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WO1999034803A1 - Compositions et traitements convenant pour les lesions tissulaires liees a la reperfusion d'une ischemie ou dues a des endotoxines - Google Patents

Compositions et traitements convenant pour les lesions tissulaires liees a la reperfusion d'une ischemie ou dues a des endotoxines Download PDF

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Publication number
WO1999034803A1
WO1999034803A1 PCT/US1998/027600 US9827600W WO9934803A1 WO 1999034803 A1 WO1999034803 A1 WO 1999034803A1 US 9827600 W US9827600 W US 9827600W WO 9934803 A1 WO9934803 A1 WO 9934803A1
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ischemia
reperfusion
endotoxin
adenosine
injury
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English (en)
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Constance F. Neely
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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Priority claimed from US09/004,938 external-priority patent/US6001842A/en
Application filed by University of Pennsylvania Penn filed Critical University of Pennsylvania Penn
Priority to CA002312867A priority Critical patent/CA2312867A1/fr
Priority to EP98966091A priority patent/EP1044003A4/fr
Priority to AU22069/99A priority patent/AU2206999A/en
Publication of WO1999034803A1 publication Critical patent/WO1999034803A1/fr
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    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir

Definitions

  • compositions of the present invention can be administered prior to, during or following harvesting a donor organ which will be transplanted, prior to or during a surgical procedure in which ischemia is expected, prior to angioplasty or thrombolytic therapy, or after transplantation or reperfusion of an ischemic organ following surgery, angioplasty or thrombolytic therapy. These compositions can also be used to prevent or treat ischemia- reperfusion injury in high risk patients. These compositions are also useful in preventing endotoxin induced tissue injury.
  • Nucleotides and nucleosides and their purinoceptors have been found to be important mediators in determining pulmonary vascular (PV) tone.
  • Nucleotides are autacoids; that is, they are released locally, metabolized locally by stereoselective nucleotidases, and act on their own local receptors to bring about changes in vascular tone, and neutrophil and platelet function.
  • the effects of nucleotides and nucleosides on PV tone were first described in 1929 by Drury and Szent -Gyorgi when they demonstrated that the nucleoside adenosine produced a fall in arterial pressure and a rise in pulmonary artery pressure in dogs and cats.
  • Burnstock originated the purinergic receptor hypothesis.
  • Burnstock G Handbook of Physiology - The Cardiovascular System II, 2nd Edition, Volume 2, Chapter 19, pp 567-612, 1979.
  • Adenosine-sensitive receptors referred to as P- L receptors, were characterized as having an agonist potency in the order of adenosine > AMP > ADP > ATP. These receptors were found to act via an adenylate cyclase system and were antagonized by methylxanthines . Since the original classi ication was made, P 1 receptors have been subdivided into A** , and A 2 receptors based upon their effect on adenylate cyclase, receptor affinity and radioligand binding.
  • Aj_ receptors inhibit adenylate cyclase activity.
  • High affinity A** receptors have been identified in brain, heart, lung, kidney, skin, pancreas, stomach, spinal cord, intestines, vas deferens, liver, spleen, testis, adrenergic nerve terminals, white blood cells and fat cells.
  • a 2 receptors stimulate adenylate cyclase activity.
  • a 2 receptors have been identified in coronary arteries and 2-phenylaminoadenosine (CV1808) was second only to NECA as the most potent coronary vasodilator .
  • a adenosine receptors mediate negative inotropic and negative chronotropic effects while A 2 receptors mediate coronary vasodilation. Effects of agonists and antagonists on A-* . and A 2 adenosine receptors in a variety of tissues have been reported by several different investigators. Bamifylline, a selective A** , adenosine receptor antagonist, has been demonstrated to prevent thromboxane release in the lung, including immunologically sensitized lungs. Berti et al . Pharmacol . Res . 22:143-150, 1990; Berti et al . Arzneum
  • 5,248,678 disclose a method of treating comatose patients to increase arousal and alertness as measured by the Glascow Coma
  • Patent 4,772,607 disclose diallyl analogs of xanthine which act as adenosine antagonists displaying an increased affinity for adenosine A ⁇ receptors in particular. Use of these analogs as CNS stimulant cognition activators, antifibrillatory agents and bronchodilators is taught .
  • Rzeszotarski et al also disclose a number of xanthine derivatives which are potent adenosine A-* , receptor antagonists for use as bronchodilators and cardiotonics .
  • Patent 5,175,290 disclosing related xanthine derivatives, it is suggested that selectivity of these compounds for the adenosine A 1 receptor may not be responsible for their therapeutic effects.
  • a method of treating cells having a reduced apical Cl- conductance by contacting these cells with a selective adenosine A-* . receptor antagonist has been disclosed as a treatment for cystic fibrosis.
  • Pollard et al . U.S. Patent 5,366,977.
  • Adenosine attenuates ischemia-reperfusion injury of the heart upon administration prior to ischemia or reperfusion. Ely, S et al . , J " . Thorac Cardiovasc Surg
  • Adenosine also serves as the primary substrate for ATP synthesis by the purine salvage pathway.
