USE OF AMINO ACIDS FOR THE TREATMENT AND PROPHYLAXIS OF PATHOGENIC EFFECT CAUSED BY INTRACELLULAR CALCIUM OVERLOAD

Abstract: The present invention provides a method and composition for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload comprising administering to a mammal at least one amino carboxylic acid or its physiologically acceptable salt in an amount effective to treat calcium overload. By effectively treating intracellular calcium overload, the present invention can advantageously prevent cell lysis and irreversible cell damage, especially in mammals suffering from a disease condition associated with or resulting from insufficient oxygenation.

Description: USE OF AMINO ACIDS FOR THE TREATMENT AND PROPHYLAXIS OF PATHOGENIC EFFECT CAUSED BY INTRACELLULAR CALCIUM OVERLOAD BACKGROUND OF THE INVENTION The present invention relates to a method and composition for treating and preventing pathogenic effects in mammals caused by intracellular calcium overload. Calcium overload occurs in the tissue and organs of mammals suffering from a disease condition associated with or resulting from insufficient tissue oxygenation. By treating intracellular calcium overload, the present invention also effectively prevents irreversible cell damage and cell lysis in cells transiently deprived of oxygen. Healthy cells regulate free cytosolic calcium concentrations by limiting influx of the cation across the cell's plasma membrane, sequestering free calcium, and pumping calcium ions out of the cytosol . When a cell becomes ischemic, insufficient free energy exists to operate the ion pumps. As calcium accumulates in the cytosol, degradative enzymes become activated and begin to further affect the cell's ability to regulate calcium. Calcium activated enzymes, e.g., phospholipases, break down the cell's membranes, making them even "leakier" to calcium. Additional enzymes, e.g., proteases, also attack the molecular pumps. When oxygen is restored to the tissue, free radical oxygen species are produced that can further damage these systems. If the combined effects of the enzymes and the free radical oxygen species becomes severe enough, the cell will not recover and maintain acceptable levels of calcium, even if it successfully re-energizes when circulation is reestablished. The cell has become irreversibly damaged and will ultimately die from an overload of calcium. This pathogenic sequence might be repeated millions of times in the first several hours following a transient interruption in blood supply to the heart or brain. Neuron damage, following a stroke or cardiac arrest, and myocyte damage, following coronary artery occlusion, are two examples of such cell damage. When essential cellular constituents leak out of such damaged cells, the cell is referred to as lysed and, of course, is irreparable. Various treatments have been studied for treating or preventing calcium mediated cellular damage to reduce the likelihood of cell lysis during and following a transient period of oxygen deprivation. The most common treatments involve administering chemical compounds that either limit entry of calcium ions into the cell (i.e., plasma membrane channel blockers) or antagonize the calcium activated enzymes by binding to intracellular proteins like calmodulin. These treatments, however, are not restricted to the damaged cells. They can affect the function of normal, healthy cells and cause a number of adverse side effects . More selective methods are, therefore, needed to treat or prevent calcium mediated damage in cells deprived of oxygen, while avoiding these adverse side effects. SUMMARY OF THE INVENTION It is an object of the present invention to treat or prevent the pathogenic effects in a mammal caused by intracellular calcium overload. It is another object to prevent irreversible cell damage or hypoxic or post-hypoxic lysis in a mammal that has suffered from a disease condition associated with or resulting from insufficient tissue oxygenation. It is a more specific object of the present invention to provide methods and compositions for preventing lysis of such cells and thereby reducing the extent of organ damage in a mammal which has suffered from anoxia, hypoxia or ischemia, such as that which occurs in cardiac arrest, pulmonary embolus, renal artery occlusion, coronary artery occlusion, occlusive stroke, hemorrhagic stroke, adult respiratory distress syndrome, neonatal respiratory distress syndrome, suffocation, or profound anemia. