WO2006026304A1 - Cardioprotective formulations and uses thereof - Google Patents

Abstract

The invention relates to methods and pharmaceutical compositions relating to tissue preservation, which are particularly valuable as cardioprotective modalities.

Description

CARDIOPROTECTIVE FORMULATIONS AND USES THEREOF

Inventors: Jose Rafael Lopez Padrino (Attorney Docket No.: ELMOOIO-PCT)

Field of the Invention

[0001] This invention relates to live tissue preservation and more specifically, to the reduction of cell injury such as ischemic injury e.g., during cardiac surgery.

BACKGROUND OF THE INVENTION

[0002] Considerable efforts have been directed towards the development of methodologies for the preservation of myocardial cardiovascular tissues critical to the improvement of successful cardiovascular surgery. Myocardial protection plays a pivotal role in open-heart surgery, including revascularization (e.g., in coronary artery surgery), and repairs (e.g., in aortic and mitral valves replacement) as well as heart transplantation.

[0003] Ischemia and/or hypothermia during cardiovascular surgery may lead to abnormalities of tissue volume regulation, lack of high-energy substrate availability, reduced capacity for post-ischemic oxidative metabolism, depressed availability of high energy phosphate precursors, and potential damage by oxygen-induced free radical-mediated oxidant injury (Wechsler and Abd-Elfattah, Card. Surg. 5:251-255, 1990). In addition, myocardial protective strategies are geared to prevent reperfusion injury upon resolution of the coronary occlusion and the ultimate release of the aortic cross-clamp.

[0004] The literature is replete with strategies for avoiding or minimizing iatrogenic injury induced by cardiopulmonary bypass or by surgically imposed ischemia (see e.g., Takaba and Inhoue, Ann. Thorac. Cardiovasc. Surg. 6:3-8, 2000). The infusion of cardioplegic solutions through the heart tissues to stop electrical and mechanical activities for sufficiently long periods of time while preserving myocardial integrity has been the subject of much investigation. Ideally, the effects of such solutions should be totally and rapidly reversible, so that the heart resumes substantially normal functional activity once the cardioplegic solution has been replaced by blood. Acting as a selective perfusion agent, cardioplegic solutions can alter or inhibit ischemic injury by virtue of hypothermia and asystole. Vinten-Johansen and Thourani describe the use of cardioplegia to prevent reperfusion injury (by altering delivery parameters and the composition of the solution) using various adjunctive agents and pharmacological therapies for which cardioplegia solutions serve as a delivery means (Extra Corpor. Technol. 32:8-48, 2000).

[0005] In commonly used cardioplegic solutions, electromechanical arrest of the heart is achieved by depolarization of the cell membrane by the high concentration of potassium. Unfortunately, depolarization has also been implicated in the ischemic injury associated with presently used solutions (e.g., "the St. Thomas' Hospital cardioplegic solution") resulting in significant deterioration of myocardial function upon reperfusion, often exacerbating undesired post-operative conditions (see, e.g., Cleveland et al., Ann. Thorac. Surg. 61:760- 768, 1996).

[0006] Membrane depolarization has been implicated in the impaired intracellular

Ca²⁺ homeostasis observed in myocardial cells exposed to hyperkalemic challenge (Lopez et al, J. Thorac. Cardiovasc. Surg. 112:820-831, 1996; Lopez et al. , Am. J. Physiol. 270:H1384-H1389, 1996; Jovanovi et al. Eur. J. Pharmacol. 298:63-69, 1996; Jovanovi et al. Ann. Thorac Surg. 63:153-161, 1997; Jovanovi etal., Ann. Thorac. Surg. 64:588-589, 1997). K⁺-induced Ca²⁺ loading is undesirable as it can impair the contraction and relaxation of a cell, perturb normal membrane excitation, and induce abnormal gene expression (Tani, Ann. Rev. Physiol. 52:543-559, 1990; Kloner et al, J. Card. Surg. 9:397-402, 1994; McKenney et al., J. Am. Coll. Cardiol. 24:1189-1194, 1994; Jovanovi et al., Ann. Thorac. Surg. 65:586- 591, 1994).

[0007] Persistently high levels of Ca²⁺ within the cytosol have been found to lead to irreversible cellular hypercontracture, and ultimately cell death, due to the inability of the cross-bridge cycling to relax (Tani, Ann. Rev. Physiol. 52:543-559, 1990). Cell shortening is one accepted indicator of the level of cellular injury associated with hyperkalemic induced elevation of cytosolic Ca²⁺ concentration.

[0008] Altered contractile function may be expressed either in terms of the interval wherein the myocyte remains contracted or in terms of the physical length over which the myocyte may contract. Muscle cell contraction is an energy dependent mechanism subject to interference on a number of levels. One regulating mechanism for contraction is myocyte Ca²⁺-dependent, actomyosin Mg²⁺-dependent ATPase which consumes ATP during cross- bridge cycling with actin during contraction. Dysfunction under this mechanism due to a loss of energy reserves is important when one realizes that in the rat heart, for example, it is estimated that cross bridge cycling can consume 80% of the ATP produced (Ebus, et al, J. Physiol. (Lond.) 492:675-87 1996).

[0009] Postulated mechanisms whereby myocyte protection may be achieved include increasing the availability of cellular ATP (Murry et al, Cir. Res. 66:913-31, 1990, PyIe et al., Am. J. Physiol. Heart Circ. Physiol. 279:H1941-48, 2000), opening of ATP sensitive K⁺ channels (Gross et al, Cir. Res. 70:223-33 1992), or closure of L-type Ca²⁺ channels, thereby reducing Ca²⁺ influx. (Reimer, et al, Ann. NY Acad. Sci. 723:99-115 1994).