  • R- PIA selective A adenosine receptor agonist
  • Adenosine has also been reported to act upon adenosine P x receptors in the pulmonary vascular bed to induce vasoconstriction and vasodilation. Neely et al . J “ . Pharmacol . and Exp . Therap . , 250 (1) : 170-176 , 1989.
  • ATP- sensitive purinoceptors are present on pulmonary arteries and veins which produce arterial and venular constriction of pulmonary vessels. Liu et al . J “ . Pharmacol . Exp . Ther . 251:1204-1210, 1989. It is believed that following ischemia and reperfusion, ectonucleotidase activity is reduced.
  • a reduction in 5 ' nucleotidase activity can result in a decrease in the concentration of adenosine at A 2 adenosine receptors and an increase in ATP at P 2X purinoceptors, resulting in an increase in pulmonary vascular tone and edema formation.
  • ATP-sensitive (P 2 ) purinoceptors Based upon potency profiles of structural analogues for ATP, ATP-sensitive (P 2 ) purinoceptors have been subclassified into P 2X and P 2Y purinoceptors. With few exceptions, P 2X receptors are located on vascular smooth muscle cells and mediate vasoconstriction and P 2y receptors are located on endothelial cells and mediate vasodilation. Burnstock, G. and Kennedy, C, Gen . Pharmac . 16:433-440, 1985; Ralevic et al . Br . J.
  • An agonist potency profile for P 2Y purinoceptors is 2- MeSATP>ATP> ⁇ , ⁇ -MeATP, ⁇ , ⁇ -MeATP.
  • P 2Y purinoceptors are antagonized by the P 2Y receptor antagonist reactive blue 2. Hopwood, A.M. and Burnstock, G. , E. J. Pharmacol . 136:49-54, 1987; Burnstock, G. and Warland, J.J.I. , Br . J. Pharmacol .
  • Diadenosine pentaphosphate and hexaphosphate derivatives mimic the contractile effects of ⁇ , ⁇ -MeATP in the bladder and vas deferens and a synthetic ⁇ , ⁇ - adenine dinucleotide, P 1 , ⁇ 5 diadenosine 5 ' pentaphosphate (AP 5 A) desensitizes P 2X receptors suggesting these compounds act on P 2X receptors.
  • AP 5 A diadenosine 5 ' pentaphosphate
  • PPADS 2 ', 4 ' -disulfonic acid
  • Ischemia-reperfusion injury of the lung occurs after lung transplantation, pulmonary thromboembolectomy or cardiopulmonary bypass.
  • Egan TM et al . , Lung transplantation . Curr Probl Surg 26:675-751, 1989; Levinson RM, et al., Am Rev Resp Dis 134:1241-1245, 1986; Kuratani T, et al., J " Thorac Cardiovas Surg 103:564-568, 1992.
  • Ischemia- reperfusion injury of the lung also occurs after ischemia and reperfusion of distant organs, for example the intestines.
  • Schmeling DJ et al . , Surg 106:195-201, 1989.
  • two hours of ischemia followed by three hours of reperfusion produced structural and functional abnormalities that did not occur with ischemia alone.
  • Murata T et al . , Am Kev Resp Dis
  • compositions comprising an A-* . adenosine receptor antagonist and/or a P 2X purinoceptor antagonist are useful in the prevention and treatment of ischemia-reperfusion injury following organ transplantation, resulting from surgical procedures, following angioplasty or thrombolytic therapy and associated with certain injuries or disease states. These compositions have also been found to be useful in preventing endotoxin-related tissue injury.
  • An object of the present invention is to provide a method of preventing or inhibiting ischemia-reperfusion organ injury comprising administering to an animal an effective amount of an A adenosine receptor antagonist .
  • Another object of the invention is to provide compositions comprising an A 1 adenosine receptor antagonist useful in the prevention or treatment of ischemia-reperfusion organ injury resulting from transplantation, surgical procedures, angioplasty or thrombolytic therapy, or certain disease states.
  • Another object of the present invention is to provide a method of preventing or inhibiting ischemia-reperfusion organ injury comprising administering to an animal an effective amount of a P 2X purinoceptor antagonist.
  • Another object of the present invention is to provide a method of inhibiting endotoxin-related tissue injury which comprises administering to an animal an effective amount of an A-* , adenosine receptor antagonist.
  • Another object of the present invention is to provide a method of inhibiting endotoxin-related tissue injury which comprises administering to an animal an effective amount of a P 2X purinoceptor antagonist .
  • ATP which is released during ischemia, is metabolized to adenosine by species-specific ectonucleotidases located on endothelial and vascular smooth muscle cells.
  • ATP and adenosine act on specific extracellular receptors, adenosine-sensitive P x and ATP-sensitive P purinoceptors located on a number of cell types including endothelial and vascular smooth muscle cells, neutrophils, and platelets. These cells are important in the pathophysiology of ischemia- reperfusion injury of organs.
