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WO2006026304A1 - Formulations cardioprotectrices et utilisations correspondantes - Google Patents

Formulations cardioprotectrices et utilisations correspondantes Download PDF

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Publication number
WO2006026304A1
WO2006026304A1 PCT/US2005/030128 US2005030128W WO2006026304A1 WO 2006026304 A1 WO2006026304 A1 WO 2006026304A1 US 2005030128 W US2005030128 W US 2005030128W WO 2006026304 A1 WO2006026304 A1 WO 2006026304A1
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Prior art keywords
carnitine
carnosine
concentration
therapeutically effective
effective amount
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José Rafael LOPEZ PADRINO
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Ivax LLC
Laboratorios Elmor SA
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Ivax LLC
Laboratorios Elmor SA
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine

Definitions

  • 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.
  • 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.
  • revascularization e.g., in coronary artery surgery
  • repairs e.g., in aortic and mitral valves replacement
  • 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).
  • myocardial protective strategies are geared to prevent reperfusion injury upon resolution of the coronary occlusion and the ultimate release of the aortic cross-clamp.
  • 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).
  • K + -induced Ca 2+ 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).
  • 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 Ca 2+ -dependent, actomyosin Mg 2+ -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).
  • 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 Ca 2+ channels, thereby reducing Ca 2+ influx. (Reimer, et al, Ann. NY Acad. Sci. 723:99-115 1994).
  • 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 , Arzneiffenforschung 30:2140-2143, 1980; Alabovskii et al, BMl. Ekps. Biol. Med. 127:290-294, 1999) or L-carnitine (Nakagawa et al, Thorac. Cardiovasc. St ⁇ -g 42:85-89, 1984; Nemoto et al, Ann.
  • cardioplegic solutions containing either L-carnosine or L-carnitine did not reduce the Ca 2+ concentration to a level sufficient to reduce the extent and/or prevent cell damage.
  • 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").
  • myocyte injury can be reduced by the method.
  • surgery associated injury can be reduced by providing the stabilizer and the enhancer.
  • the present invention provides a method for reducing myocardial injury during ischemic cardiovascular surgery (e.g., open-heart surgery and heart transplantation).
  • 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 Ca 2+ that otherwise occurs in cells/tissues exposed to high concentrations of K + .
  • compositions for reducing myocardial injury during cardiovascular surgery e.g., open-heart surgery and heart transplantation are provided.
  • Figure 1 is a diagrammatic representation depicting the intracellular concentration of Ca 2+ in isolated cardiomyocytes in the absence (Tyrode) and presence of 16 mM K .
  • Exposure to K + -challenge significantly increased [Ca 2+ ] to 2,064 ⁇ 160 nM, (n 18) 0? ⁇ 0.001).
  • FIG. 2 is a diagrammatic representation depicting the intracellular concentration of Ca 2+ 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.
  • Figure 3 is a representation of fluorescent tracings showing that the effect of L- carnosine on K + -induced Ca 2+ 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.
  • Figure 4 is a diagrammatic representation depicting the effect of L-carnitine on
  • Figure 5 is a representation of fluorescent tracings showing that the effect of L- carnitine on K + -induced Ca 2+ 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.
  • 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).
  • FIG. 7 is a diagrammatic representation depicting the effect of L-carnosine plus L-carnitine on K + -induced Ca 2+ loading.
  • the mean cytosolic concentration of Ca 2+ 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).
  • 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).
  • Figure 9 is a diagrammatic representation depicting the intracellular Ca 2+ concentration of cardiomyocytes that are exposed to Tyrode solution (Tyrode), or to Tyrode solution containing L-carnosine and L-carnitine (Mixture).
  • 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.
  • the compositions and methods described herein are useful to reduce ischemic damage such as the damage observed as a result of hyperkalemic cardioplegia.
  • 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.
  • the tissue damage to be reduced is myocyte tissue damage, such as surgery-associated myocyte tissue damage.
  • myocyte tissue denotes any tissue containing a myocyte cell such as, for example, arteries, or cardiac tissue.
  • the terms “damage” and “injury” are used interchangeably.
  • 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).
  • 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.
  • myocardial protective strategies are geared to prevent reperfusion injury upon resolution of the coronary occlusion and ultimate release of the aortic cross-clamp.
  • 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.
  • cardioplegia 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.
  • cardioplegic solution 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.
  • 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).
  • hyperkalemic cardioplegic solution is a hyperkalemic cardioplegic solution.
  • hyperkalemic encompasses that concentration of potassium ions (K + ) capable of myocyte membrane depolarization resulting in Ca 2+ influx induction, and in the case of cardiomyocytes, cardiac arrhythmias and cardiac arrest.
  • 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.
  • 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.