[0010] Not surprisingly, investigators have sought to identify cardioprotective moieties capable of preventing or countering K⁺-induced Ca ⁺ loading to reduce the risk of cellular damage inherent to ischemia and hypothermia. Studies aimed at improving the cardioprotective quality of cardioplegic solutions have investigated cardioplegic solutions containing either L-carnosine (Gercken et al , Arzneimittelforschung 30:2140-2143, 1980; Alabovskii et al, BMl. Ekps. Biol. Med. 127:290-294, 1999) or L-carnitine (Nakagawa et al, Thorac. Cardiovasc. Stø-g 42:85-89, 1984; Nemoto et al, Ann. Thorac. Sw. 71:254-259, 2001). Despite some initial optimism due to the observation of some enhanced cardioprotective qualities (e.g., reduction of calcium loading), cardioplegic solutions containing either L-carnosine or L-carnitine did not reduce the Ca²⁺ concentration to a level sufficient to reduce the extent and/or prevent cell damage.

[0011] Therefore, there remains a dire need for novel approaches to cardioplegia that overcome the limitations and disadvantages of the compositions and methodologies available to practitioners at the present time. Ideally, such approaches should have enhanced cardioprotective qualities to reduce myocyte cell damage.

BRIEF SUMMARY OF THE INVENTION

[0012] The inventors have discovered that a desirable cardioprotective effect can be obtained by the administration of (a) a moiety capable of maintaining intracellular pH and acting as an antioxidant, as well as (b) a moiety capable of enhancing intracellular energy levels. The compositions and methods taught herein when used in conjunction with cardioplegia can inhibit K⁺-induced Ca²⁺ loading and reduce cellular damage as measured indirectly by cell shortening. [0013] Thus, the present invention provides a method for reducing cell injury comprising providing therapeutically effective concentrations of (a) a moiety capable of maintaining intracellular pH and acting as an antioxidant (hereinafter, the "stabilizer"), as well as (b) a moiety capable of enhancing cellular energy levels (hereinafter, the "enhancer"). In one embodiment, myocyte injury can be reduced by the method. In another embodiment, surgery associated injury can be reduced by providing the stabilizer and the enhancer. In a specific embodiment, the present invention provides a method for reducing myocardial injury during ischemic cardiovascular surgery (e.g., open-heart surgery and heart transplantation).

[0014] The present invention also sets forth novel compositions for reducing cell injury (e.g., myocyte injury), comprising a stabilizer and an enhancer that, when used in conjunction with the administration of cardioplegic solutions, can significantly diminish the intracellular concentration of Ca²⁺ that otherwise occurs in cells/tissues exposed to high concentrations of K⁺. In one embodiment, compositions for reducing myocardial injury during cardiovascular surgery (e.g., open-heart surgery and heart transplantation) are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will become more fully understood from the detailed description given below. The accompanying figures are given by way of illustration only, and thus are not limiting of the present invention.

[0016] Figure 1 is a diagrammatic representation depicting the intracellular concentration of Ca²⁺ in isolated cardiomyocytes in the absence (Tyrode) and presence of 16 mM K . The mean cytosolic concentration of Ca in control cardiomyocytes (Tyrode) was 108 ± 1.41 nM, (n=18). Exposure to K⁺-challenge significantly increased [Ca²⁺] to 2,064 ± 160 nM, (n=18) 0?<0.001).

[0017] Figure 2 is a diagrammatic representation depicting the intracellular concentration of Ca²⁺ in control myocytes (Tyrode), in myocytes that were exposed to a hyperkalemic solution (K⁺), and in myocytes that were exposed to a hyperkalemic solution containing L-carnosine. L-carnosine diminished the K⁺-induced Ca²⁺-loading from 2,064 ± 160 nM to 1,486 ± 43 nM, (n=15) (p< 0.001). [0018] Figure 3 is a representation of fluorescent tracings showing that the effect of L- carnosine on K⁺-induced Ca²⁺ loading is reversible. The tracings represent the relative fluorescence of myocytes measured first in the presence of a solution containing 16 mM K⁺ (A), then in a solution containing 16 mM K⁺ and 0.5 mM L-carnosine (B), and last in the Tyrode solution containing 16 mM KCl.

[0019] Figure 4 is a diagrammatic representation depicting the effect of L-carnitine on

K⁺-induced Ca²⁺ loading. The intracellular concentration of Ca²⁺ was measured in control myocytes (Tyrode), in myocytes that had been exposed to a Tyrode solution containing 16 mM K⁺ (K⁺), and in myocytes that had been exposed to a Tyrode solution containing 1 mM L-carnitine. L-carnitine diminished the K⁺-induced Ca²⁺ loading from 2,064 ± 160 nM to 1,234 ± 40 nM, (n=16) (pO.OOl).

[0020] Figure 5 is a representation of fluorescent tracings showing that the effect of L- carnitine on K⁺-induced Ca²⁺ loading is reversible. The tracings represent the relative fluorescence emitted from control myocytes which were exposed first to Tyrode solution (Tyrode), then to Tyrode solution containing 16 mM KCl (K⁺), and last to Tyrode solution containing 16 mM KCl and 1 mM L-carnitine.

[0021] Figure 6 is a diagrammatic representation showing the mean cytosolic concentration of Ca ⁺ in myocytes in the absence (Tyrode), and in the presence of L-carnosine + L-carnitine (Mixture).

[0022] Figure 7 is a diagrammatic representation depicting the effect of L-carnosine plus L-carnitine on K⁺-induced Ca²⁺ loading. The mean cytosolic concentration of Ca²⁺ was measured in control myocytes (Tyrode), in myocytes that were exposed to a hyperkalemic Tyrode solution (K⁺), and in myocytes that were exposed to a hyperkalemic Tyrode solution containing L-carnosine + L-carnitine (Mixture).

