Field of invention

The present invention is directed to alpha-ketoglutarate containing infusible energy substrate preparations and the uses thereof. The preparations are advantageous by their capacity in improving the ischemic tolerance of organs interrupted from their regular blood flow and in the improvement of the recovery of such organs.

Background of the invention

It has been shown that the amino acid metabolism is of a large importance in the tolerance of ischemic damages in the heart. The metabolism of amino acids is also an important factor for the recovery of hearts subjected to global ischemia from a myocardial infarction or a surgical trauma. There are several studies of how an infused supply of certain amino acids lead to myocardial protection from ischemic damages, see e.g. J Thorac Cardiovasc Surg, Vol. 101, 1991, pages 23-32, (PL Julia et al.) J Thorac Cardiovasc Surg, Vol. 100, 1990, pages 888-895, J Thorac Cardiovasc Surg, Vol. 105, 1993, pages 513-19 and the references cited therein.

In cardiac surgery it has become state of the art to infuse cardioplegic solutions to stop the heart and reduce its energy consumption, while protecting the heart from ischemic damages when the blood flow is temporarily interrupted during the surgical procedures

Amino acid enriched cardioplegic solutions are described by e.g. the U.S. patent 4,988,515 wherein an improved glutamate/aspartate containing cardioplegic solution is described. This document shows how a cardioplegic solution with a specific calcium ion concentration range and osmolality range improves cardiac recovery from ischemia after interrupting the blood flow to dogs' hearts. Concentrated aqueous solutions of the amino acids, calcium ion and glucose are mixed with other standard additives and a cardioplegia compatible diluent or carrier, such as blood, stroma free haemoglobin, oxygenated plasma, crystalloids and fluorocarbons. The study in J. Thor. Cardiovasc. Surg., Vol. 104, No. 4, 1992, pages

1141-7, ( F Beyersdorf et al.) demonstrates how a glutamate/aspartate enriched blood cardioplegical solution treatment may salvage hearts thought to be irreversibly damaged after a cardiac arrest.

Certain studies has also shown that branched-chain amino acids in a cardioplegic solution have a protective effect against ischemic damages and they have also been suggested as a supplement to nutritional regimens for cardiac patients, as in the European patent EP 0283 513. The U.S. patent 4,415,556 discloses a solution containing potassium alpha-ketoglutarate that improves the ischemic tolerance of the heart during cardiac surgery when the blood supply is interrupted. A better energy status is demonstrated after 300 minutes ischemia at 23°C with a cardioplegic solution containing 1 mmol potassium alpha-ketoglutarate in a histidine/tryptophane buffer with the addition of conventional electrolytes and a sugar and a polyol. The herein described HTK -s olution (histidine-tryptophane-ketoglutarate) is disclosed in several articles as a state of the art cardioplegic solution for increasing the ischemic tolerance during heart surgery. According to the article in Pflugers Archiv, Vol. 415, 1989, pages 269-275 (E Krohn et al.) the HTK- solution is intended to be used clinically, immediately after aortic cross clamping when a cold solution (4 to 8°C) is infused into the coronary system for 6 to 10 min. During this aerobic equilibrium period the heart muscle will be arrested, the myocardial temperature decreases from 37°C to 10°C and the θ2-consumption decreases from about 7 to 0.1 ml/min/100 g. The low content of alpha-ketoglutarate comprised in the HTK-solutions might be regarded as attempts to recreate the original intracellular conditions in the heart after it has been interrupted from its normal blood supply.

In British J. Int. Care, February 1994, page 41, it is suggested that it is time to re-examine the possibility of the cardioprotective effects of alpha- ketoglutarate and glutamine in appropriate patients.

The German patent application DE 39 43424 teaches a parenteral composition of alpha-ketoacid analogues to certain essential amino acids to be used as a nutrient supply for patients with renal insufficiency that cannot tolerate amino acids. The solutions can contain 15 g/1 of alpha-ketoglutarate. In the European Patent EP 318446 a nutritional composition containing 5 to 25 g/litre of alpha-ketoglutarate is disclosed to improve the negative nitrogen balance and protein catabolism that occurs in postoperative and post traumatic states.

The International patent application WO 93/23027 discloses an infusible composition providing more than 0.25 g/kg body weight/ day of alpha-ketoglutarate to critically ill patients in order to improve the protein synthesis capacity and to maintain the energy level in skeletal muscle tissue. In a suitable composition according to this application the alpha-ketoglutarate

amount exceeds 17.5 g/1, preferably more than 25 g/1 in a 1 1 amino acid solution per day dose, which for a 70 kg patient will correspond to more than 0.25 g/kg bw and day when administered with conventional total parenteral nutrition constituents. This disclosure is generally directed to the treatment of such critically ill patients having more than a 50% depletion of their glutamine pool in the skeletal muscles, who suffer from severe complications like sepsis and multiple organ failure. It does, accordingly, not reveal anything about the specific energy substrate requirements in the post-traumatic heart or any other vital organs subjected to ischemia, for example, during bypass surgery or during preservation of organs for transplantation.

