Methods of Treating Cardiac Reperfusion Disease

CLAIM OF PRIORITY

This application claims the benefit under 35 USC §H9(e) to U.S. Provisional Patent Application Serial No. 60/748,247, filed on December 7, 2005, the entire contents of which are hereby incorporated by reference.

TECHNICALFIELD

This invention relates to the treatment of cardiac reperfusion disease using a cardiac autoantigen, e.g., troponin.

BACKGROUND

Myocardial infarction linked to myocardial ischemia-reperfusion (MI/R) is one of the most common causes of death in both the western and developing worlds (Cohn et al., 1997. Circulation 95:766; Buja, 2005. Cardiovasc Pathol 14:170). Myocardial ischemia-reperfusion (MI/R) injury is a potent stimulus for tissue destruction and subsequent cardiac failure. Myocardial infarction associated with MI/R injury is the most common cause of death in the western world and the developing world. Recent evidence shown that acute MI/R is associated with an intense inflammatory reaction including neutrophils and monocytes(Frantz et al., 2005. Curr Pharm Des 11 :1279). Inflammation plays an important role both in acute extension of injury and repair of the myocardium.

Following ischemia, most cells in the heart, including endothelial cells, may produce interleukin-1 β (IL- lβ), IL-6 and tumor necrosis factor α (TNF-α) that can directly induce cell death as well as contribute to vessel wall injury, hemorrhage, edema and tissue necrosis(Hallenbeck, 2002. Nat Med 8:1363). Immunoregulatory cytokines such as IL-IO and transforming growth factor βl (TGF-βl) can modulate immune processes and inhibit the expression of inflammatory ThI type responses as well as inflammation in general (Weiner, 2001. Immunol Rev 182:207). IL-10 is preferentially produced by TrI type regulatory T-cells (Groux, 2001. Microbes Infect 3:883) and TGF-β is preferentially produced by Th3 type regulatory cells which suppress both ThI and Th2 cells (Weiner, 2001. Immunol Rev 182:207; Weiner, 2001. Nat Immunol 2:671).

During the course of the disease, different cardiac proteins are released into the bloodstream. One such protein is troponin, which is synthesized exclusively in myocardial cells (Apple, 2001. Cardiovasc Toxicol 1:93; Panteghini, 2000. Clin Biochem 33:16). The cardiac troponins form part of the regulatory mechanism for muscle contraction and a high concentration of troponin in the bloodstream reflects not only cardiac ischemia, but left ventricular (LV) mass, which is a strong predictor of cardiovascular death in this population. Thus, the level of isoforms of troponin T (cTnT) in the bloodstream is a very sensitive marker of myocardial injury (Lowbeer et al., 2002. Nephrol Dial Transplant 17:2178).

SUMMARY

At least in part, the present invention is based upon the discovery that administration of a cardiac autoantigen, e.g., troponin, either prior to, during, or after an ischemic injury, decreasing cardiac inflammation and myocardial damage.

Described herein are methods for treating or preventing cardiac reperfusion diseases in a subject. The methods include administering to the subject a therapeutically effective amount of a composition comprising troponin polypeptide. In some embodiments, the administration is mucosal. Mucosal administration can be, e.g., oral or nasal, e.g., using a metered dose inhaler, a dry powder inhaler, a nebulizer, or a mask. In some embodiments, the composition also includes one or both of a carrier and an adjuvant, hi some embodiments, the composition includes one or both of cardiac-specific troponin T (cTnT) and cardiac specific troponin I (cTnl).

Cardiac reperfusion diseases include, but are not limited to, myocardial infarction, surgically-induced ischemia/reperfusion disease, stable angina, or unstable angina. In some embodiments, the subject has had an ischemic cardiac injury, e.g., within the past 12-24 hours, or is at risk of having an ischemic cardiac injury. In some embodiments, the ischemic cardiac injury is due to acute coronary artery occlusion, severe arterial stenosis, atherosclerosis, embolism, coronary spasm, or a surgical procedure, e.g., any procedure that causes or carries a risk of ischemic injury to the heart, e.g., heart surgery, lung surgery, spinal surgery, brain surgery, abdominal surgery, or organ transplantation surgery (e.g., cardiac, lung, pancreas or liver transplantation), or vascular surgery, e.g., angioplasty (including elective angioplasty), coronary artery bypass graft, and surgery involving cardiac bypass.

In general, a cardiac reperfusion disease that can be treated or prevented by a method described herein that includes administering troponin is a disease that is generally associated with an increase in circulating troponin levels in the blood. In general, the methods described herein can be used to treat such diseases before troponin levels are increased. hi some embodiments, the subject is at risk of having an ischemic cardiac injury because they are scheduled for a surgical procedure, e.g., any surgical procedure that causes or carries a risk of ischemic injury to the heart, e.g., heart surgery, lung surgery, spinal surgery, brain surgery, abdominal surgery, or organ transplantation surgery (e.g., cardiac, lung, pancreas or liver transplantation) or vascular surgery, e.g., angioplasty (including elective angioplasty), coronary artery bypass graft, and surgery involving cardiac bypass. In some embodiments, the subject is scheduled for surgery in 1, 2, 4, 6, 8, 12, 24, or 48 hours, or 3, 4, 5, 6, or 7 days, or two weeks or more.

Thus, the methods described herein can be used to treat cardiac diseases including stable or unstable angina, myocardial infarction, and surgically-induced (e.g., ischemia that is intentionally or involuntarily induced during a surgical procedure) ischemia/reperfusion disease, such as those caused by or related to acute coronary artery occlusion, severe arterial stenosis, atherosclerosis, embolism, coronary spasm, and surgical procedures, e.g., heart surgery, lung surgery, spinal surgery, brain surgery, abdominal surgery, or organ transplantation surgery (e.g., cardiac, lung, pancreas or liver transplantation), vascular surgery, e.g., angioplasty (including elective angioplasty), coronary artery bypass graft, and surgery involving cardiac bypass, or any other procedure that causes or carries a risk of ischemic injury to the heart.

In some embodiments, the subject is selected on the basis of having had or being at risk for having a cardiac reperfusion disease, or is selected on the basis of needing a surgical procedure that causes or carries a risk of ischemic injury to the heart. Such procedures can include cardiac transplantation or vascular surgery.

Also included are methods of enhancing the survival or function of a transplanted heart. The methods include (a) providing a heart from a donor; (b) transplanting the heart into a recipient; and (c) before, during, and/or after step (b), administering to the recipient a therapeutically effective amount of a therapeutic

composition including a troponin polypeptide, wherein the administration of the therapeutic composition is sufficient to enhance survival or function of the heart after transplantation of the organ to the recipient.

Also within the invention is the use of troponin polypeptides in the manufacture of a medicament for the treatment or prevention of cardiac reperfusϊon disease. The medicament can be used in a method for treating or preventing cardiac reperfusion disease in a patient suffering from or at risk for cardiac reperfusion disease. The medicament can also be used in a method of organ transplantation, e.g., to reduce the risk of or prevent ischemia-reperfusion injury. The medicament can also be used in a method of performing vascular surgery or angioplasty on a patient. The medicament can also be used in a method of treating or preventing the effects of cardiac ischemia in a patient. The medicament can be in any form described herein, and can be administered alone or in combination with one or more supplemental therapeutic modalities, e.g., as described herein.

Also within the methods described herein is a method of treating a subject that includes providing the subject with an inhaler device that comprises one or more doses of a troponin composition described herein, and instructing the subject to self- administer one or more doses, e.g., upon occurrence of a cardiac reperfusion disease- related event, e.g., a one or more symptoms of cardiac ischemia, e.g., chest pain (angina), neck or jaw pain, arm pain, clammy skin, shortness of breath, and/or nausea and vomiting; or upon instructions from a health care provider to do so.

One of skill in the art will appreciate that the methods described herein can equally be used with other cardiac autoantigens in place of, or in addition to, troponin. For example, other cardiac autoantigens include cardiac myosin, SlOO protein, beta polypeptide (S-IOOb), heat shock proteins (HSPs), e.g., HSP60, HSP70, HSP20, HSP25, HSP27, alphaB-crystallin (CryAB), and Desmin.

The term "subject" is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary applications are clearly included in the methods described herein. In general, a subject is a mammal, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.