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of instrumentalities and combinations particularly pointed out in the appended claims. To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the invention comprises a method for treating or preventing pathogenic effects in mammal caused by intracellular calcium overload comprising administering to the mammal at least one amino carboxylic acid or its physiologically acceptable salt in an amount effective to treat intracellular calcium overload. The present invention also relates to a pharmaceutical composition for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload comprising an amount of an amino carboxylic acid or its physiologically acceptable salt effective to treat intracellular calcium overload together with a pharmaceutically acceptable carrier. Reference will now be made in detail to preferred embodiments of the invention. DETAILED DESCRIPTION OF THE INVENTION An aspect of the present invention contemplates treating a mammal suffering with a disease condition resulting from insufficient tissue oxygenation by administrating one or more therapeutic amino carboxylic acid agents to alleviate the toxic effects of calcium overload on hypoxic and/or post hypoxic cells, anoxic and/or postanoxic cells, and ischemic and/or postischemic cells. The therapeutic amino carboxylic acid of the present invention can treat such cells by entering their interiors. It is believed that these acids are transported into the cell in association with sodium and sodium dependent, plasma membrane transport systems. Once inside, the acids can treat the effects of calcium overload. Some of the therapeutic amino carboxylic acid agents of the present invention are selective for certain sodium dependent transport ("symport") proteins. In one embodiment, the administered therapeutic agent is a substrate for the "GLY" transport system. The GLY transport system is a sodium-dependent amino carboxylic acid transport protein widely expressed in terminally differentiated mammalian cells. See H.N. Christensen, Physiological Reviews, Volume 70, p. 43-77, 1990, hereby incorporated by reference. The preferred substrate for the GLY symport is glycine. The amino carboxylic acid substrates for the sodium dependent "A" transport system include metabolizable model substrates, alanine or proline, or non-metabolizable substrates, 2-aminoisobutyric acid or N-methyl-alpha-aminoisobutyric acid. The preferred substrate for the A symport is alanine. The amino carboxylic acid substrate for the sodium dependent "ASC" transport system is serine. Both the A and ASC transport systems are expressed in terminally differentiated mammalian cells. See, H.N. Christensen, supra. The substrate for another sodium-dependent amino carboxylic acid transport protein, the "N" transport system, which expresses specific terminally differentiated mammalian cells like hepatocyte, H.N. Christensen supra, includes amino carboxylic acids selected from histidine, asparagine, or glutamine. The preferred substrate for the N symport is histidine. Some of the amino carboxylic acids of the present invention can also stimulate the hepatic urea cycle in a mammal. This is important in situations where administration of a particular acid exceeds hyper-physiological amounts, creating potential toxicity concerns. With the hepatic cycle stimulating acids of the invention, however, one can advantageously increase dosage amounts of an amino carboxylic acid, in some cases above the hyper-physiological amount. The preferred hepatic cycle stimulating amino carboxylic acid is arginine. In accordance with another aspect of the present invention, a pharmaceutical composition is provided containing: (a) at least one substrate for the GLY transport system; (b) at least one substrate for the A transport system; (c) at least one substrate for the ASC transport system; (d) at least one substrate for the N transport system; and (e) at least one stimulator or activator of the hepatic urea cycle. The preferred composition comprises a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine. Additional therapeutically