A PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING ISCHEMIC CELL DAMAGE AND METHODS FOR THE USE AND PREPARATION THEREOF

Abstract: A pharmaceutical composition for preventing and treating ischemic cell damage, which composition contains a physiologically acceptable, water- soluble salt of phosphoenolpyruvic acid and a physiologically acceptable, water-soluble salt of adenosine-5'-triphosphoric acid in a mutual ratio of from 5:1 to 1000:1, preferably from 10:1 to 500:1, calculated on a molar basis. A method of preventing and treating ischemic cell damage by administering such a composition is also disclosed as well as a method for the preparation of said composition by dissolving said salts in a physiologically acceptable aqueous liquid, adjusting the pH value of the solution, sterilizing and, when desired, freeze-drying.

Description: APHARMACEUTICALCOMPOSITIONFORPREVENTINGANDTREATINGISCHEMIC CE£LDAMAGEAND METHODS FORTHE USEAND PREPARATION THEREOF The present invention relates to a pharmaceutical com¬ position. More specificly, the invention relates to a pharmaceutical composition for preventing and treating ischemic cell damage. The invention also relates to a method for preventing and treating ischemic cell damage in mammals, including humans, or in organs removed therefrom. Ischem c damage to the body cells of a mammal occurs when the flow of blood in a part of the body is impeded to such an extent that the supply of oxygen to the cells of said part of the body ceases or is greatly reduced. The reason why the cell requires oxygen is because the pre¬ dominant part (about 92 % ) of adenos ne-5 ' -tri phosphate (ATP), necessary to the survival of the cell , is formed by a mechanism which involves an aerobic oxida-tion. In the event of an oxygen deficiency, the ATP-content of the cell falls, the resultant injury to the cell being initially re¬ versible, i.e. the damage can be stopped when the ATP- content of the cell returns to more normal values, and in time becomes irreversible, resulting in the death of the cell and subsequently (with an increase in the number of dead cells) the death of the organ. In order to reduce the extent of ischemic cell damage, it is feasible to think that the ATP-content of the cells could be increased by supplying ATP directly to the cells, thereby to avoid the aerobic forming reaction for ATP. It has been found, however, that exogenous ATP cannot penetrate into the : cells to the extent required to markedly ease the ischemic damage. In accordance with the present invention it has now surprisingly been found that an introduction of relatively small quantities of ATP enables another substance, namely a salt of phosphoenolpyruvic acid, to be introduced into the cell in larger quantities simultaneously with ATP, where said substance then contributes to the conversion of adenosine diphosphate (ADP) present in the cell to ATP in one single reaction step which does not require the presence of oxygen. Accordingly, the present invention relates to a pharmaceutical composition for preventing and treating ischemic cell damage, said composition being characterized in that it includes a physiologically acceptable, water- soluble salt of phosphoenolpyruvic acid (which salt is here¬ inafter referred to as PEP) and a physiologically acceptable, water-soluble salt of adenosine-5 ' -triphosphoric acid (which salt.is hereinafter referred to as ATP) in a mutual ratio of 5:1 to 1000:1, preferably from 10:1 to 500:1, calculated on a molar basis. As examples of physiologically acceptable salts of the two compounds can be mentioned sodium salts, magnesium salts or ammonium salts and salts with physiologically acceptable organic amines, such as tri s (hydroxy ethyl )aminomethane (called tris), methyl glucamine, arginine, or itine or histiαirie.. Conveniently, the molar ratio between PEP and ATP can, for example, be greater than 20:1, for example greater than 50:1, and can be lower than 400:1, e.g. lower than 300:1, e.g. lower than 200:1. With the composition according to the invention there is obtained with the two incorporated components a syn- ergistic effect. This effect cannot be obtained with solely one of the components. Neither can such an advantageous effect, free from complications, be obtained with PEP in combination with other additives. In addition to the salts of phosphoenolpyruvic acid and of adenosine-5 ' -triphosphoric acid the pharmaceutical composi tion according to the invention comprises agents for establishing physiological conditions and, when necessary, water as a carrier. For the purpose of administration, the composition according to the invention is in the form of a sterile solu- tion for parenteral administration and organ perfusion, but is preferably stored in a freeze-dried form because of the unfavourable stability properties of the solutions. Agents for establishing physiological conditions are, for instance, buffer substances, ions for establishing correct osmolality and, when necessary, col loid-osmotical ly active substances, such as albumin and clinical dextrans, i v t v a 40. 