiac specific genes, including murine α-and β-myosin heavy chain (MHC), sarco(endo)plasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), skeletal α-actin, and GAPDH (internal control) or 2) cytokine genes, including interleukin-lβ (IL-I β), IL- 6, tumor necrosis factor-β (TNF-α), and GAPDH (BD Biosciences, San Jose, CA). Unhybridized RNA was digested with RNase, and the protected fragments were separated by polyacrylamide gel electrophoresis. The detection and quantitation of the individual protected fragments were accomplished by Phosphorimager densitometry (Molecular Dynamics, Sunnyvale, CA). The densitometry values for each mRNA species were normalized to the GAPDH mRNA signal from the same sample to correct for variations in RNA loading.

[0079] Experimental groups. The mice to be analyzed after 4 wk of reperfusion were randomly assigned to one of four experimental groups: 1) a sham-operated group not exposed to I/R and injected daily for 4 wk with control saline solution (Sham + saline), 2) a group exposed to I/R and treated with daily injections of saline for 4 wk (I/R + saline), 3) a sham-operated group not exposed to I/R and injected daily with bucillamine for 4 wk (Sham + bucillamine), and 4) a group exposed to I/R and treated with daily injections of bucillamine for 4 wk (I/R + bucillamine). Mice to be analyzed for infarct size after euthanasia at 48 h were randomly assigned to two groups: 1) a group assigned to I/R that received bucillamine as described above for 2 days of I/R (I/R + bucillamine) and 2) a group assigned to I/R that received saline as described above for 2 days of I/R (I/R + saline).

[0080] Data analysis. All data were expressed as means ± SE. Statistical comparisons between treatment groups were performed by two-way ANOVA with Bonferroni's post test (GraphPad Software, San Diego, CA). A P < 0.05 was considered statistically significant.

RESULTS

[0081] Infarct size. Mice receiving bucillamine or saline (control) were euthanized after 48 h of reperfusion for the determination of infarct size. Individual values of infarct size as a percentage of the left ventricle weight are plotted in Fig. 1. The area- at risk measurements were 37 + 5% (saline) versus 36 ± 6% (bucillamine). The infarct size measurements were 14 ± 6% (saline) versus 15 ± 8% (bucillamine). There were no significant differences or trends between the two groups.

[0082] The 4-wk experimental model. There were no apparent adverse reactions to bucillamine during the 4-wk treatment period in any of the treated mice. The gain in body weight was similar and did not differ statistically among the experimental groups (Fig. 2A). All mice exposed to I/R had visible infarcts postmortem. Twenty- one of the 22 mice originally receiving injections survived the 4-wk treatment interval. One mouse that had undergone I/R injury and had received saline injections died from cardiac rupture 5 days after surgery. The final animal numbers per group were as follows: Sham + saline (n = 6), I/R + saline (n = 5), Sham + bucillamine (n = 4), and I/R + bucillamine (n = 6). There were no statistical differences in lung weights or liver weights between the different groups.

[0083] Cardiac mass. Figure 25 shows the cardiac mass, as defined by the heart weight-to-body weight ratio, in the four groups of mice. There was a significant increase in cardiac mass (13%, P < 0.05) in the I/R + saline group compared with the Sham + saline group 4 wk after I/R exposure. Bucillamine had no effect on cardiac mass in the sham-operated mice. The increase in heart weight-to-body weight ratio in response to I/R was attenuated in the I/R + bucillamine group and was statistically indistinguishable from the Sham + bucillamine group. Thus bucillamine provided a long-term protective effect against the cardiac hypertrophic response to injury following I/R injury.