active agents can be co- administered with the amino carboxylic acids of the present invention. Examples of classes of agents which may complement the action of the therapeutic amino carboxylic acids of the present invention include: (1) a plasma volume expander, such as dextran; (2) a water soluble magnesium salt, such as magnesium chloride; (3) a thrombolytic enzyme, such as streptokinase; (4) scavengers of toxic oxygen metabolites, such as superoxide dismutase and/or catalase; (5) a xanthine oxidase inhibitor, such as allopurinol; and (6) a substance which binds free iron, such as desferrioxamine. The amino carboxylic acids contemplated for use in the present invention include the amino acids that have been established as protein constituents, especially alpha-amino acids. Amino acids can be obtained in various ways, including hydrolysis of a protein or fermentation of glucose, for example. Amino acids have also been created in the laboratory by passing an electric discharge through a mixture of ammonia, methane, and water vapor. Hawley, "The Condensed Chemical Dictionary," 10th Ed., 1981, pp. 48-49. The amino carboxylic acids of the present invention can be administered in intravenous infusions after a hypoxic, anoxic, or ischemic condition occurs to treat intracellular calcium overload and prevent irreversible cell injury and cell lysis. For example, the amino carboxylic acids of the present invention can be administered by intravenous infusion immediately after a cerebral infarction, a myocardial infarction, asphyxia, or cardiopulmonary arrest. Alternatively, the acids can be administered by intravenous or intraarterial infusion concurrently and in association with thrombolytic therapy. The therapeutic agents of the present invention can be used prophylactically in surgical settings where circulation to an organ or organ system is deliberately interrupted, e.g. coronary artery bypass surgery, tissue grafting, endarterectomy, angioplasty, etc. The present invention also contemplates adding the amino carboxylic acids to a cardioplegia solution for organ perfusion, and to perfusion and preservative solutions for organ transplantation. The acids of the present invention can be administered either individually, in mixtures with one another, or together with a physiologically suitable carrier or vehicle. If appropriate, the acids may be administered in the form of a physiologically acceptable salt, for example, an acid addition salt. A preferred carrier is 5% dextrose in water or half-strength normal saline, buffered to pH 7.4 with a physiologically acceptable buffer substance. In administering an amino carboxylic acid of the present invention, a loading dose is given at the start of treatment. Thereafter, a maintenance dose is to be administered either continuously or intermittently in order to maintain optimal levels of the acid in the blood. The timing and amount of the maintenance dose can be determined by intermittently monitoring the levels of the acid in the blood. The amount of amino carboxylic acid administered as a loading or maintenance dose will depend upon the particular acid employed, the number of acids administered, and the method of application. Due to the potentially toxic effects of hyper-physiological concentrations of amino carboxylic acids in the blood, the acids are typically administered so as to attain a blood plasma concentration of no greater than 200 to 300 milligrams total free amino carboxylic acid per deciliter (100 ml) of blood. As mentioned above, however, if an hepatic cycle stimulating amino carboxylic acid of the present invention is co- administered, it may be possible to increase the dosage amount . In accordance with a preferred embodiment of the present invention, there is provided a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine administered by intravenous infusion into a 70 kg mammal. In this embodiment, the acids can be dissolved in one liter of a suitable water based carrier to a concentration of between 0.5-0.7% glycine (0.6% being preferred) , between 0.6-0.9% proline (0.8% being preferred) , between 0.8-1.2% histidine (1.0% being preferred) , between 0.5-0.8% serine (0.7% being preferred) , between 0.5-0.7% alanine (0.6% being preferred) , between 0.3-0.5% glutamic acid (0.4% being