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EP0741566A1 - Utilisation d'acides amines dans le traitement et la prophylaxie des effets pathogenes de l'accumulation calcique intracellulaire - Google Patents

Utilisation d'acides amines dans le traitement et la prophylaxie des effets pathogenes de l'accumulation calcique intracellulaire

Info

Publication number
EP0741566A1
EP0741566A1 EP95907481A EP95907481A EP0741566A1 EP 0741566 A1 EP0741566 A1 EP 0741566A1 EP 95907481 A EP95907481 A EP 95907481A EP 95907481 A EP95907481 A EP 95907481A EP 0741566 A1 EP0741566 A1 EP 0741566A1
Authority
EP
European Patent Office
Prior art keywords
carboxylic acid
amino carboxylic
transport system
mammal
substrate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95907481A
Other languages
German (de)
English (en)
Inventor
Robert D. Pearlstein
Richard S. Kramer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leigh Biotechnology Inc
Original Assignee
Leigh Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leigh Biotechnology Inc filed Critical Leigh Biotechnology Inc
Publication of EP0741566A1 publication Critical patent/EP0741566A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • 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.
  • the present invention also effectively prevents irreversible cell damage and cell lysis in cells transiently deprived of oxygen.
  • a cell becomes ischemic insufficient free energy exists to operate the ion pumps.
  • 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.
  • 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
  • myocyte damage following coronary artery occlusion
  • myocyte damage following coronary artery occlusion
  • lysed When essential cellular constituents leak out of such damaged cells, the cell is referred to as lysed and, of course, is irreparable.
  • anoxia hypoxia or ischemia
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • composition containing:
  • 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.
  • classes of agents which may complement the action of the therapeutic amino carboxylic acids of the present invention include:
  • a plasma volume expander such as dextran
  • thrombolytic enzyme such as streptokinase
  • scavengers of toxic oxygen metabolites such as superoxide dismutase and/or catalase
  • a xanthine oxidase inhibitor such as allopurinol
  • 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.
  • 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.
  • 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 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.
  • 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 .
  • a mixture of glycine, proline, histidine, serine, alanine, glutamic acid, glutamine, and arginine administered by intravenous infusion into a 70 kg mammal.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 1 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.
  • 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 ⁇ 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:
  • 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:
  • amino carboxylic acids of the present invention i.e. , glycine and the mixture
  • 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:
  • 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.
  • 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) .
  • Hepatocytes were incubated anaerobically for 90 minutes, then washed and resuspended in fresh medium containing either no additives (for the untreated cells) , chlorpromazine (25 ⁇ 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
  • 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.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

Procédé et préparation permettant de traiter ou de prévenir chez les mammifères les effets pathogènes de l'accumulation calcique intracellulaire, consistant à administrer à un mammifère au moins un acide aminocarboxylique ou son sel physiologiquement acceptable, en une quantité permettant de traiter l'accumulation calcique intracellulaire. Par le traitement efficace de ce dysfonctionnement, la présente invention permet de prévenir la lyse cellulaire et les endommagements irréversibles des cellules, notamment chez des mammifères présentant des états pathologiques associés à une oxygénation insuffisante ou en résultant.
EP95907481A 1994-01-24 1995-01-23 Utilisation d'acides amines dans le traitement et la prophylaxie des effets pathogenes de l'accumulation calcique intracellulaire Withdrawn EP0741566A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US18841194A 1994-01-24 1994-01-24
US188411 1994-01-24
PCT/US1995/000811 WO1995019768A1 (fr) 1994-01-24 1995-01-23 Utilisation d'acides amines dans le traitement et la prophylaxie des effets pathogenes de l'accumulation calcique intracellulaire

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EP0741566A1 true EP0741566A1 (fr) 1996-11-13

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EP (1) EP0741566A1 (fr)
AU (1) AU1569895A (fr)
CA (1) CA2181923A1 (fr)
WO (1) WO1995019768A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5859001A (en) 1996-01-11 1999-01-12 University Of Florida Research Foundation, Inc. Neuroprotective effects of polycyclic phenolic compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9519768A1 *

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AU1569895A (en) 1995-08-08
CA2181923A1 (fr) 1995-07-27
WO1995019768A1 (fr) 1995-07-27

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