  • a number of complex events occur after ischemia and reperfusion, including the release of cytokines and chemoattractants, activation of neutrophils, adherence of neutrophils to endothelial cells, and the release of oxygen radicals and vasoactive substances, including thromboxane.
  • thromboxane Following ischemia and reperfusion of the rabbit lung, an increase in thromboxane is associated with an increase in pulmonary vascular tone, alterations in endothelial cell tight junctions and pulmonary edema formation.
  • Zamora CA et al . , J Appl Physiol 74:224-229,
  • Adenosine via its effects on specific adenosine receptors A 1 and A 2 , effects pulmonary vascular tone, Neely CF, et al., J " . Pharmacol Exp Ther 250 (1) : 170-176 , 1989; platelet function, Hourani SMO, Cusack NJ, Actions and Structure
  • Adenosine produces vasoconstriction in the feline lung vasculature by acting on A x adenosine receptors which induce the release of thromboxane.
  • adenosine By acting on A 2 adenosine receptors, adenosine produces vasodilation, inhibition of oxygen radical release from neutrophils and platelet aggregation, and a decrease in endothelial cell permeability.
  • Adenosine-sensitive A-* . and A 2 receptors play important roles in ischemia-reperfusion injury of organs following transplantation, during certain surgical procedures, following angioplasty and thrombolytic therapy, and following shock or trauma.
  • Adenosine, selective A x adenosine receptor agonists (when administered prior to ischemia) , and brief periods of ischemia (preconditioning ischemia) have been shown to attenuate ischemia-reperfusion injury of the heart.
  • Such treatments are also believed to attenuate ischemia- reperfusion injury of the lung.
  • ischemia- reperfusion injury of the lung For example, in lung transplant operations, it is possible for a surgeon to subject a lung to brief periods of ischemia prior to removing the lung from a donor.
  • the effects of preconditioning are brief.
  • a more effective treatment would be to administer a drug into the lung which would stay in the lung until transplantation takes place.
  • a x adenosine receptors mediate the effects of preconditioning, it has been proposed that administration of a very hydrophobic A ⁇ adenosine receptor agonist such as R-PIA could prove useful in improving the outcome of organ function following transplantation.
  • administration of an A-* a very hydrophobic A ⁇ adenosine receptor agonist
  • adenosine receptor agonist can result in several unwanted side effects, including decreased heart rate and myocardial contractility, bronchospasm and a decrease in urine output resulting from decreased kidney function. It has now been found that administering a selective A 1 adenosine receptor antagonist attenuates ischemia-reperfusion injury more effectively and without the unwanted side effects.
  • a method of preventing ischemia-reperfusion organ injury wherein an animal, preferably a human, is administered an effective amount of a selective A x adenosine receptor antagonist at a selected time prior to a surgical procedure in which ischemia is expected to occur so that the organ injury is prevented.
  • effective amount refers to a concentration of a selective A 1 adenosine receptor antagonist which is sufficient to interfere with the action of adenosine upon this receptor.
  • selected time refers to an amount of time which is sufficient to allow a selective adenosine A** .
  • the selected time be prior to ischemia.
  • the preferred selected time is from about 20 to 30 minutes, more preferably 30 minutes, prior to ischemia. If the antagonist is administered intravenously, the selected time may be longer, for example, 30 minutes to an hour prior to ischemia.
  • Surgical procedures for which this method is useful include harvesting donor organs for transplantation.
  • surgical procedures and organs at risk of ischemia reperfusion injury during these procedures include, but are not limited, brain injury during carotid artery surgery, cerebral vascular surgery and surgery of the heart and aorta; brain, spinal cord, intestine and kidney injury during surgery of the thoracic aorta and kidney injury during abdominal aortic surgery; lung injury following thromboembolectomy or the use of cardiopulmonary bypass during lung and heart surgery; heart injury following revascularization (coronary artery bypass graft surgery) ; kidney injury following surgery on renal arteries; intestinal injury following surgery on the mesenteric arteries; and skin injury following harvesting of a skin graft.
  • a x adenosine receptor antagonist be administered prior to the surgical procedure
  • administration of the A x adenosine receptor antagonist after the onset of ischemia but pri or to or during reperfusion has also been found to inhibit tissue injury re lat ed to ischemia- reperfus ion .
  • Selective A x adenosine receptor antagonists can also be administered prior to or following angioplasty or thrombolytic therapy to prevent or inhibit tissue or organ injury caused by ischemia followed by reperfusion.
  • an effective amount of an A adenosine receptor antagonist is administered to a patient at a selected time prior to or following an angioplasty procedure or prior to or following administration of a thrombolytic agent such as urokinase so that, upon lysis of the clot, tissue injury related to ischemia followed by reperfusion is prevented.