preferred) , between 0.3-0.5% glutamine (0.4% being preferred) , and between 0.4-0.6% arginine (0.5% being preferred) . A large volume loading dose of from about 2 to 3 ml per kg of body weight is first administered over a period of 30 minutes. Thereafter, a maintenance dose of about 1 to 2 ml per kg of body weight per hour is administered for 4 to 6 hours. Further administration of the therapeutic solution is determined by monitoring the blood amino carboxylic acid levels in order to maintain a total blood concentration of total amino carboxylic acids of between 200 to 300 mg per deciliter (100 ml) of blood. Treatment can continue for at least 12 hours after tissue oxygenation is normalized. The suitability of the therapeutic acids of the present invention for treating calcium overload of cells and for preventing irreversible cell damage and cell lysis can be predicted from the following examples. EXAMPLE 1 Hepatic cells were isolated from rat livers by the method described in P.O. Seglen, "Preparation of Rat Liver Cells", Experimental Cell Research Vol. 82, pp. 391-398, 1973, with several modifications. The liver was initially perfused through the v. porta with 250 ml of a 37°C, oxygen saturated buffered salt solution (BSS : 144 πiM NaCl, 5 mM KCl, 5 mM NaHC0 3 , 1 mM NaH 3 P0 4 and 10 mM HEPES, pH 7.4) containing 10 mM a EGTA, followed by 300 ml of warm, oxygenated BSS containing 50 mg collagenase (Worthington Type II, 175 units/ mg) and 1.3 mM CaCl_ . Both perfusates were introduced in situ at a rate of 20 ml/minute. The liver was dispersed by several passages through a glass pipette tip (4 mm aperture) , subsequently filtered through two layers of a 250 micron Nylon mesh, and diluted in 50 ml of a chilled suspension medium of the BSS containing 1.3 mM CaCl_, 1.3 mM MgCl_, 20 mM glucose, and 5 mM pyruvate. The crude suspension was purified by differential centrifugation (100 g per 2 minutes) . The purified cell suspension, consisting primarily of liver parenchymal cells, was washed three times with the cold suspension medium. The final pellet was resuspended at a concentration of 20 mg cell protein (Biuret assay) per milliliter of suspension medium and incubated aerobically for 1 hour at 24°C in a 50 ml polypropylene centrifuge tube mounted horizontally on the rocker tray of a shaking water bath (30 strokes per minute) . At the end of this incubation period, hepatocyte viability was assessed by trypan blue exclusion. Suspensions exhibiting less than 80% viability were discarded. Samples of the purified cell suspension were then transferred to two 50 ml polypropylene test tubes. One of these tubes was purged of oxygen by flushing the gas phase with argon and then permitting the cells to consume the residual oxygen in the medium. Anaerobiosis was attained within 3 minutes as determined polarographically. This anaerobic suspension was maintained in a 37°C. water bath without agitation. The other test tube was supplemented with oxygen by flushing the gas phase with pure oxygen. Diffusion of oxygen from the gas phase to this aerobic reference cell suspension was encouraged by orienting the tube in such a fashion as to maximize surface area and by gently agitating the suspension in the 37°C. water bath. After 90 minutes, both suspension were removed from the water bath and the cells were washed two times with an ice cold suspension buffer and then resuspended in a warm, aerated suspension medium to a concentration of 10 mg cell protein. A one milliliter sample of the anaerobic suspension was added to 4 ml of warm, aerated suspension medium containing either no additives (for the untreated cells) , or a single amino carboxylic acid at a concentration of 5 mM. Cell viability was assessed in each test population at the end of a subsequent 90 minute aerobic incubation at 37°C by measuring the fraction of cytosolic lactate dehydrogenase activity retained intracellularly. The results of these viability assessments are reported in Table 1 as follows: The data in Table 1 demonstrates the effectiveness of the amino carboxylic acids of the present invention (e.g., glycine, histidine, asparagine, glutamine, glutamate, alanine, arginine, glycine, proline and serine) in preventing loss of cell viability normally associated with a transient period of oxygen