  • 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”.
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • 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.
  • 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.
  • L-carnosine denotes the dipepetide carnosine, ( ⁇ -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 ( ⁇ -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).
  • Enhancers 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.
  • 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 ⁇ -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.
  • 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).
  • the combination of a stabilizer and enhancer can diminish the accumulation of intracellular Ca 2+ in myocytes to a level that is lower than that attained by supplying either a stabilizer or an enhancer individually.
  • the inventors have discovered that the K + - induced Ca 2+ loading can be inhibited resulting in intracellular Ca 2+ concentrations below the critical levels correlating with cell injury.
  • the administration of exogenous L-carnosine and L-carnitine in a hyperkalemic cardioplegic solution can diminish the accumulation of intracellular Ca 2+ in myocytes to a level lower than that attained by solutions including either L-carnosine or L-carnitine alone.
  • 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
  • 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.
  • cardiac enzymes e.g., CK, CK-mb enzymes
  • Examples of therapeutically effective amounts of enhancer include (a) 0.001,
  • Examples of therapeutically effective amounts of stabilizer include (a) 0.3, 0.4,
  • 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.
  • L-carnosine and L-carnitine in combination can be administered prophylactically to reduce myocyte tissue damage prior to administering cardioplegia to a patient.
  • L-carnosine and L-carnitine can be administered to a patient in the cardioplegic solution in combination.
  • L-carnosine and/or L-carnitine preparations can also be administered before and after surgery to extend the cardioprotective effect.
  • 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, MgCl 2 10Mm, CaCl 2 , 1.2 mM, NaHCO 3 adjusted to pH 7.4, or to oxygenated blood which is cooled and diluted with the high K+ solution.
  • Preparations of the stabilizer, the enhancer, and cardioplegic preparations 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.
  • the mixture can be administered directly into the coronary ostia (antegrade)(see, e.g., Baretti et al., Thorac. Cardiovasc. Surg. 50: 25-30, 2002).
  • 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.
  • a stabilizer e.g., L- carnosine
  • an enhancer e.g., L-carnitine
  • 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.
  • the routes of administration of cardioplegic solutions are antegrade or retrograde infusions.
  • 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.
  • 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.
  • a stabilizer e.g., L-carnosine
  • an enhancer e.g., L-carnitine
  • 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).
  • 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.
  • 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.
  • 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.
  • cardioprotective solutions e.g., cardioplegic solutions, e.g., hyperkalemia cardioplegic solutions further comprising an enhancer and a stabilizer.
  • 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.
  • An example of the cardioplegic composition of the present invention comprises a stabilizer (e.g., L-carnosine) and an enhancer (e.g., L-carnitine).
  • a stabilizer e.g., L-carnosine
  • 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.
  • compositions of the present invention are optionally formulated in a
  • compositions according to the invention may contain more than one type of stabilizer or enhancer, as well any other pharmacologically active ingredient.
  • 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.
  • polymers may be added according to standard methodologies in the art for sustained release of a given element.
  • 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).
  • a pharmaceutically acceptable carrier such as saline solution and water (e.g., 4:1)
  • 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.
  • pharmaceutically acceptable carrier(s) such as a diluent or an excipient.
  • 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.
  • 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.
  • 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.
  • 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 Ca 2+ in myocytes according to the invention.
  • 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).
  • 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.
  • the fluorescent probe Fura-2AM is available from Molecular Probes
  • 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).
  • 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.
  • Cell shortening is a normal response of the contractile machinery in response to an elevation of intracellular calcium concentration (above 1 ⁇ M).
  • intracellular calcium concentration above 1 ⁇ M
  • 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).
  • cell shortening is used as an independent parameter to infer intracellular calcium concentration.
  • 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.
  • L-carnitine like L-carnosine, reversibly affects the K + -induced rise in intracellular Ca 2+ (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 + .
  • 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 Ca 2+ 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 Ca 2+ was below the threshold concentration required for contraction.
  • 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.
  • a stabilizer e.g., L-carnitine
  • an enhancer e.g., L-carnosine