[0084] Cardiac function. Figures 3A and B show M-mode echo tracings from individual mice representative of each experimental group. The echo measurement data compiled for each experimental group are shown in Table 1. I/R injury caused a statistically significant increase in the left ventricular endsystolic diameter in the I/R + saline group compared with the Sham + saline group. The end-diastolic diameter tended to increase in the I/R + saline group compared with the Sham + saline group, but the results did not reach statistical significance. There were no differences in ventricular dimensions between the saline- and bucillamine-treated sham-operated mice. Neither end-diastolic diameter nor end-systolic diameter was statistically different between the I/R + bucillamine group and either of the two Sham groups. No significant differences were seen in septal or posterior wall thicknesses in any group, although there was a trend toward an increase in posterior wall thickness in the I/R + saline group compared with the Sham + saline group. Table 1. CARDIAC DIMENSIONS AND FUNCTIONAL PARAMETERS

Treatment LVDd, LVDs IVSd, PWd HR, n PS, % Groups mm mm mm mm beats/min

Presurgery 21 2.9±0 1 1.310.1 0.96+0 02 0.9410.03 5612 639+9

28-day Postsurgery

Saline

Sham 6 3.2±0 2 1.5±0.2 1.0210 03 0.8710.07 5513 677+4

I/R 5 3.8±0 3^* 2.510.3 *# 0.9510 02 0.9910.06 3213^*" 679+8

Bucillamine

Sham 4 2.9±0 2 1.410.1 1.0410 05 0.9510.06 5112 668+20

I/R 6 3.3±0 2 2.010.2 1.02+0 06 0.8710.04 4114 668+7

Values are means + SE for each animal group; n, number of animals in each group. Left ventricular dimensions were obtained from short-axis two-dimensional-guided M-mode echoes. Measurements were obtained over 3 separate contractile cycles and then averaged to obtain mean values for each animal before obtaining the group means. Left ventricular internal diameter (LVD) was measured at end diastole (d) and end systole (s). Interventricular septal (IVS) and posterior wall (PW) thicknesses were measured at end diastole (d). Heart rate (HR) was calculated from diastole-to-diastole intervals. Percent fractional shortening (FS) for each animal was calculated as FS = [(LVDd - LVDs)/LVDd] x 100%. #P < 0.01, ischemia-reperfusion (I/R) + saline vs. Sham + saline; *P < 0.05, I/R + saline vs. Sham + bucillamine.

[0085] Following sham operations, contractile function, measured as fractional shortening (FS), remained constant over the 4-wk interval in both the saline- and bucillamine-treated mice (Table 1). However, I/R injury resulted in a large and highly significant (67%, P < 0.01) decrease in FS in the I/R + saline mice compared with the Sham + saline mice (Fig. 4). The decrease in FS caused by I/R was significantly attenuated in the I/R + bucillamine group (P < 0.05 compared with the I/R + saline group). Therefore, long-term treatment with bucillamine preserves cardiac contractile function following I/R injury.

[0086] Fetal gene expression. Expression of fetal isoforms of several genes has been observed in pathological cardiac hypertrophy (8, 22, 33, 35, 47). For example, the expression of the natriuretic peptides, ANP and brain natriuretic peptide, the skeletal isoform of α-actin, and the β-isoform of MHC are increased. In contrast, the expression of the SERC A2a gene decreases in pathological hypertrophy. The expression of the α-isoform of MHC is also decreased, resulting in a further decrease in the α-MHC-to-β-MHC ratio. We compared the myocardial expression of these genes in saline- and myocardial-treated mice 4 wk after I/R injury.

[0087] Figures 5A-F show the effects of I/R injury, without or with bucillamine treatment, on the expression of the fetal isoforms of specific cardiac genes. The data were expressed as the abundance of each mRNA species relative to the sham-operated controls. I/R injury resulted in a significant increase in β-MHC expression in the saline-treated mice. This increase was prevented by long-term bucillamine treatment (Fig. 5_^4). I/R injury in the saline-treated mice also resulted in increases in ANP and skeletal α-actin expression (Figs. 5,B and C). Again, bucillamine prevented the increases in the expression of these genes. As shown in Fig 5D, I/R tended to decrease the expression of α-MHC in both the saline- and bucillamine-treated mice, although these results did not reach statistical significance.

[0088] However, I/R caused a statistically significant decrease in the α-MHC-to-β- MHC ratio in the saline-treated mice, which was prevented by bucillamine treatment (Fig. 5E). Finally, SERCA2a expression significantly decreased following I/R injury in the saline-treated mice, and this decrease was attenuated by bucillamine (Fig. 5F). Thus, 4 wk after I/R injury, the pathological pattern of gene expression (increased β- MHC, ANP, and skeletal α-actin; and decreased α-MHC, α-MHC-to-β-MHC ratio, and SERCA2a mRNA) appeared in saline-treated mice but was attenuated by bucillamine.