[0023] Figure 8 is a representation of fluorescent tracings showing that the effect of L- carnosine plus L-carnitine of K⁺-induced Ca ⁺ loading is reversible. The tracings represent the relative fluorescence of myocytes measured first in the presence of a hyperkalemic Tyrode solution (A), second, in the presence of the hyperkalemic solution of (A) containing L- carnosine plus L-carnitine (B), and last in the presence of Tyrode solution (C) as in (A). [0024] Figure 9 is a diagrammatic representation depicting the intracellular Ca²⁺ concentration of cardiomyocytes that are exposed to Tyrode solution (Tyrode), or to Tyrode solution containing L-carnosine and L-carnitine (Mixture).

DETAILED DESCRIPTION OF THE INVENTION

[0025] The patents and scientific literature referred to herein establish the knowledge of those with skill in the art and are. hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art- understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

[0026] Methods and pharmaceutical compositions of matter are disclosed relating to live tissue preservation, which are particularly valuable as cardioprotective modalities. The inventors have unexpectedly discovered that the administration of both a stabilizer and an enhancer in conjunction with the administration of a cardioplegic solution, significantly inhibited K⁺-induced Ca²⁺ loading which is postulated to be responsible for cell damage following cardioplegia.

[0027] The invention identifies moieties, that when used in combination, have been found to reduce tissue damage associated with surgery, e.g., for cardiovascular repair and tissue transplantation. Thus, the compositions and methods described herein are useful to reduce ischemic damage such as the damage observed as a result of hyperkalemic cardioplegia.

[0028] Without wishing to be bound to a specific mechanism, the inventors have postulated that cardioplegia-induced myocyte injury is intrinsically related to calcium loading, intracellular pH imbalance/oxidation, and intracellular energy level depletion. It is important to note that while this hypothesis may provide the mechanisms of action that underlie the invention, other teachings are contrary. However, the mechanism(s) responsible for the surprising findings discussed herein are not known, but this does not impede one of skill in the art from practicing the invention described. [0029] In view of the above, the present invention provides a method to reduce surgery-associated tissue damage by contacting the tissue with a stabilizer and an enhancer.

[0030] "Reduce" and any permutation of the verb means to prevent the occurrence of, lessen or inhibit the intensity of, ameliorate, or completely reverse surgery-associated tissue damage as compared to untreated tissue.

[0031] In some embodiments, the tissue damage to be reduced is myocyte tissue damage, such as surgery-associated myocyte tissue damage.

[0032] For the purposes of this invention, "myocyte tissue" denotes any tissue containing a myocyte cell such as, for example, arteries, or cardiac tissue. For the purposes of the present invention, the terms "damage" and "injury" are used interchangeably.

[0033] The term "surgery-associated myocyte tissue damage" is used herein to mean deviation from myocyte tissue homeostasis occurring during cardio/vascular surgery or transplant surgery, including, but not limited to iatrogenic injury induced by cardiopulmonary bypass itself or by surgically imposed ischemia. Ischemia and/or hypothermia induced damage include abnormalities of tissue volume regulation, lack of high-energy substrate availability, reduced capacity for post-ischemic oxidative metabolism, depressed availability of high energy phosphate precursors, and the potential damage done by oxygen-induced free- radical-mediated oxidant injury (see Wechsler and Abd-Elfattah, supra).

[0034] The goals of myocyte protection and specifically myocardial protection during cardiac surgery are not only to facilitate the operation by providing a quiet, bloodless field, thereby facilitating the precision of the operation, but also to avoid surgery-associate myocyte tissue injury or damage. In addition, myocardial protective strategies are geared to prevent reperfusion injury upon resolution of the coronary occlusion and ultimate release of the aortic cross-clamp.

[0035] The invention is particularly useful in those instances where for the purposes of cardio/vascular surgery or transplant surgery myocyte tissues are contacted with a cardioplegic solution during cardioplegia.

[0036] The term "cardioplegia" herein refers to a technique of myocardial preservation during cardiac surgery, usually employing infusion of a cold, potassium-laced solution ("cardioplegia solution"), sometimes mixed with blood, to achieve arrest of the myocardial fibers and reduce their oxygen consumption to nearly nothing. Techniques using warm (body temperature) blood are also used. The term "cardioplegic solution" herein refers to solutions used for myocardial preservation that vary in temperature and properties. Some of the solutions are used to chemically arrest the heart; others help prevent cardiac damage due to edema (swelling of tissue), loss of metabolites, and improper acid-base balance. A number of cardioplegic solutions are known and can be used in the present invention (see, e.g., Hearse et al., Cardioplegia, NY, Raven, 1981; En Gelman et al., A Textbook for Cardioplegia for Difficult Clinical Problems, Mr. Kosco, NY, Furata, 1992; Diasco et al., J. Thorac. Cardiovasc. Surg. 100: 910-913, 1990; Ascione et al., Eur. J. Cardio-thor. Surg. 21; 440-446, 2002; Mauney et si., Am Thorac. Surg. 60: 819-823, 1995).

[0037] One type of cardioplegic solution is a hyperkalemic cardioplegic solution. As used herein, the term "hyperkalemic" encompasses that concentration of potassium ions (K⁺) capable of myocyte membrane depolarization resulting in Ca²⁺ influx induction, and in the case of cardiomyocytes, cardiac arrhythmias and cardiac arrest.