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  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des procédés et des compositions pharmaceutiques se rapportant à la conservation des tissus, qui présentent un grand intérêt comme modalités cardioprotectrices.
PCT/US2005/030128 2004-08-25 2005-08-24 Formulations cardioprotectrices et utilisations correspondantes Ceased WO2006026304A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0060565A2 (fr) * 1981-03-18 1982-09-22 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Milieu de culture de cellules entièrement synthétique
WO1996036348A1 (fr) * 1995-05-19 1996-11-21 Farmila-Farmaceutici Milano S.R.L. Compositions pharmaceutiques et/ou dietetiques possedant une activite antioxydante et contenant de la carnosine ou ses derives et des acides amines ramifies
US20020182196A1 (en) * 2001-04-19 2002-12-05 Mccleary Edward Larry Composition and method for normalizing impaired or deteriorating neurological function
WO2002102149A1 (fr) * 2001-06-14 2002-12-27 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. Solution de conservation et de perfusion d'organes en attente d'etre transplantes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0060565A2 (fr) * 1981-03-18 1982-09-22 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Milieu de culture de cellules entièrement synthétique
WO1996036348A1 (fr) * 1995-05-19 1996-11-21 Farmila-Farmaceutici Milano S.R.L. Compositions pharmaceutiques et/ou dietetiques possedant une activite antioxydante et contenant de la carnosine ou ses derives et des acides amines ramifies
US20020182196A1 (en) * 2001-04-19 2002-12-05 Mccleary Edward Larry Composition and method for normalizing impaired or deteriorating neurological function
WO2002102149A1 (fr) * 2001-06-14 2002-12-27 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. Solution de conservation et de perfusion d'organes en attente d'etre transplantes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GERCKEN G ET AL: "MYOCARDIAL PROTECTION BY HISTIDINE AND CARNOSINE BUFFERED CARDIOPLEGIC SOLUTIONS", PFLUEGERS ARCHIV, SPRINGER VERLAG, BERLIN, DE, vol. 391, no. SUPPL 1, 1981, pages R02, XP009057336, ISSN: 0031-6768 *
NAKAGAWA T ET AL: "THE EFFECT OF L-CARNITINE ON MYOCARDIAL PROTECTION IN COLD CARDIOPLEGIA FOLLOWED BY REPERFUSION", THORACIC AND CARDIOVASCULAR SURGEON, THIEME MED. PUB., NEW YORK, NY,, US, vol. 42, no. 2, April 1994 (1994-04-01), pages 85 - 89, XP001145968, ISSN: 0171-6425 *
PREUSSE C J ET AL: "ISCHEMIA TOLERANCE AND POSTISCHEMIC RECOVERY OF THE HEART AFTER USE OF CARNOSINE AND HISTIDINE BUFFERED CARDIOPLEGIC SOLUTIONS", PFLUEGERS ARCHIV, SPRINGER VERLAG, BERLIN, DE, vol. 389, no. SUPPL, 1981, pages R01, XP009057335, ISSN: 0031-6768 *

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