[0089] Cytokine gene expression. Since redox activity early in ischemia may stimulate cytokine activation, we tested whether the protection of cardiac function by bucillamine was mediated by prolonged alterations in myocardial cytokine gene expression. No statistically significance or trend toward differences in the cardiac expression of IL-I β, IL-6, or TNF-α was seen among the experimental groups.

[0090] It is well established that early reperfusion of the infarcted myocardium results in the generation of high levels of oxidative activity. Considerable attention has been paid to possible reduction in infarct size by the acute administration of antioxidants or other anti-inflammatory measures during the early stages of reperfusion (12, 13, 25). However, there has also been evidence of the prolonged elevation of oxidative activity for weeks or months following I/R (10, 24, 40), and little is known about its importance in the long-term recovery from acute myocardial infarction. The present work demonstrates that a thiol donor antioxidant, particularly a dithiol donor, bucillamine, administered daily for 1 mo after I/R markedly attenuates ventricular remodeling in genetically normal mice. In previous studies of transgenic animals, either antioxidant capacity was impaired or there was exaggerated oxidative activity before, during, and after the initiation of ischemia.

[0091] The present study demonstrates that long-term treatment with bucillamine, a representative dithiol anti-oxidant, attenuated the increase in cardiac mass, loss of contractile function, and pathological gene expression observed in saline-treated mice 4 wk after I/R injury. The present study more specifically shows that a sustained delivery of a dithiol donor antioxidant after the onset of reperfusion in normal mice attenuates long-term I/R- induced cardiac hypertrophy, loss of cardiac contractile function, and pathological patterns of gene expression. Oxidative activity immediately at the onset of reperfusion may primarily affect cytokine release and acute inflammatory responses (27), whereas later oxidative activity may have a greater influence on genes that control myocyte function and size (2, 9). Thus the benefits of antioxidant therapy following I/R extend beyond the acute injury phase and continue during the chronic phase of myocardial remodeling.

[0092] Although oxidative stress is a determinant of infarct size in the acute response to I/R injury (12, 13, 25), there is not necessarily a consistent correlation between infarct size and the subsequent development of ventricular remodeling. As alluded to above, Vasilyev et al. (42) found that mice deficient in myeloperoxidase (an enzyme that catalyzes the generation of reactive oxygen species in leukocytes) had improved cardiac function 24 days after I/R compared with wild-type mice, despite equivalent infarct sizes. Additionally, in an ischemia model without reperfusion, Shiomi et al. (32) demonstrated that an over expression of glutathione peroxidase in transgenic mice prevented ventricular remodeling and heart failure, independently of infarct size. The data of this study shows an improvement in cardiac function following antioxidant treatment, despite equivalent infarct sizes in treated and control animals. Thus it is likely that the development of ventricular remodeling is not solely related to the amount of damage sustained during or immediately following ischemia but, rather, is strongly influenced by the evolving response to injury in the surviving myocardium

[0093] The analysis of gene expression in heart tissue from human patients has identified a pattern of abnormal myocardial gene expression that occurs with cardiac remodeling (22, 35). Similar changes are observed in rodent models (8, 47), although the magnitude of the responses of individual genes differs among species. Although it is not necessarily the case that all the changes in genetic expression have direct functional consequences, decreases in SERCA2a expression and the α-MHC to-β- MHC ratio are associated with the loss of myocyte contractility(22, 28). Furthermore, sustained decreases in SERCA2a protein levels are thought to contribute to the loss of cardiac function during the progression to heart failure (28).

[0094] Therefore, the attenuation of I/R-induced decreases in SERCA and α-MHC- to-β-MHC ratio by antioxidant therapy, as seen in this study, can be usefulness for the prevention of heart failure. Indeed, in human clinical studies, the improvement of cardiac function through β-adrenergic blockade (21) or left ventricular assist devices (44) correlated with a reversal of the pathological pattern of gene expression.