The terms "effective amount" and "effective to treat," as used herein, refer to the administration of a pharmaceutical compositions(s) described herein in an amount or concentration and for period of time including acute or chronic administration and periodic or continuous administration that is effective within the context of its administration for causing an intended effect or physiological outcome. The terms "treat" or "treatment," are used herein to describe delaying the onset of, inhibiting, or alleviating the effects of a disease or condition, e.g., a disease or condition described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. AU publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. IA is a bar graph illustrating the effect of nasal troponin treatment on proliferation of splenocytes. The bars are presented as stimulation index (s.i.), which is the ratio between number of cells follow stimulation with 100 μg troponin as number of cells without the antigen. Black bar, PBS-treated controls. Gray bars, troponin-treated animals. White bars, animals treated with troponin and Emulsome (EMU).

FIGs. IB and 1C are bar graphs illustrating cytokine release from splenocytes stimulated with Troponin; IB, Interleukin-2 (IL-2), IL-6, and IL-10; 1C, Interferon gamma (IFN-γ). Note that the scale for IB is 0-1800, while the scale for 1C is 0-18000.

FIG. ID is a bar graph illustrating the effect of nasal treatment with murine or porcine troponin on proliferation of splenocytes, as indicated.

FIGs. IE and IF are bar graphs illustrating cytokine release from splenocytes stimulated with porcine troponin (grey bars) or murine troponin (white bars); IE, IL-2, IL-6, and IL-IO; IF, IFN-γ. Note that the scale for IE is 0-700, while the scale for IF is 0-4500.

FIG. 2 A is a pair of photographs illustrating the effect of nasal troponin on heart infarct size 24 hours following MI/R surgery in an exemplary animal. At 24 hours, Triphenyl tetrazolium chloride staining was performed to assess infarct size. The lighter regions (outlines) denote the infarcted tissue. The sections presented are from the left ventricular (LV) area of representative individual mice.

FIG. 2B is a dot graph illustrating that infarct volume is significantly less in nasal troponin treated mice (n=12, p<0.005) as compared to BSA treated mice (n=12); each dot represents an individual animal.

FIG. 3 is a line graph illustrating a reduction of proliferation against troponin in the nasally treated mice following MI/R surgery. There is significant reduction of proliferation against troponin in the treated mice (filled triangles) vs. control (open triangles; p<0.03). The levels of proliferation in the treated group approach those in the untreated group (basal, X's).

FIG. 4A is a quartet of photographs illustrating that nasal tolerance to troponin protects against tissue injury. Masson trichrome staining (left panels) was performed to assess collagen deposition that is associated with tissue death (magnification x5). The lighter gray regions (blue in the original) denote collagen deposition. There is less myocardial tissue and more collagen deposition in the control group. Haematoxylin and Eosin (H&E) staining (right panels, magnification x20) demonstrate less myocardium injury in treated animals (lower panels) as compared to BSA-treated animals (top panels). Arrows identify areas of collagen deposition.

FIG. 4B is a dot graph demonstrating the results of quantification of the percentage of collagen in the left ventricle, showing significant (p<0.05) anterior wall thinning and collagen deposition 1.5 months after injury in control mice vs. troponin-treated mice, in which the myocardium appeared normal with decreased collagen deposition.

FIG. 4C is a pair of M-mode images from the myocardium of a control, BSA-treated mouse (left image) and a troponin-treated mouse (right panel). The

double-headed arrow indicates the increased gap in the LV due to myocardium damage as compare to the troponin treated animal.

FIGs. 5A-5B are a pair of images of sections of hearts from control (5A) or nasal troponin-treated (5B) mice 24 hours after MI/R injury stained for CD4+ cells. Staining shows immunoperoxidase (magnification x5) on a background of hematoxylin staining; arrows identify regions of CD4+ infiltration.

FIGs. 5C-5D are each pairs of immunofluorescence images of serial sections of hearts from control (5C) or nasal troponin-treated (5D) mice 24 hours after MI/R injury labeled using niAbs directed against CD4, CDl Ib, IFN-γ, or IL- 10 (magnification x 20). Arrows identify colocalization (yellow in original) of CD4+ cells with either IFN-γ (left panel in 5C and 5D) or IL-10 (right panel in 5C and 5D).

FIG. 6A is a bar graph illustrating the results of quantification of populations of cells expressing CD4 or CDl Ib. Results represent blinded analysis of three samples/group stained in the area of the left ventricle. Results are SEM (troponin vs. control: for CDlIb, CD4: p < 0.02)

FIG. 6B is a bar graph illustrating the quantification of CD4+ populations. Results represent blinded analysis of three samples/group stained in the area of the left ventricle. Results are in SEM (troponin vs control for IFN-γ and IL-IO = p < 0.05)

FIGs. 6C-6D are bar graphs illustrating the quantification of cytokine expression in the LV 24 hours following MI/R surgery by RT-PCR. The area of the infarct in left ventricle showed a significant reduction in the pro-inflammatory cytokines TNF-α (6C) and IL-6 (6D) (p<0.05).

FIG. 7 A is a pair of photographs showing the effect of nasal troponin on heart infarct size in EL-IO-/- mice 24 hours following MI/R surgery. At 24 hours, triphenyl tetrazolium chloride staining was performed to assess infarct size. The pale regions denote the infarcted tissue.

FIG. 7B is a dot graph showing results indicating that there is no significant difference in infarct volume between Troponin+Emu treated mice (n=6) as compared to BSA treated IL-10-/- mice (n=6).

FIG. 7C is a line graph showing results indicating that there is no significant difference in splenocyte proliferation between Troponin+Emu treated mice as compared to BSA treated IL-IO-/- mice (n=6).

FIG. 8 A is a pair of dot graphs showing the effect on infarct size of mucosal troponin given after MI/R surgery in two separate experiments. Each dot represents the results in an individual animal. Infarct volume was significantly reduced in mice that received troponin 1 hour following the MI/R surgery as compared to control mice that received BSA.

FIG. 8B is a line graph showing that mucosal troponin given after MI/R surgery reduces proliferation in response to troponin.

FIG. 8C is a pair of bar graphs showing the results of RT-PCR analysis of expression of TNF-α and IL- 12 in splenocytes from control vs. troponin-treated animals fp<0.05).

FIG. 8D is a bar graph showing the results of RT-PCR analysis of expression of TNF-α and IL-6 in the left ventricle from control vs. troponin-treated animals (p<0.05).

FIGs. 8E-F are each pairs of immunofluorescence images (magnification = x 20) of cells stained for expression of CD4, and either IFN-γ or IL-10. Arrows identify colocalization (yellow in original) of CD4+ cells with either IFN-γ or IL-IO.

FIG. 8G is a bar graph illustrating the results of quantification of cell populations from the experiments shown in FIGs. 8E-F. Results represent blinded analysis of three samples/group stained in the area of the left ventricle. Results are in SEM (troponin vs control: for IFN-I, IL-10/7 < 0.01).

FIGs. 9A-9B are graphs illustrating the results of FACS analysis of recovery of CD3+/4+ cells from total CD3+/4+ cells loaded 17.1% (I) the purity of recovered cells was 90.8% (IT) with no detectable CD3+/8+ cells. 9A, CD4+ cells; 9B, CD3+ cells.

FIGs. 9C-9D are dot graphs showing the effect of adoptive transfer of CD4+ T-cells from individual troponin tolerized animals, either wild type (9C) or IL-10-/- (9D), given one hour following the MI/R surgery on heart infarct size. FIG. 9C, Infarct volume was significantly less in mice received 1 x 10 ⁶ CD4+ T cells from troponin tolerized mice (n=5, p<0.05) as compared to mice (n=5) that received 1 x

10 ⁶ CD4+ T-cells from BSA tolerized mice. FIG. 9D, there was no significant difference in infarct volume in control mice and mice that received CD4+ cells Iran BSA-tolerized IL-IO -/- mice (n=7). Each dot represents an individual animal.

FIG. 9E is a line graph showing the effect of adoptive transfer of CD4+T- cells on splenocyte proliferation in response to troponin. Cells were taken from animals treated with BSA (filled squares) or troponin (filled triangles), or were untreated (open triangles). 24 hours after i.v. administration of the CD4+ T-cells, mice were sacrificed and their splenocytes were cultured at 0.5 x 10 cell/well with Troponin in 0.2 ml medium. For proliferation measurements, cells were pulsed with [ ³ H]Thymidine at 72 hours and radioactivity determined 16 hours later.