Immediately after a cardiac trauma (such as surgery or infarction) the heart metabolism is altered so that its substrate uptake is insufficient with respect to its oxygen consumption. This condition seems to lead to a catabolic status where the myocardial tissue utilizes endogenous substrates such as glycogen and also, to a large extent amino acids rather than free fatty acids (FFA), glucose or lactate which are the normal substrates in the energy production of the heart. The change in metabolism is most expressed during the first hours after the trauma, whereupon the metabolism is gradually normalised for about eight hours. The exact mechanism behind this change in metabolism is not clearly known, but an explanation within the context of the findings of the present invention is that the supply of intermediates to the Kreb's cycle of energy production is reduced, because a net-export of glutamine from the skeletal muscles and the heart to the kidneys and the splanchnic organs is induced by the general trauma reaction. The reduced capacity of the Kreb's cycle will lead to a reduction in the ability to utilize FFA-metabolites, while the insulin resistance leads to a low use of glucose and lactate in the myocardial tissue.

The increased utilisation of glutamate and branched-chain amino acids (BCAAs) of the heart shows the importance of amino acids for the post- traumatic myocardial energy production and that the supply of amino acids can be rate limiting for this production. It would, however, be difficult to supply the heart with large amounts of amino acids, such as glutamate or glutamine, as suggested in the European patent application EP 0483 614, since their transportation across the cellular membranes are limited. A large amount of infused glutamate, which is slowly metabolised in the body, can also lead to side effects due to its neurotransmitting capacity. The efficacy of an infusion of glutamine will also be reduced by the enzymatic activity of glutaminase.

It has been demonstrated that critically ill patients infused with alpha- ketoglutarate maintain their energy level in the skeletal muscles and recover more quickly from a postoperative catabolic status. In previous studies by the inventors it has been indicated that the availability of Kreb's cycle intermediates and their amino acid precursors may be rate-limiting for the myocardial energy production during and after cardiac surgery. In the references cited above, it is shown that certain amounts of amino acids or amino acid precursors in cardioplegic solution, can improve the cardiac recovery after traumas that may lead to ischemic damages. There is a constant demand for improved energy substrates and /or cardioplegical solutions to reduce morbidity and the need of intensive care following both cardiac traumas and other severe conditions related to a reduced blood supply to vital organs. The present invention is directed to new highly concentrated alpha-ketoglutarate containing energy substrates, uses thereof as well as methods involving said compositions. The preparations are especially useful for preventing the myocardial tissue from ischemic damages during cardiac surgery, thereby reducing the postoperative morbidity and the need for postoperative intensive care.

Description of the invention

The present invention is related to compositions providing a total parenterally infused dosage of 5 to 250 gram alpha-ketoglutarate for improving the ischemic tolerance of vital organs interrupted from their regular blood flow and for improving the recovery and /or normal metabolism of such organs. Preferably the compositions shall provide the patient with total amounts of between 5 to 50 g alpha-ketoglutarate, and more preferably from 10 to 30 g alpha-ketoglutarate.

The compositions are intended to be used as energy substrates, useful for preventing organs from damages during surgery, or other critical conditions when the blood supply to a vital organ is interrupted, such as the first phase after a myocardial infarction or for perfusion and preservation of organs that shall be transplanted.

The compositions of the according invention shall preferably be capable of providing an alpha-ketoglutarate level of about 5 to 100 mM in the fluid passing through the organ interrupted from its normal blood flow and more preferably about 10 to 50 mM.

Uses of the compositions and methods of their clinical applications also are apparent from the appended claims.

Due to the reduced stability of alpha-ketoglutarate in aqueous solutions the compositions will preferably be in a dry powdered form, prepared by methods like lyophilisation or spray drying, that can be readily reconstituted to an administerable solution, just prior to the use. It is advantageous to provide such preparations in sterilizable multi-compartment packages or kits, wherein the solid alpha-ketoglutarate and the reconstitution solution are kept apart during the storage. The person skilled in the art is familiar with numerous solutions to this type of technical problems which therefore not will be discussed in more detail.