[0095] Without wishing to be bound by any particular theory of action, bucillamine-induced attenuation of the expression of the pathological pattern of gene expression may occur through the prevention of abnormal oxidative signaling. Oxidative stress induces pathological gene expression in isolated cardiac myocytes (2). In addition, SERCA2a mRNA levels are reduced by oxidative stress in the acute phase of I/R injury (37). Thus antioxidant therapy may modulate abnormal signaling pathways that alter myocardial gene transcription following I/R injury. Finally, the prevention of adverse remodeling by bucillamine could indirectly result in a more physiological pattern of gene expression due to a decreased stress environment in the heart. [0096] Despite evidence in animal models that increased levels of oxidant stress are associated with cardiac remodeling and development of heart failure, vitamin E, a lipid-soluble antioxidant, did not reduce the incidence of heart failure either in patients with established atherosclerotic disease (20) or with recent myocardial infarction (23). Vitamin E largely exerts its antioxidant effects by preventing lipid peroxidation. However, lipid peroxidation is only one of the mechanisms by which adverse effects of oxidative stress may occur. The prevention of acute lipid peroxidation alone may not prevent left ventricular dysfunction induced by I/R (16).

[0097] In contrast, there is abundant evidence that hydrogen peroxide, a highly diffusible reactive oxygen species, functions as a signaling agent in low concentrations (30, 34) and may stimulate hypertrophy or apoptosis in cardiac myocytes (17). Bucillamine and other synthetic dithiol donors, through their ability to rapidly restore thiol groups to endogenous oxidized glutathione or thioredoxins, are extremely efficient in limiting intracellular hydrogen peroxide accumulation. In this manner thiol donors probably minimize hydrogen peroxide-mediated redox signaling (1, 43).

[0098] The present work demonstrates that chronic administration of a potent dithiol antioxidant, bucillamine, protects against long-term pathological ventricular remodeling, particular ventricular remodeling that is subsequent to I/R injury. Bucillamine reduced hypertrophy, improved contractile function, and prevented pathological expression of cardiac-specific genes. These results are compatible with the hypothesis that, following a myocardial infarction treated with reperfusion, a prolonged redox modulation of cell signaling can exert adverse effects on ventricular shape and function that are associated with abnormal myocardial gene expression.

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Claims

CLAIMSWe claim:

1. A method for inhibiting or preventing ventricular remodeling in a patient at risk of ventricular remodeling which comprises the step of administering a clinically effective amount of a dithiol anti-oxidant of formula 1 :

or a pharmaceutically acceptable salt thereof where:

Rl and R2, independently of one another, are alkyl groups, particularly lower alkyl groups having 1-6 or 1-3 carbon atoms;

R3 is carboxylic acid, carboxylate or salt thereof or an ester(-CO-OR_E) or amide (-

CO-N(RN)₂ of the carboxylate;

R4 or R5, independently of one another, are selected from the group consisting of a hydrogen, an alkyl group, an alkanoyl group, a phenyl-alkyl group or a phenylcarbonyl group and the phenyl ring in the phenyl-alkyl and phenylcarbonyl groups can be unsubstituted or substituted by at least one group selected from halogen, hydroxyl, alkyl or alkoxy;

A is -(CH2)- group and m is 0 or an integer from 1-6; and

R_E and R_N, independently of one another, are alkyl, or alkyl aryl group; to the patient at risk until the risk of ventricular remodeling is lowered to an acceptable level or until maximal benefit of administration is achieved.

2. The method of claim 1 wherein ventricular remodeling is the result of myocardial infarction, cardiomyopathy or myocarditis.

3. The method of claim 1 wherein the dithiol anti-oxidant is administered for a minimum of six hours following treatment of myocardial infarction with reperfusion.

43

4. The method of claim 3 wherein treatment is continued for two or more days after such reperfusion treatment.

5. The method of claim 3 wherein treatment is continued for at least one week after such reperfusion treatment.

6. The method of claim 3 wherein treatment is continued for at least one month after such reperfusion treatment.

7. The method of claim 1 wherein reperfusion treatment is not employed prior to administration of the dithiol anti-oxidant.

8. The method of claim 1 where administration is continued for at least one week.

9. The method of claim 1 where administration is continued for at least one month.

10. The method of any one of claims 1-9 wherein administration is continued until abnormal myocardial gene expression is no longer observed.