DETAILED DESCRIPTION

Accumulated data demonstrates that inflammation plays an important role in the pathophysiology of ischemic MI/R. However, even in the most severe ischemic insult, the process of tissue destruction may not be completed for hours or days (Frantz et al., 2005. Curr Pharm Des 11 :1279; Ren et al., 2003. Curr Drug Targets Inflamm Allergy 2:242; Hansen 1998. J MoI Cell Cardiol 30:2555). This provides an opportunity for strategies of protection to salvage the ischemic inflammation process.

Mucosal tolerance is a well established method whereby regulatory T cells can be induced to a specific antigen by nasally or orally administering the antigen; see Faria and Werner, 2005. Immunol. Rev. 206:232-59. Upon re-stimulation with the mucosally administered antigen, T-cells in mucosally tolerized animals secrete cytokines such as transforming growth factor βl (TGF-βl) or IL-10 that are potent anti-inflammatory cytokines with tissue protective properties. Nasal administration of antigen preferentially induces regulatory T-cells that secrete IL-10.

The experiments described herein demonstrate that mucosal, e.g., nasal, administration of a cardiac autoantigen, e.g., troponin, results in a decrease of myocardial inflammation and an improved outcome after MI/R injury, likely by inducing T cell-mediated active immunologic tolerance to troponin.

Nasal troponin significantly (p < 0.05) decreased infarct size by 50%, as measured 24 hours after MI/R surgery. Immunohistochemistry demonstrated increased IL-10 and reduced IFN-γ in the area surrounding the ischemic infarct following nasal treatment. Nasal troponin did not reduce infarct size in IL-10

deficient mice. Nasal troponin polypeptide was also efficacious in reducing infarct size 1.5 months after the MI/R surgery by 50% (p < 0.0001 vs. control) and improved the behavior score as measured by cardiac echo.

Transfer of CD4+ T cells from nasally troponin tolerized mice to untolerized mice 30 minutes after MI/R surgery also decreased infarct size (p<0.05 vs. control), whereas CD4+ T-cells from nasally BSA tolerized mice had no effect. These results demonstrate that IL-10-secreting CD4+ T cells induced by nasal troponin reduce injury following MI/R. Modulation of inflammation by nasal vaccination with troponin provides a novel mechanism to improve outcome after MI/R and enhance recovery.

As one theory, not meant to be limiting, nasal administration of troponin induces regulatory T-cells specific for troponin that secrete anti-inflammatory cytokines, such as IL-10, which decreases inflammation in the heart, thereby limiting myocardial damage caused by an ischemic insult.

Cardiac Autoantigens

The methods described herein include the mucosal administration of cardiac autoantigen to treat or prevent a cardiac reperfusion disease. Exemplary cardiac autoantigens include troponin, cardiac myosin, SlOO protein, beta polypeptide (SlOOb), heat shock proteins (HSPs), e.g., HSP60, HSP70, HSP20, HSP25, and HSP27, alphaB-crystallin (CryAB), and desmin.

One of skill in the art will appreciate that active fragments of the autoantigen, e.g., troponin, can also be used. An active fragment is a fragment that include a T-cell epitope. Methods are known in the art for identifying a T-cell epitope; see, e.g., Greer et al., 1992. J. Immunol. 149(3):783-8; Reijonen and Kwok, 2003. Methods 29(3):282-8. For example, serial analysis of 15-mer overlapping fragments of a troponin protein can be used.

Troponin

Troponin is synthesized exclusively in myocardial cells. Troponins form part of the muscle contractile structure along with actin and tropomyosin. There are three general forms of troponins in humans: I, T and C, which are found in all muscle types. There are two human cardiac-specific isoforms of troponins I and T, cardiac- specific troponin T (cTnT) and cardiac specific troponin I (cTnl). Commercially available immunoassay systems have been developed for their measurement (see, e.g.,

Collinson et al., 2001. Ann. Clin. Biochem. 38(Pt 5):423-49). High concentratioiss of troponin reflect cardiac ischemia.

The methods described herein include the administration of troponin polypeptides, which can be, e.g., synthetic (i.e., artificially), recombinant (i.e., expressed in an autologous system such as a transgenic animal or cultured cell), or native (i.e., purified from a native source, e.g., muscle tissue such as cardiac muscle), hi general, where the subject is human, it will be desirable to use a human troponin, but other species maybe used as well, e.g., porcine. Methods for obtaining, producing, and purifying troponins are known in the art. cTnT cTnT is also referred to as Troponin T type 2 (cardiac) (TNNT2); CMH2; TnTC; cTnT; CMDlD; and MGC3889. There are a number of variants of cTnT; the mRNA and polypeptide sequences of variant 1 can be found at GenBank Accession No. NM_000364.2 andNP_000355.2, respectively; the mRNA and polypeptide sequences of variant 2 can be found at GenBank Accession No. NM_001001430.1 and NP_001001430.1, respectively; the mRNA and polypeptide sequences of variant 3 can be found at GenBank Accession No. NM_001001431.1 andNP_001001431.1, respectively; the mRNA and polypeptide sequences of variant 4 can be found at GenBank Accession No. NMJ)Ol 001432.1 and NP_OO1001432.1, respectively. For additional information see GenelD: 7139 (available at ncbi.nlm.nih.gov/entrez/ query.fcgi?db ^:=: gene), or UniGene entry no. Hs.533613 (available at ncbi.nlm.nih.gov/entrez/query.fcgi? db=UniGene). cTnl cTnl is also referred to as Troponin I type 3 (cardiac) (TNNI3); CMH7; cTnl; TNNCl; and MGCl 16817. The mRNA and polypeptide sequences of human cTnl can be found at GenBank Accession No. NM_000363.3 and NP_000354.3, respectively; for additional information see GenelD: 7137, or UniGene entry no. Hs.512709. cTnC

CTnC is also referred to as troponin C, cardiac/slow skeletal (TNNCl), and Troponin C, Cardiac (TNC). The mRNA and polypeptide sequences of human cTnC can be found at GenBank Accession No. NM_003280 and

NP 003271, respectively; for additional information see GenelD: 7134, or

UniGene entry no. Hs.118845.

One or more of the troponin isoforms can be used in combination, e.g., two or three forms; in some embodiments, a mixture of the cardiac-specific isoforms is used. For example, a mix of three heart-specific troponins cTnT, cTnl and cTnC can be used. It has shown that a difference in each one can be used to predict heart damage (Peter et al., Forensic Science International (2005), doi: 10.1016/j.forsciint.2005.08.022).

Murine cardiac myosin

Human heart disease can be mimicked in mice using cardiac myosin as autoantigen. Murine cardiac myosin-induced myocarditis is an organ-specific autoimmune disease and mediated by CD4+ T cells that recognize a myosin-specific peptide in association with MHC class II molecules. (Penninger et al. _? APMIS 105(l):l-13 (1997))

SlOO protein, beta polypeptide (SlOOb)

The beta subunit of SlOO protein is induced in the myocardium of human subjects and an experimental rat model following myocardial infarction. (Parker et al., 1998. Can J Appl Physiol 23(4):377-89).

HSP

Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Cardiomyocytes respond to stress with the expression of different heat shock proteins (HSP). HSP60 HSP60 is induced by various stress factors. Furthermore, there is up regulation of heat shock protein 60 in myocardium of patients with chronic atrial fibrillation. (Schafler et at, 2002. Basic Res Cardiol 97(3):258-61).

Moreover, Complement activating antibodies against the HSP60 is proven to be family risk factor of coronary heart disease. (Veres et al., 2002. Eur J Clin

Invest 32(6):405-10). HSP70 The inducible 70-kD heat shock protein (Hsp70) is expressed in the myocardium in response to stress and has been linked to enhanced myocardial

resistance to depression associated with ischemia-reperfusion There was a significant correlation (PO.05) between HSP70 induction and infarct size reduction, whether produced by thermal stress or oral administration of bimoclomol (Lubbers et al., 2002. Eur. J. Pharmacol. 435(l):79-83). HSP20/25/27 HSP20/25/27 are abundant in heart and normally located in the cytoplasm of the cardiac myocytes (van de Klundert and de Jong, 1999. Eur. J.

Cell. Biol. 78(8):567-72). Increased expression of HSP27 provides significant protection from simulated I/R injury in adult canine myocytes (Vander Heide,

2002. Am. J. Physiol. Heart. Circ. Physiol. 282(3):935-41). afohaB-crystallin (CrvAB)

CryAB is abundant in heart and normally located in the cytoplasm of the cardiac myocytes. AlphaB crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning. (Eaton et al., 2001. J. MoI. Cell. Cardiol. 33(9):1659-71). Transgene overexpression of alphaB crystallin was therefore successful in diminishing the independent contributory effects of both necrosis and apoptosis on I/R-induced cell death. (Ray et al., 2001. FASEB J. 15(2):393-402)

Desmin

Desmin is the main intermediate filament (IF) protein in skeletal and heart muscle cells, and is of great importance as a part of the cytoskeleton. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. The lack of desmin causes muscle fibres to become more susceptible to damage; subsequent loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart, cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen (Carlsson and Thornell, 2001. Acta Physiol Scand 171(3):341-8).