[0038] Hyperkalemic cardioplegic solutions commonly used during cardio/vascular surgery or transplant surgery have been found to cause myocyte tissue damage. The damage has been ascribed to an increase in the intracellular Ca²⁺ concentration which hampers several cellular functions including cell contraction. Cell contraction is known to be a Ca²⁺- dependent, actomyosin Mg²⁺-dependent ATPase event which consumes ATP during cross- bridge cycling with actin during contraction. The increase in myoplasmic calcium concentration compromises the production of ATP at the mitochondria, resulting in a reduction in intracellular concentration of ATP. At the same time there is an increase in the use of ATP due to the high myoplasmic calcium concentration (the majority of intracellular calcium regulatory mechanism are ATP dependent). In parallel fashion, the increase in myoplasmic calcium elicits acidification of the intracellular medium and production of reactive oxygen species, both inducing further release of intracellular calcium.

[0039] The potential for injury is reduced by contacting the myocyte tissue susceptible to damage with therapeutically effective concentrations of (a) stabilizer, as well as (b) an enhancer. "Contacting" means providing exogenously either by in vivo administration to a patient (e.g., IV in the traditional surgery setting), or by direct contact with the tissue to be protected when used in an ex vivo setting. [0040] As used herein, the term "patient" shall refer to any mammal which may experience the benefits of the invention. Although the description focuses on applications for human use, a mammal also includes animals, and the invention is therefore useful for veterinary purposes.

[0041] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of pharmacology include, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10^th Ed., McGraw Hill Companies Inc., New York (2001). Standard reference works setting forth definitions for medical terms include, e.g., Stedman's Medical Dictionary. 26^l Ed., William & Wilkins, Baltimore, MD (1995). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention, some examples of which are described herein. Materials, reagents, and the like to which reference is made in the present description and examples are obtainable from commercial sources, unless otherwise noted.

[0042] As used in this specification, the singular forms "a", "an," and "the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

[0043] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open- ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0044] The term "stabilizer" refers to a moiety capable of maintaining intracellular pH. Stabilizers according to the present invention can also be antioxidants. Non-limiting representative examples of stabilizers according to the present invention include L-carnosine. [0045] "L-carnosine" denotes the dipepetide carnosine, (β-alanyl-L-histidine) which occurs in cardiac muscle at concentrations between 2 and 10 niM (Chan et al. , Lipids 29:461-466, 1994; Flancbaum et al , Agents Actions 31:190-196, 1990; Crush, Comp. Biochem. Physiol. 34:3-30, 1970). Tissue carnosine levels are decreased in animals with chronic infection (Fitzpatrick et al, Proc. Soc. Exp. Biol. Med. 161:404-408, 1980), following trauma (Fisher et al, Proc. Soc. Exp. Biol. Med. 158:402-405, 1978), pathologic states associated with impaired cardiac contractility (Parillo et al., Ann. Int. Med. 113:227-242, 1990). Carnosine (β-alanyl-L-histidine) and some functional equivalents thereof (such as the derivatives: homocarnosine, acetylcarnosine, acetylhomocarnosine, etc.) have been known for some time to be among the most important natural antioxidant agents (Boldyrev et al, Adv. Em. Reg., 30:175-194 , 1990; Kohen et al, Proc. Natl Acad. ScI USA, 85:3175-79, 1988; Yoshikawa et al, Biochim. Biophys. Acta, 1115:15-22, 1991).

[0046] The term "enhancer" refers to a compound capable of modulating fatty acid oxidation and thus increasing cellular ATP levels. Enhancers according to the invention may optionally modulate mitochondrial CoA/acyl-CoA ratio. Non-limiting representative examples of enhancers according to the present invention include L-carnitine.

[0047] The term "carnitine" refers to the naturally occurring amino acid that is the requisite carrier of long-chain fatty acids across the mitochondrial membrane where they undergo β-oxidation (Guertl et al, Int. J. Exp. Pathol. 81:349-372, 2000). Carnitine also modulates the intramitochondrial CoA/acyl-CoA ratio. Oxidation of long-chain fatty acids is by far the most important aerobic source of adenosine triphosphate in the mammalian heart, and adequate myocardial levels of L-carnitine are essential for normal energy production (Guertl et al, Int. J. Exp. Pathol. 81:349-372, 2000; Calvani et al, Basic Res. Cardiol 95:75- 83, 2000; Bremer, Physiol Rev. 63:1420-1480, 1983). Thus, in the absence of carnitine, β - oxidation ceases, glycogen is depleted, tryacylglycerols accumulate, and organ dysfunction results (Guertl et al, Int. J. Exp. Pathol. 81:349-372, 2000; Calvani et al, Basic Res. Cardiol. 95:75-83, 2000; Bremer, Physiol Rev. 63:1420-1480, 1983). Multiple clinical and experimental studies have documented that carnitine levels are associated with infracted myocardium (Guertl et al, Int. J. Exp. Pathol. 81:349-372, 2000; Calvani et al, Basic Res. Cardiol. 95:75-83, 2000; Bremer, Physiol. Rev. 63:1420-1480, 1983; McFalls et al, Life ScL 38:497-505, 1986), and dilated cardiomyopathy (Guertl et al, Int. J. Exp. Pathol. 81:349-372, 2000; Calvani et al, Basic Res. Cardiol 95:75-83, 2000; Bremer, Physiol Rev. 63:1420- 1480, 1983: McFalls et al., Life Sci. 38:497-505, 1986). Increasing myocardial carnitine concentration by treatment with L-carnitine has been shown to improve a number of symptoms in patients (Powell et ah, Biochem. Biophys. Res. Commun. 122:1012-1020, 1984; Fabiato, Gen. Physiol. 85:291-320, 1985).