The reconstitution solution will consist of a carrier or diluent that is suitable for a composition of the intended type of administration, that can be, for example, a cardioplegical solution, a solution to be administered to the heart just after a myocardial infarction, an infusion solution to be administered to other vital organs subjected to an insufficient blood flow, a solution to be used in connection with a surgical procedure of a vital organ, e.g. for peri- or postoperative administration and a solution useful in the storage and perfusion of organs to be transplanted. Each suitable carrier or diluent can further contain other specifically adapted, but conventional constituents in the form of energy substrates, such as carbohydrates, amino acids, polyols, buffering agents, electrolytes, stabilizers and certain trace elements, as will be disclosed in more detail below. It would also, of course, if necessary due to stability reasons, be possible to incorporate such constituents also in dry, reconstitutible form, either together with the alpha-ketoglutarate, or separately.

It is also highly important to consider the physiological interaction of administered counter ions to alpha-keto glutarate, since for example sodium ions in large amounts may be undesired and give rise to adverse effects. In fact, in certain clinical applications the amount administered sodium or potassium ions can delimit the amoimt of alpha-keto glutarate possible to supply. When large amounts of alpha-keto glutarate is desired to be administered, as in accordance with the present invention, it is therefore advantageous to select a counter ion which is physiologically acceptable, such as certain organical basic compounds and thereby exchange all or parts of the undesired counter ions. Examples of suitable counter ions are tromethamine (2-amino-2- hydroxymethyl-l,3-propanediol or THAM), ornithine, lysine, histidine and arginine.

The reconstituted solutions can be further mixed with suitable conventionally used vehicles, such as extracorporeal blood from the patient in blood cardioplegia, crystalloid solutions, oxygenated plasma and transfusion emulsions containing synthetic oxygen carriers. The parenteral infusion rate of a solution containing a total dosage of 5 to 250 g alpha-ketoglutarate intended to prevent ischemic damages in an organ will vary considerably due to the condition it shall be used for and the desired length of the dosage. Normally, the infusion rates will vary between 0.5 g/h and 100 g/h. Solutions infused to patients subjected to cardiac surgery or during the first phase after a myocardial infarction shall supply a total amount of alpha-ketoglutarate of at least 5 to 250 g, preferably between 5 to 50 g, and more preferably from 10 to 30 g, either during intermittent or continuous administration. A suitable solution for cardioplegic use can contain totally about 20 to 30 of alpha ketoglutarate and be diluted with extracorporeal blood and intermittently infused to the heart by the aorta during cardiac surgery. During cardiac surgery such a solution is infused during about 2 to 5 minutes, whereupon the flow is stopped for a period for performing the surgical procedures before repeating the infusion. In total, such an alpha-ketoglutarate dosage is administered within a couple of hours. The infusion rate for other treatments of organs in danger of obtaining ischemic damages can vary considerable to the conditions and the given dosage can continuously be administered for several days, if necessary. The alpha-ketoglutarate containing solutions, for administration to organs interrupted from their regular blood flow, can, added to their carriers or diluents, contain suitable amounts of electrolytes such as sodium, potassium, and magnesium chloride, bicarbonates and phosphates; suitable carbohydrates, such as glucose, fructose, dextrose, ribose, mannitol and other polyols; amino acids, such as aspartate, glutamate, glutamine, histidine, tryptophane and branch chained amino acids can be included in the solution; as well as suitable buffering agents, such as citrate, acetate and tromethamine (tris buffer); and also certain other bioactive constituents, such as insulin, glutathione, adenosine and antibiotics, if desirable for a specific clinical application. Besides the heart, other vital organs, such as the liver, the kidneys and the intestines can benefit from the infusion with the alpha-ketoglutarate containing solutions in, or after critical situations, with an insufficient blood supply, such as surgical procedures.

Alpha-ketoglutarate containing solutions useful for perfusion and preservation of organs shall preferably comprise carriers suitable for such fluids like oxygen carrying emulsions. In addition they may comprise other suitable energy substrates, amino acids, electrolytes, carbohydrates, buffering agents, and stabilizers.

In the following exemplifying part it is demonstrated that the infused supply of high amounts of alpha-ketoglutarate leads to an improved cardiac recovery after a surgical trauma. In these tests, it is shown that the provision of high doses of alpha-ketoglutarate increases aerobic and anaerobic metabolic rate which effects are linked to an improved myocardial recovery. It is also found that alpha-ketoglutarate has a surprisingly high capacity in making vital organs, like the heart, to withstand ischemic conditions, which can not only be explained by its property as an energy substrate, but must also be explained by an influence on the activity of the induced proteolysis and /or degradation of contractile elements in the damaged tissues.

The following tests are not intended to delimit the scope of the invention that should be comprehended by the appended claims.

Detailed description of the invention

27 patients have been included in a randomized controlled study during and shortly after cardiac surgery.

Patients in the control group received blood cardioplegia comprising blood from the extracorporeal circuit (hematocrit 25%) and Plegisol ® (from Abbott) containing 15 mmol KC1/500 ml and 5 ml sodium bicarbonate 8.4%.