Compositions

The methods described herein generally include the use of a troponin polypeptide that is incorporated into a pharmaceutical composition. Such compositions include troponin and a pharmaceutically acceptable excipient. As used

herein the term "pharmaceutically acceptable excipient" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration via the selected route.

Pharmaceutical compositions comprising troponin polypeptides and/or fragments thereof are known in the art, see, e.g., U.S. Patents Nos. 5560937, 5837680, 6025331, 6403558, 6465431, 6589936, 6586401, and 6653283, and U.S. Patent Application Publications Nos. 20020142982, 20030022870, and 20030186864, all of which are incorporated herein in their entirety.

Supplementary therapeutic agents can also be incorporated into the compositions, e.g., as described herein.

The troponin polypeptides can be incorporated into a pharmaceutical composition suitable for mucosal administration, e.g., by inhalation, oral ingestion, or absorption, e.g., via nasal, intranasal, pulmonary, buccal, sublingual, rectal, or vaginal administration. Such compositions can include an inert diluent or an edible carrier. Such dosage forms can be prepared by methods of pharmacy well known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990)).

Oral Dosage Forms

For the purpose of oral therapeutic administration, the troponin polypeptides can be incorporated with excipients and used in solid or liquid (including gel) form. Oral troponin compositions can also be prepared using an excipient.

Solid oral dosage forms include, but are not limited to^ tablets (e.g., chewable tablets), capsules, caplets, powders, pellets, granules, powder in a sachet, enteric coated tablets, enteric coated beads, and enteric coated soft gel capsules. Also included are multi-layered tablets, wherein different layers can contain different drugs. Solid dosage forms also include powders, pellets and granules that are encapsulated. The powders, pellets, and granules can be coated, e.g., with a suitable polymer or a conventional coating material to achieve, for example, greater stability in the gastrointestinal tract, or to achieve a desired rate of release. In addition, a capsule comprising the powder, pellets or granules can be further coated. A tablet or caplet can be scored to facilitate division for ease in adjusting dosage as needed.

Liquids for oral administration represent another convenient dosage form, in which case a solvent can be employed. In some embodiments, the solvent is a buffered liquid such as phosphate buffered saline (PBS). Liquid oral dosage forms can be prepared by combining the active ingredient in a suitable solvent to form a solution, suspension, syrup, or elixir of the active ingredient in the liquid.

Inhalable Dosage Forms and Delivery

Troponin compositions intended to be administered by inhalation can be prepared by combining troponin polypeptide in a suitable solvent, e.g., to form a liquid composition, or by preparing a dry powder formulation. Methods for preparing such compositions are known in the art, see, e.g., U.S. Patent No. 6,794,357.

The compositions can also include excipients described herein and known in the art to enhance uptake and delivery of the troponin to desired areas of the lung, such as enhancer compounds and surfactants, see, e.g., U.S. Patent No. 6,794,357. Methods for making and using such are known in the art. See, e.g., U.S. Patent No. 5,952,008.

Thus, for administration by inhalation, the troponin compositions can be delivered in the form of a dry powder or aerosol spray, e.g., from a metered-dose inhaler (typically a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide), a dry powder inhaler, or a nebulizer. In some embodiments, e.g., where the subject is unconscious, the troponin compositions are administered passively, e.g., via an oxygen or anesthesia mask or nasal prongs, or with another inhaled agent, e.g., an anesthetic, e.g., during surgery, e.g., during an emergency surgical procedure. A number of suitable devices are known in the art, e.g., including single dose and multi dose devices. See, e.g., U.S. Patent Nos. 5,404,871; 5,347,998; 5,284,133; 5,217,004; 5,119,806; 5,060,643; 4,664,107; 4,648,393; 3,789,843; 3,732,864; 3,636,949; 3,598,294; 3,565,070; 3,456,646; 3,456,645; 3,456,644, 3,001,524, 3,012,555, 4,259,951, 5,431,154, 5,692,496, 6,305,371, and 6,708,688.

Excipients

The troponin compositions can be incorporated with excipients or carriers suitable for administration by ingestion, inhalation or absorption, e.g., via nasal sprays or drops, or rectal or vaginal suppositories. Excipients can include binders, fillers, disintegrants, lubricants, coatings, stabilizers, penetrants, enhancers, surfactants,

sweeteners, flavors and colors. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.

Binders suitable for use in pharmaceutical compositions include, but are not limited to, corn starch, potato starch, or other starches, gum tragacanth or gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidinones, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g.,Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms described herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, dextrans, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler is typically present in from about 50 to about 99 weight percent of a pharmaceutical composition or dosage form.

Disintegrants can be used in the pharmaceutical compositions to provide tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant might disintegrate in storage, while those containing too little might not disintegrate at a desired rate or under desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form the pharmaceutical compositions and solid oral dosage forms described herein. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Disintegrants that can be used in mucosal dosage forms include, but are not limited to, agar, alginic acid, calcium carbonate, Primogel™, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, corn, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used include, but are not limited to, calcium stearate, magnesium stearate or Sterotes, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc,

hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL ^® 200, manufactured by W. R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB-O-SIL ^® (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. A glidant such as colloidal silicon dioxide can also be used.

Surfactants can include, for example, a salt of a fatty acid, a bile salt, a bile salt derivative, an alkyl glycoside, a cyclodextrin, or a phospholipid. Enhancers can be, for example, a sodium, potassium, or organic amine salt of the fatty acid, and the fatty acid is preferably capric acid or another fatty acid of 10-14 carbon atoms. See, e.g., U.S. Patent No. 5,952,008.

Troponin compositions prepared in the form of suppositories can include conventional suppository bases such as cocoa butter and other glycerides.

The pharmaceutical compositions can further comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Thus the troponin compositions described herein can be processed into an immediate release or a sustained release dosage form. Immediate release dosage forms may release the troponin in a fairly short time, for example, within a few minutes to within a few hours. Sustained release dosage forms may release the troponin over a period of several hours, for example, up to 24 hours or longer, if desired. In either case, the delivery can be controlled to be substantially at a certain predetermined rate over the period of delivery, hi some embodiments, the solid oral dosage forms can be coated with a polymeric or other known coating material(s) to achieve, for example, greater stability on the shelf or in the gastrointestinal tract, or to achieve control over drug release. Such coating techniques and materials used therein are known in the art. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid and salt buffers. For example, cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyhnethyl cellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethylethyl cellulose, and hydroxypropylmethyl cellulose acetate succinate, among others, can be used to achieve enteric coating. Mixtures of waxes, shellac, zein, ethyl cellulose, acrylic resins, cellulose acetate, silicone elastomers can

be used to achieve sustained release coating. See, for example, Remington, supra, Chapter 93, for other types of coatings, techniques and equipment.

The troponin compositions can also include penetrants appropriate to the barrier to be permeated. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives.

Compositions such as liquid solutions, suspensions, syrups, and elixirs can optionally include other additives including, but not limited to, glycerin, sorbitol, propylene glycol, sugars or other sweeteners, flavoring agents, and stabilizers. Flavoring agents can include, but are not limited to peppermint, methyl salicylate, or orange flavoring. Sweeteners can include sugars, aspartame, saccharin, sodium cyclamate and xylitol.

Adjuvants

Pharmaceutically compatible adjuvants that enhance the mucosal immune response can optionally be included as part of the composition. In some embodiments, mucosal adjuvants are used; examples include mutant Cholera toxin (CT), pertussis toxin (PT), IL-lα and IL-I β (Staats and Ennis, Jr., 1999. J. Immunol. 162:6141-6147, IL-12 (Baca-Estrada et al., 1999. J. Interf. Cyt. Res. 19(5):455-462, and heat-labile E. coli lymphotoxin (LT) (e.g., Escherigen®, available from Berna Biotech AG, Bern, CH). See, e.g., Stevceva and Ferrari, 2005. Curr. Pharm. Des. 11(6):801-811 ; Yuki and Kiyono, Rev Med Virol. 2003. 13(5):293-310); Emulsions and sub-micron emulsions (U.S. Pat. Nos. 5,716,637 and 5,961,971); and IVX-908 (see, e.g., Plante et al., 2001. Vaccine 20:218-225, and Jones et al., 2004. Vaccine 22:3691-3697). Other adjuvants are known in the art, see, e.g.. Faria and Weiner, 2005. Immunol. Rev. 206:232-59; Lowell et al., 1997. J. Infect. Dis. 175(2):292-301; Cox and Coulter, 1997. Vaccine 15(3):248-256.