[0048] The terms "L-carnosine" and "L-carnitine" as used herein include functional equivalents (i.e., a biochemical moiety that possesses a biological activity, either functional or structural, that is substantially similar to the biological activity of the entity of which it is said to be a functional equivalent). Functional equivalents include naturally occurring as well as synthetic or semi-synthetic derivatives, including modifications such as covalently linked carbohydrates, and additional functions such as, for example, moieties not normally part of the molecule to which it is a functional equivalent. Such functional equivalents may improve stability, absorption, biological half life, pharmacokinetic absorption, adsorption, and the like. Functional equivalents may alternatively decrease the toxicity, eliminate or attenuate any undesirable side effects. From the foregoing, those skilled in the art will recognize that enhancers and stabilizers can include pharmaceutically acceptable salts thereof (see Remington's Pharmaceutical Sciences, 18^th Ed., Gennaro, Mack Publishing Co., Easton, PA 1990).

[0049] The combination of a stabilizer and enhancer can diminish the accumulation of intracellular Ca²⁺ in myocytes to a level that is lower than that attained by supplying either a stabilizer or an enhancer individually. Moreover, the inventors have discovered that the K⁺- induced Ca²⁺ loading can be inhibited resulting in intracellular Ca²⁺ concentrations below the critical levels correlating with cell injury. Hence, in certain embodiments, the administration of exogenous L-carnosine and L-carnitine in a hyperkalemic cardioplegic solution can diminish the accumulation of intracellular Ca²⁺ in myocytes to a level lower than that attained by solutions including either L-carnosine or L-carnitine alone.

[0050] As used herein, "therapeutically effective amount" denotes treatments at dosages and for periods of time effective to reduce surgery-associated myocyte tissue damage. Reduction of damage may be detected by the restoration (either in full or in part) of myocyte tissue homeostasis, or in the case of patients, by an improvement of the symptomology described above. A variety of parameters are available to evaluate restoration (either in full or in part) of myocyte tissue homeostasis both qualitatively and quantitatively

(e.g., reduced intracellular calcium levels, intracellular pH buffering, ATP maintenance, cell viability, contractile function, etc.). In patients, the practitioner may directly ascertain the cardioprotection by measurement of the ejection volume and heart frequency and indirectly by determination of cardiac enzymes (e.g., CK, CK-mb enzymes) that denote cardiac tissue damage.

[0051] Examples of therapeutically effective amounts of enhancer include (a) 0.001,

0.002, 0.003, 0.004, 0.005, 0.01, 0,02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, to 1.0 mM; (b) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, to 1.0 niM; (c) 0.5, 0.6, 0.7, 0.8, 0.9, to 1.0 mM and (d) 0.5 mM.

[0052] Examples of therapeutically effective amounts of stabilizer include (a) 0.3, 0.4,

0.5, 0.6, 0.7, 0.8, 0.9, to 1.0 mM; (b) 0.5, 0.6, 0.7, 0.8, 0.9, to 1.0 mM; (c); 0.8, 0.9, to 1.0 mM; and, (d) 1.O mM.

[0053] One of skill in the field will appreciate that the use of the stabilizer and enhancer according to the invention may be supplied prior to, at the same time as, or after the administration of, the cardioplegic solution. Administration at the same time includes administration substantially simultaneously even if not contemporaneous in the strictest sense.

[0054] L-carnosine and L-carnitine in combination can be administered prophylactically to reduce myocyte tissue damage prior to administering cardioplegia to a patient. Alternatively, L-carnosine and L-carnitine can be administered to a patient in the cardioplegic solution in combination. As an example of the present invention, L-carnosine and/or L-carnitine preparations can also be administered before and after surgery to extend the cardioprotective effect.

[0055] In circulatory arrest cases, for example, a single dose of 20-30 mL/kg of cardioplegic solution is usually administered upon aortic cross-clamping, and no maintenance cardioplegia is given thereafter unless the circulatory arrest time exceeds 50-60 minutes. The mixture of L-carnosine and L-carnitine could be added either to a modified St Thomas solution (NaCl HOmM, KCl 1OmM, MgCl₂ 10Mm, CaCl₂, 1.2 mM, NaHCO₃ adjusted to pH 7.4, or to oxygenated blood which is cooled and diluted with the high K+ solution.

[0056] Preparations of the stabilizer, the enhancer, and cardioplegic preparations, alone, or in any combinations, can be administered to a patient by conventional methodologies known in the field. The timing of administration may be either before, during, or after surgery or a combination thereof. As an example, the mixture can be administered directly into the coronary ostia (antegrade)(see, e.g., Baretti et al., Thorac. Cardiovasc. Surg. 50: 25-30, 2002).

[0057] An example of the present invention is the combination of a stabilizer (e.g., L- carnosine) and an enhancer (e.g., L-carnitine) as an adjunct to crystalloid and blood cardioplegia, wherein the cardioplegia may be warm or cold.

[0058] Myocardial protection depends on homogeneous distribution of cardioplegic solution to all regions of the heart to minimize the potential risk of post ischemic myocardial dysfunction and damage. Typically, the routes of administration of cardioplegic solutions are antegrade or retrograde infusions.

[0059] The term "antegrade infusion" refers to the technique for infusing cardioplegic solution via normal circulatory pathways during cardiopulmonary bypass. The solution is infused through a catheter placed near the aortic cross clamp and goes directly into the coronary arteries. The term "retrograde infusion" herein refers to the technique for infusing cardioplegic solution, whereby a catheter is inserted through the right atrial wall, and the solution is infused through the coronary sinus and coronary veins. Both warm and cold cardioplegia may be given either antegrade or retrograde. The infusion of cardioplegic solutions may be intermittent or continuous.

[0060] The present invention also provides methods for administering cardioplegic solutions containing a stabilizer (e.g., L-carnosine) and an enhancer (e.g., L-carnitine) via retrograde and antegrade routes of infusion, wherein the infusion may be continuous or intermittent.