  • a thrombolytic agent such as urokinase
  • P 2X purinoceptors have also been identified on large and small pulmonary arteries and large pulmonary veins. During ischemia-reperfusion injury, it is believed that ectonucleotidase activity is reduced resulting in an increase in ATP at the P 2X purinoceptors. This increase has been associated with an increase in vascular tone and edema formation. It has now been found that antagonists of P 2X purinoceptors, such as PPADS, selectively antagonize the vasoconstrictive responses of the P 2X receptors to selective P 2X purinoceptor agonists such as , ⁇ -MeATP, at low, baseline pulmonary vascular tone.
  • PPADS selectively antagonize the vasoconstrictive responses of the P 2X receptors to selective P 2X purinoceptor agonists such as , ⁇ -MeATP, at low, baseline pulmonary vascular tone.
  • Such antagonists had no effect on the vasodilator response of adenosine, ATP or the selective P 2y purinoceptor agonist 2-MeSATP at elevated pulmonary vascular tone. Accordingly, it is believed that administration of an effective amount P 2X purinoceptor antagonist administered at a selected time prior to, during, or following a surgical procedure in which ischemia is expected to occur can also be administered to prevent ischemia-reperfusion injury.
  • effective amount it is meant a concentration of a P 2X purinoceptor antagonist sufficient to inhibit the vasoconstrictive response of the P 2X receptor.
  • selected time it is meant as time which is sufficient to allow a P 2X purinoceptor to bind to the P 2X purinoceptor in the tissue or organ so that tissue injury relating to ischemia-reperfusion is decreased.
  • These antagonists can also be administered prior to or following angioplasty or thrombolytic therapy to prevent ischemia- reperfusion injury related to these procedures.
  • a selective A-* . adenosine receptor antagonist and a P 2 purinoceptor antagonist may also be useful in preventing or treating ischemia-reperfusion injury.
  • a single compound which antagonizes both the A-* . adenosine receptor and the P 2X purinoceptor may also be used.
  • Methods of the present invention are also useful in treating ischemia-reperfusion organ injury in high risk patients. Injuries or conditions such as bowel ischemia and reperfusion, sepsis, anaphylaxis, hemorrhagic shock and trauma can result in ischemia-reperfusion organ injury.
  • Ischemia- reperfusion injury of the organs is also associated with vasculitis and autoimmune disease.
  • cerebral air embolisms which can occur following diving and decompression are associated with ischemia-reperfusion injury of the brain.
  • the brain of newborns may be at risk of ischemia reperfusion.
  • patients suffering from such injuries or conditions are defined as "high risk” patients.
  • the methods of the present invention are useful in preventing the nephropathy, retinopathy and neuropathy of diabetes which is secondary to ischemia-reperfusion injury.
  • the intact-chest spontaneously breathing cat animal model has been used to create ischemia-reperfusion injury of the lung which is morphometrically similar to this injury in other species and reproducible quantitatively.
  • catheters are placed in the left lower lobe artery and vein in the lungs of intact -chest , spontaneously breathing cats.
  • the lobar artery catheter is of the type which allows for isolation of the left lower lobe, preferably the catheter is a triple lumen catheter with a proximal balloon.
  • the left lower lobe is perfused with blood withdrawn from the aorta at a constant flow rate with the use of a peristaltic pump. However, blood flow can be stopped for a given period of time by stopping the pump.
  • Ventilation and blood flow are interrupted for a period of time and then resumed.
  • Lung injury following these periods of ischemia and reperfusion is characterized by the presence of leukocytes, red blood cells, macrophages and edema in the alveoli, as compared to controls.
  • the morphological changes produced by two hours of ischemia followed by two hours of reperfusion were similar to those described by others in other species, including rats, rabbits and dogs.
  • Zamora CA et al . , J Appl Physiol 74:224-229 , 1993; Murata T, et al . , Am Rev Resp Dis 146:1048-1053, 1992; Hamvas
  • selective A x adenosine receptor antagonists administered prior to the period of ischemia and during reperfusion markedly attenuate the alveolar injury resulting from ischemia followed by reperfusion.
  • selective A x adenosine receptor antagonist refers to antagonists which bind preferentially to the A x adenosine receptor and do not affect the A 2 adenosine receptor.
  • Examples of antagonists selective for A ⁇ adenosine receptors include, but are not limited to, alkyl xanthines such as 8-cyclopentyl-l, 3-dipropylxanthine (DPCPX), xanthine amine cogener (XAC) , xanthine carboxylic cogener (XCC) , 1,3- dipropyl-8- (3-noradamantyl) xanthine (KW 3902), 1 , 3 -dipropyl- 8- (dicyclopropylmethyl) xanthine (KF 15372), 1, 3-dipropyl-8- (3- oxocyclopentyl xanthine (KFM 19), l-propyl-3- (4-amino-3- iodophenethyl) -8-cyclopentylxanthine (BW-A844U) , 1, 3-dipropyl- 8-sulfophenylx
  • Antibodies raised against the A ⁇ adenosine receptor which selectively target and bind to this receptor can also be used as A 1 adenosine receptor antagonists. Such antibodies targeted to the A x adenosine receptor can be produced routinely in accordance with well known methods of antibody production. It was also found that P 2X purinoceptor antagonists administered prior to the period of ischemia markedly attenuate the alveolar injury resulting from ischemia followed by reperfusion. An example of a selective P 2X purinoceptor antagonists is PPADS . Additional specific pharmacological antagonists of purinoceptors have been described by Humphrey PPA, et al . Naunyn-Schmied . Arch .