Dosage, toxicity and therapeutic efficacy

Dosage, toxicity and therapeutic efficacy of the troponin compositions can be determined by standard pharmaceutical procedures in cell cultures (e.g., of cells taken from an animal after mucosal administration of a troponin) or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅ O (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD _5O /ED ₅ o. Compositions which exhibit high therapeutic indices are preferred.

While troponin compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage and, thereby, reduce side effects.

The data obtained from the cell cultures (e.g., of cells taken from an animal after mucosal administration of a troponin) and animal studies can be used in formulating a range of dosage for use in humans. The dosage of troponin compositions lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any mucosal troponin compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from assays of cell cultures (e.g., of cells taken from an animal after mucosal administration of a troponin). A dose may be formulated in animal models to achieve a desired circulating plasma concentration of IL-IO or TGF-β, or of regulatory cells, in the range that includes the IC ₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of IL-IO or TGF-β in plasma can be measured by methods known in the art, for example, by ELISA. Levels of regulatory cells can be measured by methods known in the art, for example, by flow cytometry-based methods.

A therapeutically effective amount of a troponin may depend on the form selected, the mode of delivery, and the condition to be treated. For instance, single dose amounts in the range of approximately 0.1 to 10 mg maybe administered; in some embodiments, about 1-3, or 1-2 mgmay be administered. In some embodiments, e.g., pediatric subjects, about 1-2 mg of troponin can be administered.

In some embodiments, the troponin compositions are administered once. In some embodiments, the compositions can be administered from one or more times per day to one or more times per week; including once every other day. The troponin compositions can be administered, e.g., for one or more days, e.g., about 10 to 14 days or longer. In some embodiments, e.g., in subjects who are at risk of having a heart attack, an appropriate dosing schedule can be multiple times per week, e.g., two,

29

three, or more times per week, or once a week, once every two weeks, every three weeks, or once a month.

In some embodiments, e.g., in subjects who have already suffered an ischemic cardiac injury, administration as soon as possible after the injury is preferable, e.g., with at least a first treatment being administered within about one to two hours after the injury. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds can include a single treatment or a series of treatments.

Kits and Dosage Packs

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Kits can also be provided that include a troponin composition as an already prepared mucosal dosage form ready for administration or, alternatively, can include a troponin composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral dosage form. When the kit includes a troponin composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for mucosal administration), the kit can also optionally include a reconstituting solvent. In this case, the constituting or reconstituting solvent is combined with the active ingredient to provide a mucosal dosage form of the active ingredient, e.g., as described herein.

The dosage forms can be unit dosage forms wherein the dosage form is intended to deliver one therapeutic dose per administration, e.g., one tablet is equal to one dose.

Combination Therapies

In some embodiments, the methods described herein include combination treatments with a supplemental therapeutic modality. Generally, the supplemental therapeutic modality will be a treatment that is accepted as useful in the treatment of a cardiac ischemic disease. For example, the troponin can be co-administered with a supplemental therapeutic agent (i.e., a non-troponin-containing composition), e.g., in the same composition or a different composition. Suitable therapeutic agents include

Aspirin, e.g., 300-325 mg; Low-Molecular-Weight Heparin (LMWH); heparin; thrombolytic agents, e.g., Streptokinase, TPA (Tissue Plasminogen Activator), and Reteplase; Beta-blockers; Angiotensin converting enzyme (ACE) inhibitors; direct thrombin inhibitors, e.g., bivalirudin; intravenous nitroglycerin; and magnesium. hi order to reduce the degree of inactivation of orally administered troponin in the stomach of the treated subject, an antacid can be administered simultaneously with the immunoglobulin, which neutralizes the otherwise acidic character of the gut. Thus in some embodiments, the troponin is administered orally with an antacid, e.g., aluminum hydroxide or magnesium hydroxide such as MAALOX ^® antacid or MYLANTA ^® antacid, or an H2 blocker, such as cimetidine or ranitidine. One of skill in the art will appreciate that the dose of antiacid administered in conjunction with a troponin depends on the particular antacid used. When the antacid is MYLANTA ^® antacid in liquid form, between 15 ml and 30 ml can be administered, e.g., about 15 ml. When the cimetidine H2 blocker is used, between about 400 and 800 mg per day can be used.

Alternatively or in addition, the second therapeutic modality can be a non- pharmaceutical intervention, e.g., an invasive or non-invasive surgical intervention. Suitable interventions include, but are not limited to, angiography, angioplasty (e.g., primary angioplasty or percutanous transluminal angioplasty (PTCA)); and/or cardiac surgery (e.g., coronary artery bypass grafting (CABG)).

Cardiac Reperfusion Disease

Cardiac reperfusion disease is a leading cause of death in North America and is predicted to become more prevalent as the population ages (Scroggins, 2001. J. Infus. Nurs., 24:263-267). Ischemia and subsequent reperfusion can lead to myocardial injury through a variety of mechanisms. See, e.g., US Patent Application Publication No. 2005/0215533.

Currently prevalent therapies for ischemic heart disease are directed at the rapid restoration of blood flow to the ischemic region. However, during reperfusion the heart undergoes further damage due in large part to the generation of reactive oxygen species (ROS), e.g., superoxide anion, elevated levels of which can be detected only minutes after the reintroduction of oxygen to ischemic tissues (Bolli et al, 1995. J. Clin. Invest., 96:1066-1084). ROS have been shown to be key mediators of cellular and myocardial injury, causing lipid peroxidation and apoptosis (Siwik et

al., 1999. Circ. Res., 85:147-153; Halmosi et al., 2001. MoI. Pharmacol., 59:1497- 1505).

In general, the methods described herein are useful for the treatment and prevention of cardiac reperfusion disease in subjects who have, or are at risk for having, a cardiac reperfusion disease. Cardiac reperfusion diseases include stable or unstable angina, myocardial infarction, and surgically-induced ischemia/reperfusion disease. In general, cardiac reperfusion diseases are associated with an ischemic cardiac injury, such as those caused by or related to acute coronary artery occlusion, severe arterial stenosis, atherosclerosis, embolism, coronary spasm, and surgical procedures, e.g., cardiac transplantation, vascular surgery, e.g., angioplasty (including elective angioplasty), coronary artery bypass graft, and surgery involving cardiac bypass, or any other procedure that causes or carries a risk of ischemic injury to the heart.

Methods of diagnosing ischemic cardiac injury are known in the art and include the administration of tests such as electrocardiograms (EKG, ECG), chest X- rays, blood tests (e.g., for so-called cardiac enzymes such as creatine kinase (CK), CK-myocardial band (CK-MB), troponin and myoglobin), stress tests, cardiac catheterization and coronary angiograms.

A diagnosis of myocardial infarction can be made under the World Health Organization diagnosis guidelines (Tunstall-Pedoe et al., 1994. Circulation 90:583), or, alternatively, under the Clinical Definitions of Myocardial Infarction as Determined by the Joint European Society of Cardiology/American College of Cardiology Committee (2000. Myocardial infarction redefined: a consensus document of The Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction. J Am Coll Cardiol 36:959).

Methods for identifying subjects who are at risk of having an ischemic cardiac injury are also known. Risk factors include atherosclerosis, male gender, female gender after menopause, age, cholesterol, smoking, hypertension, diabetes, obesity, hyperlipidemia, stress, a poor diet, excess alcohol intake, use of crack cocaine, and family history of coronary artery disease and/or ischaemic heart disease, cardiac bypass surgery or coronary angioplasty, e.g., in a parent or sibling. In addition, those subjects who need or may need a surgical procedure that causes or carries a risk of

ischemic injury to the heart can be considered to be at risk of having an ischemic cardiac injury.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Cytokine Profile Following Nasal Administration of Troponin

This Example describes an assay in which troponin polypeptide was selected as a subject for tolerance.