[0061] The method or the present invention may be used in conjunction with any myocardial protective strategies that are designed to provide continuity of the operation, to avoid unnecessary ischemia and cardioplegic overdose, to allow for aortic clamping as soon as cardiopulmonary bypass is started, to permit aortic unclamping and discontinuation of bypass shortly after the technical procedure is completed, and/or to minimize the ration of ischemia and cardiopulmonary bypass (see e.g., Buckberg, Ann. Thorac. Surg. 60:805-814, 1995). [0062] The protective properties of the methods described herein are useful for heart transplantation purposes. The success of heart transplantation depends upon satisfactory function of the new heart after implantation in the recipient. One factor contributing to a successful transplant is heart preservation while it is ex vivo, i.e., while it is being transported from the donor to the recipient. It is rare, indeed, that a donor and recipient will be at the same medical facility. Therefore, the ability to preserve donor hearts while transporting them long distances is crucial to successful heart transplantation.

[0063] Another aspect of the present invention features compositions with protective properties comprising an enhancer and a stabilizer. Useful pharmaceutical solutions are cardioprotective solutions, e.g., cardioplegic solutions, e.g., hyperkalemia cardioplegic solutions further comprising an enhancer and a stabilizer. The meaning of the terms used is identical as for the first aspect of the invention.

[0064] An example of the cardioplegic composition of the present invention comprises a stabilizer (e.g., L-carnosine) and an enhancer (e.g., L-carnitine). Such compositions can be useful to reduce surgery-associated myocyte tissue damage. Compositions like this can be useful in a variety of surgical settings as discussed above.

[0065] Compositions of the present invention are optionally formulated in a

"pharmaceutically acceptable vehicle" with any of the well known pharmaceutically acceptable carriers, including diluents and excipients (see Remington's Pharmaceutical Sciences, 18^th Ed., Gennaro, Mack Publishing Co., Easton, PA 1990 and Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type of pharmaceutically acceptable carrier/vehicle employed in generating the compositions of the invention will vary depending upon the mode of administration of the composition to a mammal, generally "pharmaceutically acceptable carriers" are physiologically inert and non¬ toxic. Compositions according to the invention may contain more than one type of stabilizer or enhancer, as well any other pharmacologically active ingredient.

[0066] The compositions of the present invention may be provided in a pharmaceutically acceptable vehicle using formulation methods known to those of ordinary skill in the art. The compositions of the invention can be administered by standard routes (e.g., intravenous) and by administration directly into the coronary ostia (antegrade) (see, Baretti et al., Thorac Cardiovasc. Surg. 50:25-30, 2002) routes. The compositions of the invention include those suitable for intravenous injection. In addition, polymers may be added according to standard methodologies in the art for sustained release of a given element.

[0067] Stabilizers and enhancers may be further combined in a composition with a pharmaceutically acceptable carrier, such as saline solution and water (e.g., 4:1), and delivered via any methods known to those skilled in the art. They may also be diluted in blood withdrawn from the same patient (e.g., 4:1).

[0068] The formulations of the compositions of the invention may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques as discussed above. Such techniques include the step of bringing into association the compositions of the invention and the pharmaceutically acceptable carrier(s), such as a diluent or an excipient.

[0069] Suitable liquid compositions of the present invention comprise the active ingredient in an "aqueous pharmaceutically acceptable vehicle," such as, for example, isotonic saline, bacteriostatic water, and other types of vehicles that are well known in the art.

[0070] Compositions suitable for parenteral administration include aqueous and non¬ aqueous sterile injection solutions which may contain conventional pharmaceutical antioxidants, stabilizers, buffers, bacteriostats, and solutes, which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0071] The following examples are intended to further illustrate certain embodiments of the invention, and are not intended to limit the scope of the invention. It is expected that certain changes or modifications to the invention herein described may be effected by those skilled in the art without departing from the true spirit and scope thereof as set forth in the claims and the accompanying specification. EXAMPLES

[0072] The present non-limiting examples are provided to illustrate the use of a representative stabilizer (L-carnosine) and a representative enhancer (L-carnitine) in diminishing the accumulation of intracellular Ca²⁺ in myocytes according to the invention.

Example 1

[0073] Isolation of Single Ventricular Cardiomyocytes.

[0074] A preparation of single ventricular myocytes, devoid of vascular and neuronal elements, was made from rat hearts as follows. Adult rats (Instituto Venezulano de Investigaciones Cientificas (IVIC)) were anesthetized with pentobarbital, and the ventricular cardiomyocytes were isolated according to methods known in the art. Hearts were removed and perfused at 37 ⁰C with Medium 199 (Sigma, St. Louis, MO), Ca²⁺-ethylene glycol-B/S (β-aminoethyl ether) N,N,N',N',tetraacetic acid-buffered low Ca²⁺ medium containing pronase E (8 mg/100 mL), proteinase K (1.7 mg/100 mL), bovine albumin (0.1 g/100 rnL), and 200 μM CaCl₂. The ventricles were cut into fragments, and single cells were isolated by gently stirring the tissue in a solution containing pronase E, proteinase K, and collagenase (5 mg/10 mL).

Example 2

[0075] Measurement of Intracellular [Ca²⁺] .

[0076] Relaxed, rod-shaped cardiomyocytes having clear striations and a smooth surface were loaded with the esterified form of the fluorescent probe Fura 2- AM (3 μM) that had been dissolved in dimethylsulfoxide (Molecular Probes, OR, USA) at 37⁰C for 30 minutes. Once the loading procedure was completed, the bathing solution was changed several times to remove the extracellular Fura 2-AM. The cardiomyocytes loaded with the fluorescent probe were transferred into a temperature-regulated chamber (37⁰C) mounted on the stage of an inverted epifluorescence microscope (Attoflor Ratio Vision, Atto Instruments, Rockville, MD, USA, or Nikon, model Eclipse TE300, Nikon Corporation, Tokyo, Japan). Cardiomyocytes were locally perfused with Tyrode solution at 37°C. Excitation ultraviolet light wavelengths (340 nm and 380 nm) were selected using interference filters (Omega Optical, Vermont, USA) and a dichroic mirror, and the emitted light was filtered at 510 nm. Fluorescent signals obtained at 340 and 380 were measures every 200 msec with a photomultiplier tube and the data stored in a personal computer for data processing and analysis.