  • Antibodies raised against the P 2X purinoceptor which selectively target and bind to this receptor can also be used as selective P 2X purinoceptor antagonists. Such antibodies targeted to the P 2X purinoceptor can be produced routinely in accordance with well known methods of antibody production.
  • Lung pathology in ischemia-reperfusion injury animals included alveolar and perivascular edema, margination of PMNs along the venular endothelium, and alveolar infiltration of neutrophils, macrophages, and red blood cells. These parameters were reduced in a highly significant manner to control levels by intralobar infusion of XAC into the lower left lobe prior to ischemia and reperfusion, by intravenous DPCPX administration prior to ischemia or after 2 hours of ischemia and 1 hour of reperfusion, by intravenous BAM administration prior to ischemia and by intravenous PPADS administration prior to ischemia.
  • adenosine receptors 1 hour after reperfusion prevented progression of all other injury parameters past the 1 hour reperfusion level .
  • Results from electron microscopy verified the morphometric observations and clearly identifying histopathological changes in the 2 hour ischemia-2 hour reperfusion group such as interstitial edema of alveolar parenchyma including thickening of alveolar septae, endothelial cell and type I cell swelling and damage, and ruptured blood-air barriers resulting in interstitial and alveolar hemorrhage.
  • lung parenchyma from animals treated with an A x adenosine receptor antagonist could not be distinguished from the control lungs and the alveolar capillary endothelium and type I cell lining appeared generally normal and did not differ from perfused control lungs that showed no signs of alveolar injury.
  • compositions comprising a selective A ⁇ adenosine receptor antagonist and/or P 2X purinoceptor antagonist are effective in preventing tissue injury related to endotoxin.
  • Endotoxin produces organ damage directly by inducing the release of cytotoxic substances including nitric oxide and thromboxane from cells, such as endothelial cells.
  • Endotoxin produces organ damage indirectly by inducing the release of cytotoxic substances from circulating and resident cells, including macrophages, which act locally to produce organ damage or as blood borne substances which induce distal organ damage.
  • Endotoxin also induces the release of mediators such as nitric oxide and prostaglandins which can produce a profound shock state and organ hypoperfusion. Mayeux PR, J. Tox. and Environ . Heal th
  • Endotoxin induces tissue injury or dysfunction in a number of organs or tissues including, but not limited to, the lung, heart, eye, intestines, peritoneum, liver, stomach, ear, blood vessels, nerves, skin, brain and spinal cord, sinus and nose, kidney, testis and epididymis, ovary, fallopian tube, prostate, placenta, bronchi, and gum tissue. See, e.g., Lew WY, et al . , Am. J. Physiol . 272:H2989- H2993, 1997; Goddard CM, et al . , Am. J. Physiol .
  • vasculitis associated with autoimmune diseases and endothelial proliferation associated with diabetic organ injury can produce ischemia-reperfusion organ injury.
  • Hypoxia reduced oxygen tension
  • ischemia reduced oxygen delivery to an organ
  • endotoxin produces a transient rise in pulmonary artery pressure within 30-60 minutes, pulmonary capillary leak associated with ultrastructural changes in pulmonary capillary endothelial cells and microthrombosis by 60 minutes and a late pulmonary hypertension which lasts for several hours.
  • the mechanisms of these pathophysiological changes in the lung following endotoxin are not completely understood.
  • the acute, transient rise in pulmonary artery pressure following endotoxin is associated with increased lymph and blood levels of thromboxane and can be attenuated with cyclooxygenase inhibitors, thromboxane synthesis inhibitors and thromboxane receptor antagonists.
  • Lung pathology in endotoxin- induced injury included perivascular and peribronchial edema and hemorrhage, thickened alveolar septae, margination of neutrophils along the venular endothelium, alveolar infiltration of neutrophils and macrophages, and alveolar hemorrhagic necrosis.
  • pretreatment with a composition comprising a selective A x adenosine receptor antagonist and/or P 2X purinoceptor was found to significantly reduce the percent of alveoli injured (defined as the presence of two or more inflammatory cells or red blood cells, or edematous fluid) following administration of endotoxin in cats.
  • Parameters relating to endotoxin injury were reduced in a highly significant manner to control levels by intravenous DPCPX administration prior to endotoxin administration, by intravenous infusion of BAM prior to and throughout endotoxin administration, and by intravenous PPADS administration prior to endotoxin administration.
  • Coadministration of the adenosine receptor antagonist, BAM, and the P 2X purinoceptor antagonist, PPADS is also useful in protecting the tissue from injury.