11 jr j ι / i w y ι v« u i * j u « π n l —. l tion is adjusted as necessary with the aid of a base or an acid (which will provide phys ologically acceptable salts) to obtain a physiologically acceptable pH (which is selects in dependence upon quantity and area of application, for example within the pH-range of 3 - 7.6, such as 4 - 7). Sut sequent to being sterilized, e.g. by sterile filtration, t) e.g solution, such as a solution of conventional composition The pH can be adjusted with the use of bases or acids which provide physiologically acceptable salts, e.g. in¬ organic bases, preferably alkaline metal hydroxides, such as sodium hydroxide or potassium hydroxide, or organic bases, e.g. basic a ino acids, such as arginine or ornitine or tris or methyl gl ucamine or histidine or..inorganic acids, such as HCl o tion hereto in accordance with the aforegiven molar stoichiometric relationships. In accordance with a further method of preparation, a sterile PEP and ATP in solid state (e.g. in freeze-dried form) are dry-mixed in the aforegiven mutual quantity ratio, whereafter the mixture is conveniently pre-packed in dosage units, such as bottles containing a quantity of the mixture corresponding to a normal dose. Prior to being used, a dosage quantity of the mixture is then dissolved in a given volume of a sterile, physiologically acceptable aqueous liquid, e.g. an isotonic buffer solution, such as a tris buffer solution or a phosphate buffer solution. In accordance with one embodiment of the invention, the composition may also contain magnesium ions, for example in a stoichio etric relationship of about 1:1 in relation to ATP. These ions may be supplied, for example, by introducing a physiologically acceptable magnesium salt, preferably MgCl 2 5 in a measured quantity at a suitable stage in the preparation process, e.g. added to a buffer solution in which a powderous mixture of PEP and ATP is dissolved, or by dissolving the magnesium salt in a physrόlδgtcally iso¬ tonic saline solution in conjunction with dissolving PEP and ATP therein. The composition according to the invention is ad¬ ministered in the form of a sterile solution for parenteral administration and organ perfusion, to prevent and treat ischemic cell damage (preferably in humans but also in other mammals). An important area of application for the composition according to the invention is its use as a per¬ fusion and preservation solution for organs, e.g. in open heart surgery, and organ transplantations. Another important area of application is the use of the composition as an in¬ fusion solution for directed infusion through cathethers for treating ischemic brain and heart damage as result of. heart failure, drowning, an overdose of certain types of pharmaceutical preparations, such as soporifics etc. The active substance concentration of the solutions in¬ tended for administration varies in dependence upon the distance from the site at which the solutions are ad¬ ministered ' to the location of the ischemic cell damage to be treated. Suitably, the concentration of the solution at the location of application remote from the location of the damage is such as to achieve an extracellular concentration of ATP at the injured location of an order of magnitude of 1 - 4 % , preferably 2 - 3 %, of the intracellular physio¬ logical concentration of ATP in the body cells. This means, for examnle, in the case of perfusion solutions and pre¬ servation solutions that the concentration of such solu¬ tions with respect to PEP should be such that the organ (e.g. a heart) obtains during the whole of the perfusion period a total of, for example, 20 - 60 mmoles PEP. If the total perfusion volume is calculated to be one litre, the solution may contain for example 20 - 60 mmoles PEP per litre. If the solution volume totals two litres, the solu¬ tion may correspondingly contain 10 - 30 mmoles PEP per litre. The amount of ATP is selected in relation to the amount of PEP, so as to obtain the aforegiven mutual molar relationshi p. When treating ischemic cell damage in organs- by infusion, the dosage may be, for example, 0.1-2, e.g. 0.5-1, mole PEP per kilo body weight, the amount of ATP being selected in relation to the amount of PEP so as to fulfil the aforegiven molar stoi chi ometry. For example, in the case of a directed dosing of PEP of 1 mmole/ kilo body weight to a patient weighing 75 kilos, said patient obtains ' a total of 75 mmoles PEP (and ATP in relation thereto). This can be administered with a 50 ml solution, the PEP concentration of which is 1500 mmoles per litre. With respect to infusion solutions for directed infusion through catheters, a higher concentration is suit¬ ably selected, to compensate for dilution and losses from the infusion site to the target organ. Thus, the amounts, in which the composition according to the invention is administered will vary with the area of