Prior to studies in the model, the cytokine profile was investigated in female C57BL/6 mice, purchased from Jackson laboratory (BAR Harbor, ME). The mice were 8-10 weeks of age. Cytokine production was induced by mucosal administration in C57BL/6 mice nasally administrated Porcine troponin (a mixture of the three heart- specific troponins, cTnT, cTnl, and cTnC (Porcine, SIGMA)), with or without Th2 adjuvant (Emulsome™) three times every other day. As a control, the mice received bovine serum albumin (BSA) protein (Sigma) together with Emulsome™ or PBS. Two days after the last treatment, the mice were immunized with 100 μg troponin polypeptide mixed 1:1 in CFA (complete Freund's adjuvant) and splenocytes were taken ten days later for in vitro assays.

For the proliferation and cytokine assays, the spleen cells were pooled and cultured in 96- well plates at 5x10 ⁵ and 10 ⁶ cell/ml respectively in serum free medium, X-VTVO 20 (Biowhittaker, Walkersville, MD). To measure cytokines, culture supernatants were collected at 24 hours for IL-2 and IL-4, at 40 hours for IL-6, IL-10 and DFN-γ, and at 72 hours for TGF-β. Quantitative ELISAs for IL-2, IL-4, IL-6, IL- 10 and IFN-γ were performed using paired mAb specific for the corresponding cytokines, following the manufacturer's recommendations (BD Biosciences Pharmingen, San Diego, CA). TGF-β was determined as previously described (Inobe et al., 1998. Eur J Immunol 28:2780).

For proliferation assays, the cells were pulsed with thymidine at 72 hours, and radioactivity was measured 16 hours later (Inobe et al., 1998. Eur J Immunol 28:2780).

All continuous and ordinal data described herein are expressed as mean + SEM. by one way ANOVA followed with Duncan test (infarct size). Values of p fess than 0.05 were considered statistically significant.

Nasally treated mice showed a reduction in total cell proliferation as compared to control mice (Fig. IA). The optimal dose per mouse was found to be 40 μg. As one theory, the tolerance to troponin might work by decreasing of pro-inflammatosy cytokines such as INF-γ and IL-6 which play an important role in heart ischemia. A possible role of Th2 or Tr-I regulatory cells might be through an increase in secretion of the anti-inflammatory cytokine IL-IO (Fig. IB).

As shown in Figs. IA- lChttp://www.iimmunol.org.ezpl.harvard.edu/cgi/content/full/ 171/12/6549 - FL mice nasally treated with troponin showed a reduction in proliferation (IA) and secretion of the proinflammatory cytokines IFN-γ and IL-6 (1B-1C) (p < 0.03). The optimal dose per mouse was found to be 40 μg. Administration of troponin in combination with an emulsome-based mucosal adjuvant (Lowell et al., 1997, supra) resulted in a greater decrease in poliferation (IA, open bar) and a greater reduction of IL-6 (IB, open bar) as compared to troponin alone (grey bar) (ρ<0.05). There was a slight increase in the level of the anti-inflammatory cytokine IL-10 after troponin administration, but this was not statistically significant compared to control. No active TGF-β was detected.

In order to investigate whether the response to an endogenous (in this case, murine) troponin would be similar to the response seen with porcine troponin, the effect of nasal administration of murine and porcine troponins was compared. Mice were nasally treated with 40 μg of murine or porcine troponin or BSA on days 1, 3, and 5. On day 7, they were immunized with porcine troponin in CFA. Ten days after immunization, spleens were removed and stimulated in vitro with 5 or 50 μg of porcine Troponin. As shown in Fig. ID-F, mice nasally treated with porcine or murine troponin showed a similar reduction in proliferation (ID) and secretion of the proinflammatory cytokines IFN-γ and IL-6 (IE-F) (p < 0.05). These results indicate that the effect obtained with porcine troponin results from a specific immune response that protects against inflammation the endogenous troponin.

Example 2. Mucosal Tolerance to Troponin Reduces Tissue Damage after MVR Surgery

To investigate whether nasal administration of troponin affected tissue damage after ischemia insult, C57B16 mice were randomly divided into two groups (n = 12 per group) and were administered either troponin or BSA (control) nasally 3 times over a week. Two days after the last treatment, the animals were anesthetized wifl. 2% isoflurane in 70% N ₂ and balanced O ₂ by a facemask, and heart infarcts were produced by 1 hour of MI, followed by reperfusion.

Briefly, the proximal LAD was identified and a 8.0 suture (Ethicon) was placed around the artery and surrounding myocardium. Regional left ventricular ischemia was induced for 1 hour by ligation of the left ascending coronary artery (LAD). At the end of the ischemia period, the ligature was loosened and reperfusion was achieved. The incision was closed and the animals were allowed to recover (see, e.g., MeIo et al., 2002. Circulation 105:602-607). hi all experiments animals underwent surgery, were scored, and the infarct volume measured in blinded fashion.

Morphometric determination of infarct size was performed as follows. Twenty-four hours after reperfusion, the LAD was re-ligated and 0.3 to 0.4 mL of 1% Evans Blue in PBS (pH 7.4) was retrogradely injected into the heart to delineate the nonischemic area. The heart was excised and rinsed in ice-cold PBS. Five to six biventricular sections of similar thickness (about 2 mm) were made perpendicular to the long axis of the heart and incubated in 1% triphenyl tetrazolium chloride (TTC, Sigma Chemicals) in PBS (pH 7.4) for 15 minutes at 37°C and photographed on both sides. Area at risk (AAR) and infarct area were delineated by means of an image analysis system (M4; Imaging Research, St. Catherine's, Ontario, Canada) and calculated using the formula: percentage of infarct per heart LV volume. AAR was calculated as the left ventricular area excluding Evans Blue dye after ligation of the LAD. Infarct area was calculated as the risk area that becomes necrotic as distinguished by TTC staining or Masson Trichrome staining. The cumulative areas of ischemic infarct for all sections for each heart were used for comparisons. Infarct size was expressed as the ratio of infarct area to AAR (Pachori et al., 2004. Proc. Natl. Acad. Sci. U.S.A. 101:12282-12287; MeIo et al., 2002. Circulation 105:602-607; and Nwogu et al., 2001. Circulation 104:2216-2221).

As is shown in Figs. 2A and B, nasal administration of troponin before MWR injury reduced ischemic infarct size by 50 % at 24 hours following the MI/R surgery (from 12.4 ± 2% to 6.3 ± 1.4%, p < 0.05). There was no difference between the level of the ischemic infarct size when troponin was given with or without Th2 adjuvant To investigate the functional impact of mucosal tolerance, the level of reactivity of splenocyte cells to troponin was measured 24 hours after the surgery. The level of troponin is known to correlate with the level of myocardial disease in vivo both in mice and humans (Chu et al., 2002. Wise. Med. J. 101:40-48). As shown in Fig.3, there was a significant reduction in proliferation against troponin, in nasal troponin treated animals mice vs. controls (5,658 ± 282 CPM vs. 1,386 ± 84 CPM , p<0.03)- Following nasal troponin the level of proliferation against troponin was similar to basal levels (842 ± 43 CPM). The level of proliferation against troponin, which is; indicative of immune reactivity against troponin, in treated mice is similar to the basal level.

These data demonstrate that the administration of troponin during MI/R induction can modulate the immune response to this heart antigen. This modulation of the immune response includes a relative increase in troponin-specific regulatory T-cells with anti-inflammatory properties (i.e., increased IL-10, decreased IFN-γ and IL-6). This is demonstrated by the significantly higher levels of proliferation in the control group vs. troponin treated groups as shown in Fig. 3, and a decrease of proinflammatory cytokine, IL-6, IFN-γ and TNF-α as shown in Figs. 6A-6D. In other models of ischemia, IFN-γ parallels iNOS activity (24), which can be associated with enhanced tissue damage after ischemic injury. This modulation correlated directly with a reduction in infarct size (Figs. 2A and 2B).

These data demonstrate that nasal administration of troponin prior to MI/R injury modulates the cellular immune response to this cardiac antigen, an effect which was associated with a reduction in infarct size (Figs. 2A and 2B). Furthermore, these data support the use of reactivity against troponin as an indicator of inflammation following MI/R.there is a significant reduction of proliferation against troponin, which indicates a reduction in the pro-inflammatory response against troponin in the treated mice vs. control (p<0.03).

Example 3. Mucosal Administration of Troponin Improves Survival and Heart Function after MI/R Surgery

To investigate the long-term consequence of troponin administration on the myocardium following FR injury, troponin was administered nasally 3 times over a weeklong period as described above. Control animals received PBS. Each group contained 10 animals. Two days after the last treatment, ischemia was induced in the PBS and troponin-treated animals by one hour of MI/R using a8-0 nylon filament as described herein. In sham animals, the chest was opened as in the treated animals, but no ischemia was induced. Echocardiographic analyses were then performed at six weeks after injury to assess heart damage and function.