  • Compounds selective for both the A x adenosine receptor antagonist and the P 2X purinoceptor antagonists can also be used. Electron microscopical results show thickening of alveolar septae in lungs of endotoxin treated cats due to interstitial edema and cellular infiltration. There was transmigration of red blood cells across the blood-air barrier and their phagocytosis by alveolar macrophages.
  • alveolar type II cells (alveolar septal cells) of endotoxin treated cats sported increased numbers of tall, distinct microvilli and some type II cells protruded into the alveolar lumen from a pedunculated attachment. These changes in type II cells were not observed in treated animals or controls.
  • compositions to inhibit endotoxin-mediated cytotoxicity of other organs or tissues can be routinely determined in similar fashion in cell culture assays.
  • skin epithelial cells can be grown in culture, exposed to compositions of the present invention and then challenged with endotoxin.
  • compositions are also provided which are useful in the prevention and/or treatment of organ injury in an animal, preferably a human, resulting from ischemia followed by reperfusion.
  • Such compositions comprise either a selective adenosine A receptor antagonist, preferably XAC, DPCPX, bamifylline or N-0861, or a P 2X purinoceptor antagonist such as PPADS. It is preferred that these compositions be administered prior to ischemia, preferably 30 minutes prior to ischemia. However, compositions may be administered after ischemia but prior to or during reperfusion if required due to the condition causing the injury. It is preferred that these compositions be administered by intravenous bolus injection or infusion directly to the organ.
  • compositions may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, but are not limited to, saline, water or dextrose and water, cyclodextrins or similar sugar solutions and low dose sodium hydroxide (approximately 0.2 N) solutions.
  • compositions are also useful in inhibiting tissue injury relating to thromboxane release.
  • Thromboxane is an important mediator of anaphylaxis and the reaction to protamine treatment (used to reverse the effect of heparin in cardiac catheterization laboratories and operating rooms) .
  • Thromboxane is also an important mediator of acute renal failure.
  • a adenosine receptor antagonists may also be useful in the prevention of contrast dye or drug induced acute renal failure.
  • different forms of acute lung injury are associated with thromboxane release.
  • A- L adenosine receptor antagonists may also be beneficial for the prevention and early treatment of acute lung injury following aspiration or smoke inhalation or associated with cerebral hemorrhage, air embolism, pancreatitis, amniotic fluid embolism, near drowning, ionizing radiation, multiple transfusions, and bacterial, viral, fungal, mycobacterial , mycoplasmal, and pneumocystic pneumonias.
  • a x adenosine receptor antagonists may be beneficial for the prevention and early treatment of acute lung injury associated with certain drugs, including, but not limited to, cancer therapies such as bleomycin and mitomycin C; antibiotics such as nitrofurantoin and sulfa drugs; antiinflammatory agents such as aspirin, methotrexate and nonsteroidal antiinflammatory agents; cardiac medications such as amiodarone, procaineamide and tocainide; and narcotics such as heroine, methadone, morphine and propoxyphene . Lung toxicity caused by other agents such as oxygen and tocolytics may also be prevented by administration of an A x adenosine receptor antagonist in accordance with the present invention.
  • cancer therapies such as bleomycin and mitomycin C
  • antibiotics such as nitrofurantoin and sulfa drugs
  • antiinflammatory agents such as aspirin, methotrexate and nonsteroidal antiinflammatory agents
  • cardiac medications such as amiodarone, procaineamide and to
  • Example 1 Ischemia-reperfusion model
  • the cats are anesthetized with pentobarbital sodium, 10 mg/kg, IV, and are strapped in the supine position to a fluoroscopic table.
  • the cats spontaneously breathe room air enriched with oxygen through a cuffed endotracheal tube.
  • a specially designed 6F triple lumen balloon perfusion catheter is passed, under fluoroscopic guidance, from an external jugular vein into the arterial branch to the left lower lung lobe. After the lobar artery is vascularly isolated by distension of the balloon cuff on the catheter and the cat is heparinized (1000 U/kg, IV) , the lobe is perfused with blood withdrawn from the femoral artery through the catheter lumen immediately beyond the balloon cuff.
  • Perfusion pressure in the lobar artery is measured through the third lumen, 5 mm distal to the perfusion port .
  • the lobe is perfused with a Harvard model 1210 peristaltic pump, and the perfusion rate is adjusted so that arterial pressure in the perfused lobe approximates mean pressure in the main pulmonary artery and is not changed during an experiment.
  • Flow rates in the left lower lobe range from 35-45 ml/min.
  • Left atrial pressure is measured with a transseptally placed double lumen catheter.
  • Aortic pressure is measured with a catheter, inserted into the aorta by way of a femoral artery. All vascular pressures are measured with Gould transducers zeroed at right atrial level, and mean pressures, obtained by electronic integration, are recorded on a Gould recorder.
  • the animals are allowed to recover for one hour.
  • the Harvard peristaltic pump is stopped and the circuit is attached to the femoral vein catheter.