deprivation. This data also demonstrates the unexpected superiority of amino carboxylic acids of the present invention to be transported into the cell, in association with sodium and by the activity of sodium dependent, plasma membrane transport systems as described above to preserve cell vitality as compared with other amino carboxylic acids like leucine, valine, isoleucine, and tyrosine, which are not substrates for the sodium dependent, plasma membrane transport systems. EXAMPLE 2 Using the procedure set forth in Example 1, the effectiveness of chlorpromazine, a calcium antagonist and widely recognized cytoprotectant, in preserving cell viability following a 90 minute period of oxygen deprivation was compared to amino carboxylic acids of the present invention, i.e., glycine and a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (for the untreated cells) , chlorpromazine (25 Λ mM) , glycine (20 mM or 150 mg per 100 ml) , or the mentioned mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of the cytosolic enzyme, lactate dehydrogenase, retained intracellularly following a 90 minute aerobic incubation. A minimum of four observations were made and recorded. The results are reported in Table 2 as the mean surviving fraction with standard deviation as follows: TABLE 2 TREATMENT MEAN SURVIVING FRACTION Untreated .342 ±.067 Chlorpromazine .567 ± .055 Glycine .698 ± .065 Mixture .578 ± .092 The data from Table 2 demonstrates the effectiveness of an amino carboxylic acid of glycine and the mixture of amino carboxylic acids in preserving cell viability following a period of oxygen deprivation. It also demonstrates that either embodiment of the present invention provides comparable or superior protection against cell lysis then the well known calcium antagonist, chlorpromazine. EXAMPLE 3 Using the procedure set forth in Example 1, the effectiveness of trifluoperazine, another widely recognized cytoprotectant and calcium antagonist, in preserving cell viability following a 90 minute period of oxygen deprivation was compared to glycine and the mixture of amino carboxylic acids containing the same acids as in Example 2. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (untreated cells) , trifluperazine (25 A mM) , glycine (20 mM or 150 mg per 100 ml) , or a mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of lactate dehydrogenase retained intracellularly following a 90 minute aerobic incubation. A minimum of four observations were made and recorded. The results are reported in Table 3 as mean surviving fraction as follows: TABLE 3 TREATMENT MEAN SURVIVING FRACTION Untreated .384 ± .007 Trifluoperazine .550 + .061 Glycine .739 ± .014 Mixture .626 ± .033 As seen from the data of Table 3, the amino carboxylic acids of the present invention (i.e. , glycine and the mixture) demonstrated superior ability in preserving cell viability following a period of oxygen deprivation as compared with the known calcium antagonist, trifluoperazine. EXAMPLE 4 Using the procedure set forth in Example 1, the effectiveness of neomycin sulfate, a calcium channel blocker, in preserving cell viability following a 90 minute period of oxygen deprivation was compared to glycine and the mixture of amino carboxylic acids mentioned above. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (for the untreated cells) , neomycin sulfate (10 mM) , glycine (20 mM or 150 mg per 100 ml) , or the mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of lactate dehydrogenase retained intracellularly following a 90 minute aerobic incubation. A minimum of four observations were made and recorded. The results are reported in Table 4 as the mean surviving fraction as follows: TABLE 4 TREATMENT MEAN SURVIVING FRACTION Untreated .384 ± .040 Neomycin Sulfate .566 ± .043 Glycine .694 ± .033 Mixture .578 ± .044 The data from Table 4 demonstrates the ability of the amino carboxylic acids of the present invention to provide comparable or superior protection against cell lysis as compared with the well known calcium channel blocker, neomycin sulfate. EXAMPLE 5 Using the procedure set forth in Example 1, the effectiveness of alpha-tocopherol, an antioxidant and free radical scavenging cytoprotectant, in preserving cell viability following a 90 minute period of oxygen deprivation was compared to glycine and the mixture of amino carboxylic acids. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (for the untreated cells) , alpha tocopherol succinate (100 mg per 100 ml) , glycine (20 mM or 150 mg per 100 ml) , or the mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of lactate dehydrogenase retained intracellularly following a 90 minute aerobic incubation. A minimum of 4 observations were made and recorded. The results were reported in Table 5 as the mean surviving fraction as follows: TABLE 5 TREATMENT MEAN SURVIVING FRACTION Untreated .352 ± .050 alpha-tocopherol .421 ± .087 Glycine .749 ± .020 Mixture .635 ± .029 The data from Table 5 demonstrates the superiority of the amino carboxylic acids of the present invention in preserving cell viability as compared with alpha-tocopherol. EXAMPLE 6 Using the procedure set forth in Example 1, the effectiveness of desferrioxamine, a scavenger of delocalized iron and well known cytoprotectant, in preserving cell viability following a 90 minute period of oxygen deprivation was compared to glycine and the amino carboxylic acid mixture. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in a fresh medium containing either no additives (for the untreated cells) , desferrioxamine (25 A mM) , glycine (20 mM or 150 mg per 100 ml) , or the mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of lactate dehydrogenase retained intracellularly following a 90 minute aerobic incubation. A minimum of 4 observations were made and recorded. The results are reported in Table 6 as the mean surviving fraction as follows : TABLE 6 TREATMENT MEAN SURVIVING FRACTION Untreated .363 ± .022 Desferrioxamine .393 ± .029 Glycine 696 ± .038 Mixture .582 ± .051 The data in Table 6 clearly demonstrates the superiority of the amino carboxylic acids of the present invention in preserving cell viability as compared with desferrioxamine. EXAMPLE 7 Using a modification of the procedure set forth in Example 1, the effect of amino carboxylic acids of the present invention (glycine and the amino carboxylic acid mixture) on intracellular calcium levels was assessed by monitoring the activity of glycogen phosphorylase "a" in cells retaining intracellular enzymes 90 minutes after oxygen deprivation. Glycogen phosphorylase (E.C.2.4.1.1) is converted from the "b" form to the "a" form by phosphorylase kinase. Kinase is stimulated by increased intracellular calcium concentration via calmodulin. The measured activity of phosphorylase "a" in isolated cells has been shown to be directly related to cytosolic calcium concentration by Long and Moore, as published in J. Pharmacal. Expt. Thera. , Vol. 238, pp. 186-191, 1986. Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (for the untreated cells) , chlorpromazine (25 Λ mM) , glycine (20 mM or 150 mg per 100 ml) , or the mixture of amino carboxylic acids (total amino carboxylic acid concentration was less than 5 mM or 65 mg per 100 ml) . Cell viability was assessed as the fraction of lactate dehydrogenase retained intracellularly following a 90 minute aerobic incubation. Intracellular calcium activity was assessed in these post-anaerobic cells by the method described in Long and Moore, supra, and expressed in terms of glycogen phosphorylase a activity. The activity is determined by the number of nmols of PO .. liberated from a standard amount of glucose-1-phosphate in the presence of glycogen and caffeine, following the addition of 1 million viable, post-anaerobic cells to a lysing buffer. A minimum of four observations were made and recorded. The results are reported in Table 7 as mean surviving fraction as follows: TABLE 7 TREATMENT GLYCOGEN PHOSPHORYLASE A ACTIVITY Untreated 165.5 ± 49.2 Chlorpromazine 129.1 ± 36.5 Glycine 124.8 ± 22.9 Mixture 131.8 ± 44.7 The data in Table 7 demonstrates the effectiveness of an amino carboxylic acids of the present invention in reducing intracellular calcium activity following a period of oxygen deprivation. The data also demonstrates that the present invention provides comparable or better inhibition of calmodulin mediated processes in post-anaerobic cells as compared with the calcium antagonist, chlorpromazine. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Thus it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims or their equivalents.