[0077] An estimate of the diastolic Ca²⁺ concentration, as a function of Fura 2-AM fluorescence, was calculated according to the equation:

[Ca²⁺] = K_d[F-F_min/F_max-F]

where [Ca²⁺] is the resting cytosolic Ca²⁺, K_d is the dissociation constant of Fura- AM-Ca ⁺ complex, and F is the intensity of the fluorescence. The saturating fluorescence of the dye F_max was obtained by lysing the cells with digitonin (70 μg/mL); the fluorescence level for the dye in the absence of Ca²⁺ (F_mi_n) was obtained by adding excess of EGTAA with sufficient TRIS to shift the pH to 8, allowing the EGTA to chelate Ca²⁺ more efficiently. A Kd of 224 nM was used for Fura-2AM (see e.g., Tsien, et al., J. Cell. Biol. 94:325-334, 1982).

[0078] The fluorescent probe Fura-2AM is available from Molecular Probes

(Molecular Probes, OR, USA); L-carnosine and L-carnitine are available from Sigma Chemical Company (St. Louis, MO, USA). All other reagents used in the experiments are available from SIGMA Chemical Company (St. Louis, MO, USA).

Example 3

[0079] L-Carnitine Plus L-Carnosine Protect Cadiomyocytes Against Ca²⁺ Overload

Induced By Hyperkalemia.

[0080] Results are expressed as mean ± standard error; n refers to the number of experiments. Significant differences were determined using the Student T-test. Ap value less than 0.05 was considered significant.

[0081] Single ventricular cardiomyocytes were isolated from rat hearts, loaded with a

Ca²⁺ sensitive fluorescent probe, and imaged by digital epifluorescent microscopy as described supra. The emitted fluorescence of the probe, a measure of the intracellular Ca²⁺ concentration, was recorded from single myocytes during hyperkalemic challenges in the absence and presence of L-carnosine and L-carnitine, alone or in combination to assess the protective effectiveness of this agent. [0082] Single cardiomyocytes were prepared as described above, and bathed at 37°C in Tyrode solution containing 136.5 mM NaCl, 1.8 niM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES-NaOH, pH 7.4. A K⁺ challenge was induced by adding 16 mM KCl (Jovanovi et al, Ann. Thorac. Surg. 64:588-589, 1997).

[0083] To assess the cardioprotective effect of L-carnitine or L-carnosine, alone or in combination, the concentration of intracellular calcium was determined in separate experiments wherein the isolated myocytes were first loaded with Fura-2AM, then exposed to Tyrode solution, Tyrode solution containing 16 mM KCl, Tyrode solution containing 16 mM KCl and 0.5mM L-carnosine, Tyrode solution containing 16 mM KCl and 1 mM L-carnitine, or Tyrode solution containing 16mM KCl, 0.5 mM L-carnosine and 1 mM L-carnitine.

[0084] The effect of hyperkalemic challenge on the concentration of cytosolic Ca²⁺ is shown in Figure 1. The Ca²⁺ concentration in myocytes that had been exposed to Tyrode solution alone (Tyrode) was determined to be 108 ± 1.41 nM (n=18). Exposing cardiomyocytes to Tyrode solution containing 16 mM KCl (K⁺) significantly increased the intracellular Ca²⁺ concentration to 2,064 ± 160 nM (n=18)O<0.001). The increase in intracellular Ca²⁺ was accompanied by cell shortening.

[0085] Cell shortening is a normal response of the contractile machinery in response to an elevation of intracellular calcium concentration (above 1 μM). Thus, every time that the calcium released from the sarcoplasmic reticulum achieves an intracellular concentration higher than 1 μM, the inhibitory effect of troponin C is blocked and muscle fiber is able to generate either force (isometric condition) or shortening (isotonic condition). Both cases are the result of an interaction between myosin and actin molecules (contractile protein). Here cell shortening is used as an independent parameter to infer intracellular calcium concentration. Thus, any drug that may exert a cardioprotective effect should link to intracellular calcium levels and prevent cell shortening. This appears to be the case when the cells are exposed to the hyperkalemic solution in the presence of the mixture.

[0086] The effect of L-carnosine on the increase in intracellular Ca²⁺ caused by K⁺ is shown in Figure 2. Exposing cardiomyocytes to Tyrode solution containing 16 mM KCl and 0.5 mM L-carnosine, reduced the increase in intracellular Ca²⁺ that had resulted from the hyperkalemic challenge from 2,064 ± 160 nM (K⁺) to 1,486 ± 43 nM (L-carnosine + K⁺) (n=15) (pO.001). The presence of L-carnosine did not prevent myocyte shortening, possibly because the intracellular concentration of Ca²⁺ exceeded the contracture threshold, which is about 1000 nM.

[0087] The effect of L-carnosine in decreasing the intracellular level of Ca²⁺ induced by the hyperkalemic challenge was reversible (Figure 3). The relative fluorescence emitted from the cardiomyocytes in the presence of K⁺ (A) was diminished by L-carnosine (B). The reversibility of the effect of L-carnosine is shown in (C) when the cardiomyocytes were re- exposed to Tyrode solution containing only K .

[0088] The effect of L-carnitine on the increase in intracellular Ca²⁺ induced by K⁺ is shown in Figure 4. Exposing cardiomyocytes to Tyrode solution containing 16 niM KCl and 1 niM L-carnitine reduced the increase in intracellular concentration of Ca²⁺ from 2,064 ± 160 nM (K⁺) to 1,234 ± 40 nM (L-carnitine + K⁺), (n=16) (pO.OOl). L-carnitine, like L- carnosine, did not prevent cardiomyocyte cell shortening.