Animals underwent echocardiography 6 weeks after the last I/R injury as described (Pachori et al., 2004. supra ). Mice were anesthetized with isofluorane for echocardiographic examinations. Mouse heart rates ranged from about 600 to 650 beats/min during echocardiography. Echocardiography was performed using a 6- to 15 -MHz Ultraband Intraoperative Linear Array probe and a Sonos 5500 ultrasound imaging system (Philips Ultrasound, Andover, Massachusetts). Images were obtained from both the parasternal short and long axes, saved to cine-loop utilizing native frame rates of >60 Hz, and analyzed utilizing an off-line analysis program. Endocardial borders were traced and end-systolic and end-diastolic areas and volumes were calculated. Echocardiographic acquisition and analyses were performed by a technician who was blinded to treatment groups.

Fractional shortening was measured from M-mode images and was measured from the whole myocardium. The M-mode images from which the measurements were taken are shown in Fig. 4C. Number of animals in each group were as follows: Sham: n=6, PBS, n=5, Troponin: n=6.

The results are presented in Table 1.

Table 1. Echocardiographic data

^* vs. sham (p < 0.05); * vs. PBS, p<.05. LVDd: Left ventricular diameter (dystole); PWT: posterior wall thickness; TVSW: intraventricular septal wall thickness; EF: Ejection Fraction; mVcfc: mean velocity of fractional shortening corrected for heart rate; HR: heart rate

During the six weeks, four mice died in the control group, whereas only one died in the treated group. In terms of heart damage, the mice that were subject to MI/R surgery showed severe wall thinning, as demonstrated by reduced posterior wall thickness (PWT) and intraventricular septal wall thickness (IVSW). In terms of cardiac function, reduced ejection fraction (ES%) and reduced mean velocity of fractional shortening (mVcfc) was seen. However, mice that were treated nasally with troponin showed significant improvement in these parameters, and these parameters were similar to that measured in sham animals. These results indicate a indicating nearly complete prevention of wall damage to the myocardium (Table 1 ^* ).

These results were further confirmed by histopathology. Morphological analyses by hematoxylin/eosin and Masson trichrome staining (performed as described herein) demonstrated that troponin treated animals had significantly reduced fibroses and collagen deposition as determined by Masson's trichrome staining. Exemplary results are shown in Figs. 4A; normal myocardium is dark gray (red in original), whereas fibrosed tissue appears light gray (blue in original). The data, summarized in Fig. 4B, showed nasal administration of troponin reduced ischemic infarct size by 78% at six weeks following the MI/R injury (from 30 ± 4.5% to 9.6 ± 3.5%, p < 0.005).

Therefore, nasal troponin protects from structural and functional myocardial damage.

Example 4. Immunohistochemistry of the Heart Following Mucosal Treatment

Surrounding the area of lethally damaged core of the infarct is an area of ischemic damage with partially preserved energy metabolism (Dikow et al., 2004. J. Am. Soc. Nephrol. 15(6): 1530-6). Over time, in the absence of any treatment, this area generally progresses to infarction due to ongoing excitotoxicity, post ischemic inflammation and apoptosis (Lopez-Neblina et al., 2005. J. Invest. Surg. 18:335- 350). Thus, a prime goal of therapy is to protect the myocardium from ischemia induced inflammation (Frangogiarmis et al., 2000. J Immunol 165:2798). To

determine the effect of mucosal administration of troponin, immunohistochemical analysis of cytokines was performed.

Histology was performed on animals sacrificed before, 24 hours after, or 72 hours after MVR. surgery, LV sections (following the process of fixation, the LV was cut into 7 μm sections for staining) from mice before and after surgery were fixed in 4% paraformaldehyde overnight (O/N) followed by 4.5% sucrose for 4 hours, then in 20% sucrose O/N at 4°C. The sections were then frozen in the presence of OCT (Optimal Cutting Temperature freezing glue) and stored at -7O ⁰ C until ready to be used. The staining included immunological markers for T-cells (CD4), macrophages and neutrophils (CDlIb), plus pro-inflammatory (IFN-γ) and anti-inflammatory (IL- 10 and TGF-β cytokines) as previously described (Maron et al., 2002. Circulation 106: 1708- 1715). Sections were evaluated in a blinded manner, and controls included the use of isotype-matched niAbs. Pre-absorption of anti-cytokine antibodies with the respective relevant or irrelevant cytokines (5 μg/ml, 16 hours, 4 ⁰ C) either blocked or left unchanged the results of antibody staining, respectively (Khoury et al., 1992. Journal of Experimental Medicine 176:1355).

At 24 hours the pro-inflammatory cytokine IFN-γ was reduced within the penumbra in mice receiving nasal therapy, compared to the control group. There was a decrease in the number of CD4+ cells expressing IFN-γ in the nasal troponin group (from 55 + 7% to 17 + 3% , p < 0.01) and an increase in the percentage of CD4+ cells expressing intracellular IL-10 was increased in the nasal troponin group (from 17 ± 2% to 78 + 8%, p < 0.002); see Figs. 5C-D. No difference in expression of TGF-β in the CD4+ cells was observed. Thus, animals nasally vaccinated with troponin had enhanced expression of the anti-inflammatory cytokine IL-10 and reduced expression of the pro-inflammatory cytokine TFN-γ in the ischemic region. These data suggest a role for T cells and IL-10 in the reduction of ischemic infarct size following mucosal therapy.

CD4+ and CDlIb+ cells, and cells expressing TFN-γ and IL-10, were also quantified at 24 hours. Following MI/R-surgery, a significant reduction in macrophage-type cells (CDlIb+) was detected by immunostaining of heart sections at 24 hours after surgery (from 389 + 36 to 115 + 12, p < 0.002; see Figs. 6A-B). As one theory, not meant to be limiting, these cells may act to enhance the destructive effect of infiltrating CD4+ T-cells.

RT-PCR was carried out essentially as described in Zhang et al., Molecular Vision 11 : 887-895 (2005). Briefly, total RNA was extracted from the LV using an RNeasy kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using Applied Biosystems kit. The primers and probes for the TaqMan RT- RT-PCR reactions were: Mm00443258_ml (TNFα), Mm00518984_ml (IL-23), Mm00434165_ml (IL-12), Mm00446190_ml (IL-6); performed accordingto the manufacturer's directions using an Applied Biosystems PRISM 7700 thermal cycler. The mouse actin gene, a housekeeping gene, was used to normalize each sample and each gene. The data was consistent with results from RT-PCR analysis from the infarct region following the surgery.

24 hours after injury, the nasal troponin treated group showed a significant reduction in the pro-inflammatory cytokines IL-6 and TNF-α (Figs. 6C-D, p<0.05) that was associated with the reduction in macrophages (CDl lb+ ) in the nasal troponin treated group (Fig. 3B). Taken together these data demonstrate that troponin treated animals had significantly reduced fibrosis and normalized cardiac function as compared to control animals and thus nasal troponin protects against structural and functional myocardial damage.

Example 5. No Effect of Mucosal Administration of Troponin on Tissue Damage in IL-10 ^'7' Mice

The role of IL-10 as mediator of the ischemic infarct was established by investigation of IL-10 ^"7" mice. IL-10 is an immunoregulatory cytokine that can modulate immune processes, inhibiting the expression of inflammatory ThI type responses as well as affecting antigen-presenting cell function (Weiner, 2001. Nat Immunol. 2:671-672). IL-10 has been shown to reduce inflammation in a variety of animal models including experimental autoimmune encephalomyelitis (EAE) (Bettelli et al., 1998. Journal Of Immunology 161:3299), atherosclerosis (Maron et al., 2000. FASEB J. 14(6):A1199), and stroke model in the CNS (Frenkel et al., 2003. J Immunol 171 :6549). IL-10 may deactivate macrophage like cells and thus limit their involvement in a secondary inflammatory process. Furthermore, IL-10 limits the role of glutamate cytotoxicity by inactivation of NF-kappaB (Bachis et al., 2001. J Neurosci 21 :3104), a transcription factor that modulates inflammation and key regulatory proteins in cerebral ischemia (Schneider et al., 1999. Nat Med 5:554).

Moreover, IL-IO targets the interface between the heart and periphery by preventing adhesion and extravasion of leukocytes.