Claims: What is claimed is: 1. A method for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload, comprising administering to the mammal at least one amino carboxylic acid or its physiologically acceptable salt in an amount effective to treat intracellular calcium overload. 2. The method of claim 1, wherein the amino carboxylic acid treats hypoxic, post-hypoxic, ischemic, post- ischemic, anoxic or post-anoxic cells of the mammal. 3. The method of claim 1, wherein the amino carboxylic acid enters a cell's interior to treat calcium overload. 4. The method of claim 3, wherein the amino carboxylic acid is transported into the cell's interior through a sodium dependent, plasma membrane transport system. 5. The method of claim 4, wherein said amino carboxylic acid is a substrate for a GLY transport system. 6. The method of claim 5, wherein the amino carboxylic acid is glycine. 7. The method of claim 4, wherein amino carboxylic acid is a substrate for an A transport system 8. The method of claim 7, wherein the amino carboxylic acid is selected from the group consisting of alanine, proline, aminoisobutyric acid, and N-methyl- Λ a- aminoisobutyric acid. 9. The method of claim 8, wherein the amino carboxylic acid is alanine. 10. The method of claim 4, wherein the amino carboxylic acid is a substrate for an ASC transport system. 11. The method of claim 10, wherein the amino carboxylic acid is serine. 12. The method of claim 4, wherein the amino carboxylic acid is a substrate for an N transport system. 13. The method of claim 12, wherein the amino carboxylic acid is selected from the group consisting of histidine, asparagine, and glutamine. 14. The method of claim 13, wherein the amino carboxylic acid is glutamine. 15. The method of claim 4, further comprising an amino carboxylic acid capable of stimulating the mammal's hepatic urea cycle. 16. The method of claim 15, wherein the amino carboxylic acid capable of stimulating the hepatic urea cycle is arginine. 17. The method of claim 4, wherein amino carboxylic acid is a mixture containing at least one substrate for a GLY transport system; at least one amino carboxylic acid substrate for a transport system; at least one amino carboxylic acid substrate for an ASC transport system; at least one substrate for an N transport system; and at least one amino carboxylic acid simulator of the mammal's hepatic urea cycle. 18. The method of claim 17, wherein the amino carboxylic acid mixture contains glycine, proline, histidine, serine, alanine, glutamic acid, glutamine and arginine. 19. The method of claim 18, wherein the mixture contains about 0.6% glycine, 0.8% proline, 1.0% histidine, 0.7% serine, 0.6% alanine, 0.4% glutamic acid, 0.4% glutamine, and 0.5% arginine based one liter of a suitable carrier. 20. The method of claim 1, wherein the step of administering further comprises an initial and a subsequent maintenance dosing of the amino carboxylic acid. 21. The method of claim 20, wherein the initial dosing ranges from about 2 to 3 ml/kg of body weight and the maintenance dosing ranges from about 1 to 2 ml/kg. 22. The method of claim 1, wherein the amino carboxylic acid is administered so as to attain a blood plasma concentration of no more than 300 milligrams total free amino carboxylic acid per deciliter of blood. 23. The method of claim 1, wherein the amino carboxylic acid is administered so as to attain a blood plasma concentration of no more than 200 milligrams total free amino carboxylic acid per deciliter of blood. 24. The method of claim 1, wherein the prevented pathogenic effects include preventing cell lysis and irreversible cell damage. 25. The method of claim 1, wherein the treated mammal has suffered from a disease condition associated with or resulting from insufficient tissue oxygenation. 26. The method of claim 1, further comprising administering at least one therapeutically active agent selected from the group consisting of a plasma extender, a water soluble magnesium salt, thrombolytic enzyme, a scavenger of toxic oxygen metabolites, a xanthine oxidase inhibitor, and a free iron binding substance. 27. The method of claim 1, wherein the amino carboxylic acid is selected from the group consisting of glycine, histidine, asparagine, glutamine, glutamate, alanine, arginine, cysteine, proline, serine, aminoisobutyric acid, and N-methyl- A a-amino-isobutyric acid. 28. A pharmaceutical composition for treating or preventing pathogenic effects in a mammal caused by intracellular calcium overload comprising at least one amino carboxylic acid or its physiologically acceptable salt in an amount effective to treat intracellular calcium overload together with a pharmaceutically acceptable carrier. 29. The composition of claim 28, further comprising at least one therapeutically active agents selected from the group consisting of a plasma expander, a water soluble magnesium salt, a thrombolytic enzyme, a scavenger of toxic oxygen metabolites, a xanthine oxidase inhibitor, and a free iron binding substance.

Inventors: KRAMER RICHARD S, PEARLSTEIN ROBERT D, KRAMER, RICHARD, S., PEARLSTEIN, ROBERT, D.
Applicants: LEIGH BIOTECHNOLOGY INC, LEIGH BIOTECHNOLOGY, INC.
Application year: 1995
Main classification: A61K31
IPC codes: A61K31