[0089] L-carnitine, like L-carnosine, reversibly affects the K⁺-induced rise in intracellular Ca²⁺ (Figure 5). The relative fluorescence emitted from the cardiomyocytes in the presence of K⁺ (A) was diminished by L-carnitine (B). The reversibility of the effect of L-carnitine is shown in (C) when the cardiomyocytes were re-exposed to Tyrode solution containing only K⁺.

[0090] The resting intracellular Ca²⁺ concentration (109 ± 1.14 nM ,(n=18) (pO.OOl)) was not affected by L-carnosine in combination with L-carnitine (Figure 6). Exposing cardiomyocytes to Tyrode solution containing 16 mM KCl, 0.5 mM L-carnosine and 1 mM L-carnitine, decreased the K⁺-induced increase in intracellular Ca²⁺ from 2,064 ± 160 nM to 440 ± 16 nM, (n=15) (pO.OOl).

[0091] The results show that the combination of a stabilizer (e.g., L-carnitine) and an enhancer (e.g., L-carnosine) decreased the K⁺-induced rise in intracellular Ca²⁺ to a level that is lower than that attained by L-carnosine or L-carnitine separately. More specifically, the results indicate that cardioprotective effects of L-carnosine and L-carnitine are at least additive. The combination of L-carnosine and L-carnitine prevented myocyte shortening because the intracellular lever of Ca²⁺ was below the threshold concentration required for contraction. [0092] The present findings indicate that use of cardioplegic solutions comprising a stabilizer (e.g., L-carnitine) and an enhancer (e.g., L-carnosine), provide a greater cardioprotective effect than that of cardioplegic solutions that contain only one of the two compounds.

[0093] In this disclosure there is described only some embodiments of the present invention and but a few examples of its versatility. It is to be understood that the invention is capable of use in various other combinations and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein.

Claims

What we claim is:

1. A method for providing cardioprotective effects, comprising: administering to a patient in need thereof (a) a therapeutically effective amount of a stabilizer, (b) a therapeutically effective amount of an enhancer, and (c) a cardioplegic solution, wherein:

the stabilizer is a moiety capable of maintaining intracellular pH and acting as an antioxidant and the enhancer is a moiety capable of enhancing intracellular energy levels.

2. The method of claim 1 , wherein the cardioprotective effect is reducing surgery- associated injury to myocyte tissue.

3. The method of claim 2, wherein the administering comprises contacting the myocyte tissue.

4. The method of claim 3, wherein the stabilizer is L-carnitine and the enhancer is L-carnosine.

5. The method of claim 4, wherein the therapeutically effective amount of L- carnitine is a concentration of 0.001-1.0 mM.

6. The method of claim 4, wherein the therapeutically effective amount of L- carnosine is a concentration of 0.3-1.0 mM.

7. The method of claim 4, wherein the therapeutically effective amount of L- carnitine is a concentration of 0.001-1.0 mM and the therapeutically effective amount of L-carnosine is a concentration of 0.3-1.0 mM.

8. The method of claim 7, wherein the therapeutically effective amount of L- carnitine is a concentration of 0.5 mM and the therapeutically effective amount of L-carnosine is a concentration of 1.0 mM.

9. The method of claim 3, wherein the cardioplegic solution is a hyperkalemic cardioplegic solution.

10. The method of claim 3, wherein the cardioplegic solution comprises the stabilizer and the enhancer.

11. The method of claim 10, wherein the stabilizer is L-carnitine and the enhancer is L- carnosine.

12. The method of claim 11 , wherein the therapeutically effective amount of L- carnitine is a concentration of 0.001-1.0 mM.

13. The method of claim 11 , wherein the therapeutically effective amount of L- carnosine is a concentration of 0.3-1.0 mM.

14. The method of claim 11 , wherein the therapeutically effective amount of L- carnitine is a concentration of 0.001-1.0 mM and the therapeutically effective amount of L-carnosine is a concentration of 0.3-1.0 mM.

15. The method of claim 14, wherein the therapeutically effective amount of L- carnitine is a concentration of 0.5 mM and the therapeutically effective amount of L- carnosine is a concentration of 1.0 mM.

16. The method of claim 3, wherein the contacting is before surgery.

17. The method of claim 16, further comprising: contacting the myocyte tissue after surgery with (a) a therapeutically effective amount of the stabilizer, (b) a therapeutically effective amount of the enhancer, and (c) a cardioplegic solution.

18. A composition for providing cardioprotective effects, comprising: (a) a stabilizer and (b) an enhancer, wherein:

the stabilizer is a moiety capable of maintaining intracellular pH and acting as an antioxidant and the enhancer is a moiety capable of enhancing intracellular energy levels.

19. The composition of claim 18, wherein the stabilizer is L-carnitine and the enhancer is L-carnosine.

20. The composition of claim 19, wherein the composition is a cardioplegic solution.

21. The composition of claim 20, wherein the cardioplegic solution is a hyperkalemic cardioplegic solution.

22. The composition of claim 20, wherein the concentration of L-carnitine is in the solution is 0.001-1.O mM.

23. The composition of claim 20, wherein concentration of L-carnosine in the solution is 0.3 mM-1.0 mM.

24. The composition of claim 20, wherein the concentration of L-carnitine in the solution is 0.001-1.0 mM and the concentration of L-carnosine in the solution is 0.3- LO mM.

25. The composition of claim 24, wherein the concentration of L-carnitine in the solution is 0.5 mM and the concentration of L-carnosine in the solution is 1.0 mM.