application. The invention also relates to a method of preventing and treating ischemic cell damage in mammals, including humans, or in organs removed therefrom, the method being characterized by supplying to the mammal or to the organ a therapeutically effective dose of a pharmaceutical com¬ position according to the invention. The invention will now be desribed in more detail with reference to a number of examples. EXAMPLE 1 1.6 mmole monosodium phosphoenol pyruvate and 0.01 mmole disodiumadenosi ne-5 ' -tri phosphate are dissolved, while stirring, in 100 ml of a physiological saline solution (0.9 g NaCl/ 100 ml), whereafter the pH of the solution is ad- justed to 6.4 - 7.0 by adding dropwise to said solution 1 N and 0.1 N sodium hydroxide solution. The resultant solu¬ tion is sterilized by sterile filtration, poured into 20 ml bottles and lyophilized, whereafter the bottles are sealed (all under aseptic conditions). Subsequent to dissolving the content of one bottle in a physiological aqueous solution, the resultant composition is suited for organ perfusion in experiments on rats. EXAMPLE 2 The procedure described in Example 1 is followed but with 400 mmoles of monosodium phosphoenolpyruvate and 4 mmoles of disodium-adenosi ne-5 ' -tri phosphate. Subsequent to dissolving the contents of one bottle in a physiological aqueous solution, the resultant composition is suitable for human use with directed infusion. PHARMACOLOGICAL EXPERIMENTS For these experiments there was used a model comprising an excised rat heart, substantially in accordance with Gamble et al . , Am. J. Physiol . ___ : 604612 (1970) although modified so that the abdominal aorta of the supporting rat was connected directly to the ascending aorta of the excised heart in accordance with the following: Unstarved male Sprague-Dawl ey-rats weighing about 300 g were used in the experiment. The ascending aorta of an excised rat heart was connec- ted to the abdominal aorta of a living rat ("supporting rat") functioning as a heart-lung-machine for the excised rat heart by means of a tube ("pump tube"). The coronary effluent from the excised heart was collected in a tempera- ted water-jacketed funnel and retransfused to the supporting rat. After the ischemic period, a pulsatile blood flow was established from the supporting rat to the excised heart. The temperature of the supporting rat and the excised rat heart was kept at 37° + 1°C. Rats intended for supporting function were anaesthetized by intraperitoneal administration of 5-sec . -butyl -5-ethyl - 2-thiobarbi turic acid (Inactin ^ from Byk-Gulden Lomberg Chem. Fabrik GmbH, Konstanz, West Germany) 120 g per kilo body weight. After tracheostomy the right carotis communis was cannulated for continuous blood pressure recording by an EMT 34 transducer (from Si emens-El ema AB, Solna, Sweden) and an M 34 recorder (from Si emens-Elema AB, Solna, Sweden). The right jugular vein was cannulated for transfusion. Heparin in a dose of 200 IU (international units) was ad- ministred intravenously. The supporting rat breathed air spontaneously. The abdominal aorta was cannulated and the cannula was fixed to the aorta by silk ligatures. The cannula was cut close to the aorta and rejoined by a more flexible piece of si 1 icone- ubber tubing, to permit easy clamping. The con- nections permitted blood sampling and infusion. The body temperature was recorded continuously in the abdominal cavity. The heart donors wer anaesthetized with ether. After 200 IU of heparin intraven ously the blood was rapidly collected from the abdominal aorta. Subsequent thoracotomy and ligation of the aortic arch branches, the heart-arch- preparation was excised. The time required for this pro- cedure was 3 - 5 minutes. The ascending aorta was attached to the ccinnula from the supporting rat. Electrodes for ECG-recording were placed at the apex, the right atrium and the aortic arch. During the ischemic period, the heart was kept in a water-jacketed funnel filled with plain saline solution at 37 - 38 C. The retransfusion system consisted of a peristaltic roller pump (Mul ti perpl ex 2115 from LKB-Produkter AB , Brom a, Sweden), a temperature-controlled water bath and an air-bubble trap. All connections were of silicone rubber tubing. The system was primed with 20 ml of heparinized blood collected from the heart donor and an additional rat. The retransfusion of blood was adjusted to constant blood- pressure of the supporting rat by regulating the height of the bubble trap. Normother ic ischemia was promoted by clamping the pump tube. After the ischemic period, the tube clamp was re¬ leased and a pulsative blood-flow was re-established through the coronary arteries of the excised heart. All excised hearts were subjected to 15 minutes of complete global (37°C) ischemia, calculated from the time from excision of the heart to the start of reperfusion. Re- perfusion with the pulsative arterial blood flow from the supporting rat was continued for 30 minutes, after which all hearts were immediately f eeze-cl amped with a pair of chilled tongues (in liquid nitrogen) and transferred in liquid nitro¬ gen. Freezing was instantaneous. There was administred to a group of 11 excised rat hearts (group 1) through continuous perfusion simultaneously with the start of the reperfusion, 10 ml of physiological saline solution at room temperature through the pump tube. Perfu¬ sion was continued for seven minutes. 