To investigate the role of IL-IO in reduction of infarct size following nasal troponin, 8-10 week old female IL-10 ^"7" mice (obtained from Jackson Laboratories, Bar Harbor, ME) were subjected to MI/R surgery. The experiments were performed as described above.

As shown in Figs. 7A-C, there was no significant reduction in ischemic infarct volume (7B) or proliferation to troponin (7C) following nasal tolerization with troponin as compared to vehicle treatment in IL-10 ^" ' ^" mice. Consistent with these findings was an increase in in vitro production of BFN-γ in response to troponin in nasally treated IL-10 ^" ' ^" animals.

These results demonstrate a crucial role for IL-10 in the reduction of ischemic infarct size by nasal troponin. Furthermore, the results demonstrate a linkage between protection against myocardial injury by nasal troponin and reduction in the cellular immune response to troponin that occurs in association with MI/R injury.

Example 6. Mucosal Administration of Troponin after MI/R Surgery Reduces Tissue Damage

To investigate whether nasal troponin affects tissue damage if administered after ischemia insult, troponin was administered nasally 1 hour after MI/R surgery. Control animals received BSA given nasally. Two separate experiments were performed.

As shown in Fig. 8 A, nasal administration of troponin reduced ischemic infarct size by 50% at 24 hours following the MI/R surgery (from 15.1 ± 1% to 9.1 ± 0.7%, p < 0.01). hi Experiment I, infarct size was significantly reduced (p < 0.004) in mice that received nasal troponin (n = 7) as compared to mice that received PBS (n = 4). In Experiment II, infarct size was significantly reduced (p < 0.05) in mice that received nasal troponin (n=6) as compared to mice that received PBS (n=5).

To investigate whether the reduction in infarct size was associated with reduction in splenocyte proliferation, proliferation to troponin was measured. As shown in Fig. 8B, there was significant reduction of proliferation against troponin in the treated mice vs. control (6,330 ± 430 CPM vs. 2,929 ± 120 CPM , pO.OOl). The level of proliferation against troponin in treated mice is similar to the basal level.

Furthermore, there was an elevation of regulatory CD4+ CD25+ cells in the spleens of animals that received troponin vs. those that received BSA.

RT-PCR was also carried out using splenocytes obtained 24 hours after surgery. RT-PCR was carried out essentially as described in Zhang et al., 2005. Molecular Vision 11:887-895. Briefly, total RNA was extracted from the splenocyte using an RNeasy kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using Applied Biosystems kit. The primers and probes for the TaqMan RT- RT-PCR reactions were: Mm00443258_ml (TNF-α), Mm00434165jml (IL-12); performed according to the manufacturer's directions using an Applied Biosystems PRISM 7700 thermal cycler. The mouse actin gene, a housekeeping gene, was used to normalize each sample and each gene.

Splenocytes from the nasal troponin treated group showed a significant (p<0.02) reduction in pro-inflammatory cytokines IL-12 and TNF-α (Fig. 8C) that correlated directly with the observed reduction in splenocyte proliferation (Fig. 8B). These results demonstrate a correlation between the reduction in the peripheral immune response to troponin with the reduction of ischemic infarct size (Fig. 8C).

The level of pro-inflammatory cytokines in the heart was then examined when animals were treated with nasal troponin 1 hour after MI/R injury. RT-PCR for EL-6 and TNF-α from the left ventricular infarct region 24 hours following surgery. The treated group showed a significant reduction in pro-inflammatory cytokines IL-6 and TNF-α (p<0.05)(Fig. 8D) that correlated with the reduction in ischemic infarct size and reduction in the peripheral immune response to troponin.

To further investigate the effect of nasal troponin, iramunohistochemical analysis of cytokines TFN-γ and IL-IO was performed. CD4+ cells expressing IFN-γ and IL-10 were quantified at 24 hours after MI/R injury (Figs. 8E-G). There was a significant decrease in the number of CD4+ cells expressing IFN-γ in the nasal troponin group (from 76 ± 8% to 15 ± 6% , p < 0.01) and an increased in the percentage of CD4+ cells expressing intracellular IL-10 was increased in the nasal troponin group (from 14 ± 2% to 74 ± 7%, p < 0.02). Thus, animals nasally vaccinated with troponin had enhanced expression of the anti-inflammatory cytokine IL-10 and reduced expression of the pro-inflammatory cytokine IFN-γ in the ischemic region.

These results further support a role of reactivity against troponin as an indicator of myocardial damage following ischemic cardiac injury. Nasal troponin administration resulted in a modulation of the immune response following such an injury, as evidenced by a decrease in proliferation to troponin and a reduction in infarct size.

Example 7: Adoptive Transfer of CD4+ T-cells from Mice Nasally Treated with Troponin to Untreated Mice Reduces Heart Ischemic Infarct Size When Given 1 Hour after MJJR Injury.

Mucosal antigen induces tolerance by a number of mechanisms including anergy, deletion and active cellular regulation. To investigate the role of CD4+ cells in reduction of infarct size following nasal troponin, adoptive transfer experiments were performed. C57BL/6 mice and IL-IO-/- mice were tolerized with troponin polypeptide through nasal administration as described above.

To obtain sufficient cells for adoptive transfer, two days after the last nasal treatment, mice were immunized subcutaneously with 100 μg of troponin polypeptide in CFA and draining lymph node and spleen cells were taken ten days later and stimulated in vitro with 40 μg/ml troponin polypeptide.

Mice were nasally treated with 40 μg of troponin on days 1,3,5. On day 7 they were immunized with troponin in CFA. Ten days after immunization, both lymph nodes and spleens were removed and stimulated in vitro with 40 μg of troponin in 24 well plates (1 ml in each well containing 5x10 ⁶ cells) in T-cell medium buffer (Bettelli et al., 1998. Journal Of Immunology 161:3299) for two days. On the third day, cells were split into two wells and incubated for one more day with IL-2. On day 4, the cells were harvested and CD4+ T-cells purified by negative selection using a mouse CD4+ T-cell column (R&D Systems Inc., Minneapolis, MN, Cat. No. MCD43). In brief, leukocyte suspensions are incubated with a mixture of monoclonal antibodies and then loaded onto T Cell Subset Columns. B cells, non-selected T-cells and monocytes bind to glass beads coated with antiimmunoglobulin via both F(ab) and Fc interactions. The resulting column eluate contains a highly enriched T cell subset population with virtually no B cells, monocytes, or non-selected T cells. Figs. 9A-B show the results of using FACS analysis to monitor recovery of CD3+/CD4+ cells from total CD3+/CD4+ cells. When loaded at 17.1%, the purity of recovered cells was 90.8% with no detectable CD3+/CD8+ cells.

10 ⁶ CD4+T-cells were suspended in a solution 0.2 ml DMEM TV and injected into the recipient mice. The mice were subjected to MUR surgery one hour before adoptive transfer of cells. For controls, cells from animals immunized with CFA were treated in an identical fashion.

After 4 days of culture in vitro, CD4+ T-cells were purified by negative selection and adoptively transferred to untreated mice that had been subjected to MI/R surgery one hour before the CD4+ T cell transfer. Adoptive transfer of CD4+ T-cells taken from nasal BSA treated mice that were immunized with CFA served as a control group.

As shown in Figs. 9C, heart infarct size was reduced by 72% (from 18 ± 6% to 5 ± 1.2%, p < 0.05) in animals that received CD4+ T cells from C57BL/6 (wild type) mice nasally treated with troponin, as compared to animals that received CD4+ T cells from BSA-treated mice. Immune responses to troponin following adoptive transfer were also tested. To establish that IL-IO was also crucial in these adoptive transfer experiments, CD4+ T cells were adoptively transferred from nasally treated EL- 10-/- animals. As shown in Fig. 9D, no reduction of ischemic infarct size was observed when CD4+ T cells from nasal troponin-treated IL-10-/- animals were transferred. Thus, nasal troponin reduces ischemic size via IL-10-dependent CD4+ T cells. As shown in Fig. 9E, there was a significant reduction of proliferation against troponin in the mice that received CD4+ T-cells from nasal troponin treated animals vs. controls (3,912 ± 340 CPM BSA vs. 1,693 ± 120 CPM control, p<0.03). The level of proliferation against troponin in treated mice was similar to the basal level proliferation.

These results demonstrate that nasal troponin induces troponin specific antiinflammatory CD4+ T-cells that function to protect the myocardium following MI/R injury, and like nasal troponin given 1 hour after infarct, these cells are effective given 1 hour after MI/R injury.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.