  • the femoral vein is perfused at 35 ml/min with the Harvard peristaltic pump during the period of ischemia with blood withdrawn from the aorta which normally perfuses the left lower lobe.
  • a 4F bronchial blocker is inserted into the left lower lobe bronchus and a balloon is distended with contrast dye. Ventilation to the left lower lobe is blocked during the period of ischemia.
  • the left lower lobe is perfused for two hours at a rate of 35 ml/min with the use of the Harvard peristaltic pump with blood withdrawn from the aorta and the bronchial blocker is removed.
  • Example 3 Inhibition of ischemia-reperfusion injury in the lung
  • a adenosine receptor antagonist xanthine amine congener A adenosine receptor antagonist xanthine amine congener
  • XAC 1,3 dipropyl 8-cyclopentylxanthine
  • BAM bamifylline
  • PPADS P 2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2 ' , 4 ' -disulfonic acid
  • on-line measurements of mean lobar arterial, femoral arterial, and left atrial pressures were obtained.
  • EM electron microscopy
  • DPCPX The selective A ⁇ adenosine receptor antagonists, DPCPX and Bamifylline (BAM), were administered to cats.
  • E. coli endotoxin Sigma
  • Arterial blood gases were obtained prior to endotoxin (baseline) at 15, 30 minutes, and 1 and 2 hours following the onset of the endotoxin infusion. These data were analyzed with the use of Student's t-tests for paired analysis with Bonferroni correction for multiple t-tests for comparisons within a group and ANOVA with Bonferroni correction for multiple comparisons between groups. Group means were considered statistically significant at p ⁇ 0.05.
  • a x adenosine receptor and/or P 2X purinoceptor antagonists an alveolar inflammatory cells, red blood cells and edema in cat lung after endotoxin
  • Tissue samples were also prepared for analysis by electron microscopy as described in Example 3.
  • Example 5 Inhibition of ischemia-reperfusion injury in lungs pretreated with N-0861 A separate group of animals will receive the selective A-*, adenosine receptor antagonist, N 6 endonorbornan-2- yl-9-methyladenine, N-0861, (2 mg/kg) as an intravenous bolus plus a continuous infusion of 0.2 mg/kg/min 30 minutes prior to ischemia and continued for 30 minutes or at the same dose one hour after reperfusion. Following reperfusion, lung specimens will be examined and results analyzed in accordance with Example 3.
  • a separate group of animals will receive the selective A-*, adenosine receptor antagonist, N 6 endonorbornan-2- yl-9-methyladenine, N-0861, (2 mg/kg) as an intravenous bolus plus a continuous infusion of 0.2 mg/kg/min 30 minutes prior to ischemia and continued for 30 minutes or at the same dose one hour after reperfusion. Following reperfusion, lung specimens will be examined and results
  • Example 6 Inhibition of endotoxin-induced lung injury in lungs pretreated with N-0861 A separate group of animals will receive the selective A-* .
  • adenosine receptor antagonist, N-0861 (2 mg/kg) as an intravenous bolus plus a continuous intravenous infusion of N-0861 (0.2 mg/kg/min) for 30 minutes prior to endotoxin administration.
  • This continuous intravenous infusion of N- 0861 is continued during endotoxin administration and for 1 hour after endotoxin.
  • lung specimens will be examined and results analyzed in accordance with Example 3.
  • Example 7 Assay for determining effects of x adenosine receptor antagonist and/or P 2X purinoceptor antagonist in skin epithelial cells
  • Skin epithelial cells are grown in 24 -well culture flasks in RPMI medium containing 10% fetal bovine serum or other suitable medium recommended by the supplier. Skin epithelial cells are labeled to approximately 1 hour at 37°C with 51 Cr (40 ⁇ Ci as sodium chromate/lO 6 cells, specific activity 456 mCi/mg Cr) . The cells are then washed and allowed to leak for about 1 hour at 37°C in RPMI plus 10% fetal bovine serum. The cells are then preincubated for 2 hours with 0.1 ml of phorbol 12 -myristate 13 -acetate (PMA; 1 ⁇ M) . The cells are washed again with the medium to remove the PMA.
  • PMA phorbol 12 -myristate 13 -acetate
  • the cells are then incubated with different concentrations (0.1 pg-1 ⁇ g/ml in 0.1 ml) of endotoxin in triplicate for 6 hours in the medium containing 10% fetal bovine serum and 2 U/ml adenosine deaminase .
  • concentrations 0.1 pg-1 ⁇ g/ml in 0.1 ml
  • endotoxin in triplicate for 6 hours in the medium containing 10% fetal bovine serum and 2 U/ml adenosine deaminase .
  • the uppermost 0.1 ml of supernatant is removed from the wells of the plate and counted for radioactivity in a gamma counter.