10 ml of solution according to Example 1 were administred to a further group of seven excised rat hearts (group 2) in the same manner as in group 1 , by perfusion through the pump tube. The following biochemical analyses were made: Subsequent to being frozen, the excised hearts were stored at a temperature of -70 C and then freeze-dried at a temperature beneath -4°C. Adenine-nucl eoti des and energy charge. were determined after perchloric acid extraction in the heart material using an HPLC- ethod (HPLC = High Performance Liquid Chromatography) . Thus, the analyses made it possible to accurately determine quantitatively the adenosine triphosphate (ATP), adenosi ne-di phosphate (ADP) and adenosi ne-monophosphate (AMP) enabling subsequent cal¬ culation of the energy charge (ACP, i.e. "adenylate charge potential") in accordance with the Atkinson equation (see Atkinson, D.E.: "The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modi- fiers". Biochemistry _7 (1968) 4030-4038). The enzyme creatine kinase (CK) efflux was followed as an indicator of tissue damage. The principle for determining CK in serum was in accordance with recommendations from the Scandinavian Enzyme Committee, and the enzymatic activity was expressed in . ,ukat/l. (With respect to the HPLC-method applied, re¬ ferences were made to Hartwick, R.A. and Brown, P.R. , "The performance of microparticl e chemically bonded anion- exchange. resins in the analysis of nucleotides ." , J. Chromatog. 112 (1979) 651 - 660). OMP The results are given in the following table, = the number of hearts in the group. The values are given as mean values - standard deviations When making a comparison between group 1 (control group) and group 2 (treatment group) several important differences were found : The difference in ACP is statistically significant (p < 0.02) as are also the differences in ATP/ADP-ratio (p < 0.05) and the difference in the release of creatine kinase (as a measurement of cell-leakage) in the range of 20 - 30 minutes (p < 0.02). In addition to the important biochemical differences shown in the table, the treated hearts in group 2 exhibited substantially improved contractibil ity when loaded than those in group 1, which had not been treated with the solution according to Example 1. O PI

Claims: C L A I M S 1. A pharmaceutical composition for preventing and treating ischemic cell damage, c h a r a c t e r i z e d i n that it contains a physiologically acceptable, water-soluble salt of phosphoenolpyruvic acid and a physiologically acceptable, water-soluble salt of adenosine- 5 1 -triphosphoric acid in a mutual ratio of from 5:1 to 1000:1 , preferably from 10:1 to 500: 1 , calculated on a molar basis. 2. A pharmaceutical composition according to Claim 1 , c h a r a c t e r i z e d i n that it in addition, to the salts of phosphoenolpyruvic acid and of a adenosine-5 ' -triphos¬ phoric acid comprises agents for establishing physiological conditions and, when necessary, water as a carrier. 3. A pharmaceutical composition according to Claim 1 , c h a r a c t e r i z e d i n that it exists in a freeze-dried form or in the form of a sterile solution for parenteral administration. 4. A pharmaceutical composition according to Claim 1 , c h a r a c t e r i z e d i n that it contains magnesium ions in a stoi chi ometri c relationship of about 1 :1 in relation to adenosine triphosphate. 5. A method of preventing and treating ischemic cell damage in mammals, including humans, or in organs excised therefrom, c h a r a c t e i z e d b y administering to the mammal or to said organ a therapeutical ly effective dose of a pharmaceutical composition according to Claim 1. 6. A process for the preparation of a pharmaceutical composition for preventing and treating ischemic cell damage, c h a r a c t e i z e d b y dissolving a physiologically acceptable water-soluble salt of phospho¬ enolpyruvic acid and a physiologically acceptable, water- soluble salt of adenosine-5 ' -triphosphoric acid in a mutual ratio of from 5:1 to 1000:1, preferably from 10:1 to 500:1, calculated on a molar basis, in a physiologically accept¬ able aqueous liquid, adjusting the pH of the solution as necessary with the aid of a base or an acid to obtain a physiologically acceptable pH, then sterilizing the solu¬ tion and, when desired, freeze-dryi ng the resultant solution, OMPI

Inventors: HULTMAN JAN LENNART, RONQUIST GUNNAR, HULTMAN, JAN, LENNART, RONQUIST, GUNNAR
Applicants: PHARMACIA AB, PHARMACIA AKTIEBOLAG
Application year: 1983
Main classification: A61K31
IPC codes: A61K31