11. The method of any one of claims 1-10 wherein administration employs a slow- release dosage form.

12. The method of any one of claims 1-11 wherein administration is oral or parenteral.

13. The method of any one of claims 1-12 wherein in the dithiol antioxidant of formula 1 , R3 is a carboxylic acid (-COOH), carboxylate (-COO-) or a carboxylate salt (-COCTX⁺, where X ⁺ is a pharmaceutically acceptable cation).

14. The method of any one of claims 1-13 wherein Rl and R2 are C1-C3 alkyl groups.

15. The method of any one of claims 1-14 wherein R4 and R5 are hydrogens.

16. The method of any one of claims 1-15 wherein m is 0 or 1.

17. The method of any one of claims 1-16 wherein Rl and R2 are methyl groups.

18 The method of any one of claims 1-12 wherein the dithiol anti-oxidant is bucillamine.

19. The method of any one of claims 1-12 wherein the dithiol antioxidant is the compound of formula 1 wherein m is 1, Rl and R2 are methyl groups, R4 and R5 are hydrogens and R3 is carboxylate or a salt thereof.

20. The method of any one of claims 1-12 wherein the dithiol anti-oxidant is

CH,

21. The use of a dithiol anti-oxidant of formula:

or a pharmaceutically acceptable salt thereof where:

CO-N(R_N)₂ of the carboxylate; R4 or R5, independently of one another, are selected from the group consisting of a hydrogen, an alkyl group, an alkanoyl group, a phenyl-alkyl group or a phenylcarbonyl group and the phenyl ring in the phenyl-alkyl and phenylcarbonyl groups can be unsubstituted or substituted by at least one group selected from halogen, hydroxyl, alkyl or alkoxy;

A is -(CEl)- group and m is 0 or an integer from 1-6; and

R_E and R_N, independently of one another, are alkyl, or alkylaryl group; for the reduction, inhibition or prevention of ventricular remodeling.

22. The use of a dithiol anti-oxidant of formula:

or a pharmaceutically acceptable salt thereof where:

R3 is carboxylic acid, carboxylate or salt thereof or an ester (-CO-OR_E) or amide (-

CO-N(R_N)₂ of the carboxylate;

R4 or R5, independently of one another, are selected from the group consisting of a hydrogen, an alkyl group, an alkanoyl group, a phenyl-alkylgroup or a phenylcarbonyl group and the phenyl ring in the phenyl-alkyl and phenylcarbonyl groups can be unsubstituted or substituted by at least one group selected from halogen, hydroxyl, alkyl or alkoxy;

A is -(CRZ)- group and m is O or an integer from 1-6; and

R_E and R_N, independently of one another, are alkyl, or alkylaryl group; for the reduction, inhibition or prevention of ventricular remodeling subsequent to myocardial infarction and reperfusion treatment.

23. The use of a dithiol anti-oxidant of formula:

or a pharmaceutically acceptable salt thereof where:

CO-N(R_N)₂ of the carboxylate;

A is -(CH2)- group and m is O or an integer from 1-6; and

R_E and R_N, independently of one another, are alkyl, or alkylaryl group; for the reduction, inhibition or prevention of heart failure subsequent to ventricular remodeling.

24. The use of any one of claims 21 -23 wherein in the dithiol antioxidant of formula 1 , R3 is a carboxylic acid (-C00H), carboxylate (-COO-) or a carboxylate salt (-C00^"X⁺, where X ⁺ is a pharmaceutically acceptable cation).

25. The method of any one of claims 21-23 wherein Rl and R2 are C1-C3 alkyl groups.

26. The method of any one of claims 21-23 wherein R4 and R5 are hydrogens.

27. The method of any one of claims 21 -23 wherein m is 0 or 1.

28. The method of any one of claims 21-23 wherein Rl and R2 are methyl groups.

29. The method of any one of claims 21-23 wherein the dithiol anti-oxidant is bucillamine.

30. The method of any one of claims 21-23 wherein the dithiol antioxidant is the compound of formula 1 wherein m is 1, Rl and R2 are methyl groups, R4 and R5 are hydrogens and R3 is carboxylate or a salt thereof.

31. The method of any one of claims 21 -23 wherein the dithiol anti-oxidant is

CH₃