  • a ⁇ adenosine receptor antagonist such as 1,3- dipropyl-8-cyclopentylxanthine (DPCPX; 1 nM - 10 ⁇ M) and/or a P 2X purinoceptor antagonist such as pyridoxalphosphate-6- azophenyl -2 ', 4 ' disulfonic acid (PPADS; 5-50 ⁇ M) is studied by preincubating the cells with DPCPX, PPADS or DPCPX plus PPADS for 30 to 60 minutes before challenging with endotoxin. Total releasable counts are determined by freezing and thawing of 51 Cr- labeled cells in hypotonic medium. Spontaneous release is determined from the wells containing no PMA and endotoxin. Results are expressed as:

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Abstract

L'invention concerne des traitements préventifs ou curatifs des lésions liées à la reperfusion d'une ischémie dans un organe, comprenant l'administration d'une composition contenant un antagoniste sélectif du récepteur d'adénosine A1 et/ou un antagoniste du purinorécepteur P2X. L'invention concerne également des traitements préventifs et curatifs des lésions tissulaires d'origine endotoxinique comprenant l'administration d'une composition contenant un antagoniste sélectif du récepteur d'adénosine A1 et/ou un antagoniste du purinorécepteur P2X.
PCT/US1998/027600 1998-01-09 1998-12-23 Compositions et traitements convenant pour les lesions tissulaires liees a la reperfusion d'une ischemie ou dues a des endotoxines Ceased WO1999034803A1 (fr)

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CA002312867A CA2312867A1 (fr) 1998-01-09 1998-12-23 Compositions et traitements convenant pour les lesions tissulaires liees a la reperfusion d'une ischemie ou dues a des endotoxines
EP98966091A EP1044003A4 (fr) 1998-01-09 1998-12-23 Compositions et traitements convenant pour les lesions tissulaires liees a la reperfusion d'une ischemie ou dues a des endotoxines
AU22069/99A AU2206999A (en) 1998-01-09 1998-12-23 Compositions and methods for use in ischemia-reperfusion and endotoxin-related tissue injury

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003103675A3 (fr) * 2002-06-07 2004-03-25 Endacea Inc Polytherapies destinees aux troubles lies aux purinocepteurs
EP1305023A4 (fr) * 2000-08-01 2005-02-23 Univ Virginia Utilisation d'agonistes, d'antagonistes et d'amplificateurs allosteriques selectifs du recepteur d'adenosine a1 afin de manipuler l'angiogenese
EP1747785A1 (fr) * 2005-07-28 2007-01-31 Istituto Clinico Humanitas Cyclodextrines pour la detoxification du sang
EP1429775A4 (fr) * 2001-09-06 2007-04-11 Biogen Idec Inc Procedes de traitement des maladies pulmonaires
EP2036562A1 (fr) * 2007-08-15 2009-03-18 Endacea, Inc. Antagoniste des récepteurs A1 de l'adénosine pour prévenir et traiter des blessures tissulaires et la sepsis associée à une infection par la peste

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US5733916A (en) * 1995-03-24 1998-03-31 The Trustees Of The University Of Pennsylvania Prevention and treatment of ischemia-reperfusion and endotoxin-related injury using adenosine and purino receptor antagonists

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US5504090A (en) * 1994-03-30 1996-04-02 Trustees Of The University Of Pennsylvania Compositions and methods for the prevention and treatment of ischemia-reperfusion organ injury
JPH10501122A (ja) * 1994-05-27 1998-02-03 グラクソ、グループ、リミテッド Ｐ▲下２ｘ▼レセプター（プリノセプターファミリー）
US6316423B1 (en) * 1996-04-10 2001-11-13 The United States Of America As Represented By The Departmant Of Health And Human Services Method of treating ischemic, hypoxic and anoxic brain damage

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US5733916A (en) * 1995-03-24 1998-03-31 The Trustees Of The University Of Pennsylvania Prevention and treatment of ischemia-reperfusion and endotoxin-related injury using adenosine and purino receptor antagonists

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1305023A4 (fr) * 2000-08-01 2005-02-23 Univ Virginia Utilisation d'agonistes, d'antagonistes et d'amplificateurs allosteriques selectifs du recepteur d'adenosine a1 afin de manipuler l'angiogenese
US7732484B2 (en) 2000-08-01 2010-06-08 University Of Virginia Patent Foundation Use of selective adenosine A1 receptor allosteric enhancers to manipulate angiogenesis
EP1429775A4 (fr) * 2001-09-06 2007-04-11 Biogen Idec Inc Procedes de traitement des maladies pulmonaires
WO2003103675A3 (fr) * 2002-06-07 2004-03-25 Endacea Inc Polytherapies destinees aux troubles lies aux purinocepteurs
EP1747785A1 (fr) * 2005-07-28 2007-01-31 Istituto Clinico Humanitas Cyclodextrines pour la detoxification du sang
EP2036562A1 (fr) * 2007-08-15 2009-03-18 Endacea, Inc. Antagoniste des récepteurs A1 de l'adénosine pour prévenir et traiter des blessures tissulaires et la sepsis associée à une infection par la peste

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CA2312867A1 (fr) 1999-07-15
EP1044003A1 (fr) 2000-10-18
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AU2206999A (en) 1999-07-26

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