Blood and Plegisol® were mixed at a 4:1 ratio and delivered with a pump and a heat exchanger. When the aortic clamp was applied, 1000 ml of the cardioplegic solution was delivered through a cannula in the aortic root at a rate of 260 ml/min. Another dose of 500 ml blood cardioplegia was infused after the completion of each peripheral anastomosis. The temperature of the blood cardioplegia was 8°C. After completion of the last peripheral anastomosis, the final cardioplegia dose was given with the blood cardioplegia rewarmed to 30°C. The terminal warm dose was given with at least 500 ml, or more, until the septal temperature reached 25°C. The aortic cross-clamp was thereafter released so that the heart was reperfused in a normal manner.

In the alpha-ketoglutarate allocated patients, 30 grams of alpha- ketoglutarate were added to 500 ml Plegisol®, and delivered with the blood cardioplegia, which was delivered exactly as described with the control

patients. Depending on the duration of the cardioplegic ischemia, this regimen resulted in that a total dose of 15 to 30 grams of alpha-ketoglutarate was delivered.

To evaluate the effects the blood cardioplegia content of oxygen, lactate, creatine kinase isoenzyme MB (CK-MB), and troponine-t was measured and related to the corresponding concentration of blood in the coronary sinus. Thus, blood entering the heart was compared with blood leaving the heart. When the concentration of a substance is higher in the blood entering the heart, than leaving the heart, this implies that an uptake of the measured substance has occurred during the passage through the heart. In the same way, when the concentration of a substance is higher in the blood leaving the heart, this indicates a myocardial release of the substance. In the first case, the extraction value is positive, and in the second case, it is negative. In the present context, oxygen extraction was positive indicating that oxygen was consumed by the heart. Correspondingly, the lactate extraction was negative, signifying a myocardial release. Oxygen extraction gives an estimate of the aerobic oxidation, whereas, lactate release represents anaerobic activity.

Measurements were performed at the end of each infusion of blood cardioplegia, and during the first reperfusion period (1 hour). During this period the arterial blood concentration was measured instead of blood cardioplegia concentration, since both represent blood entering the heart. Finally, arterial levels of CK-MB and troponine-t were measured every hour for 3 hours after the operation.

Figure 1 shows myocardial oxygen extraction (aerobic capacity) during cold (4 to 8) and warm (9) cardioplegia and after blood cardioplegia

(during reperfusion 10 to 12), (mean ± SEM), in control patients and in patients treated with alpha-ketoglutarate. Oxygen extraction represents aerobic oxidation in the heart and was greater during early reperfusion in the alpha- ketoglutarate treated patients. Figure 2 shows myocardial lactate release (mean ± SEM) in control patients and in patients treated with alpha-ketoglutarate, during cold (4 to 8) and warm (9) cardioplegia and after blood cardioplegia (during reperfusion 10 to 12). The release of lactate, reflecting anaerobic oxidation, was greater in the alpha-ketoglutarate treated patients, as signified by a more negative value. Figure 3 gives the blood concentration in arterial blood of ischemic marker CK-MB (representing myocardial dysfunction and membrane leak), means± SEM of the two study groups, during blood cardioplegia (2 to 9) and during 4 hours reperfusion (10 to 15). CK-MB is used as a marker for

myocardial ischemia. The lower concentrations in the alpha-ketoglutarate group indicates that alpha-ketoglutarate protects the heart from ischemia to a higher degree.

Figure 4 gives similar data for troponine-t during blood cardioplegia and reperfusion. Troponine-t is a highly cardiospecific substance signifying myocardial ischemic injury.

The results indicate that alpha ketoglutarate in the infused doses of totally 15 to 25 gram leads to a doubled myocardial aerobic capacity during blood cardioplegia and increased with 70% during the early reperfusion period. The anaerobic metabolic capacity, represented by myocardial release of lactate is demonstrated to be doubled during cardioplegia and tripled during the early reperfusion if an alpha-ketoglutarate containing solution is infused. The myocardial release of lactate had ceased within 1 hour when alpha- ketoglutarate in pharmacological doses had been added to the blood cardioplegia, while it was maintained in the control patients. This implies that aerobic metabolism was normalized quicker with alpha-ketoglutarate.

The ischemic markers creatine kinase (CK-MB) and troponine-t, which represent values proportional to the degree of ischemic cellular damages, were released to higher levels in control patients, see Figures 3 and 4. These levels were related to preceding aerobic capacity (r=0.65) and anaerobic capacity (r=0.60). The r-values represent the correlation coefficients for the oxygen extraction and the lactate release of the myocardium, 5 minutes after reperfusion, when correlated to the arterial level 4 hours later.

The highly significant difference in troponine-t levels during the reperfusion phase, as demonstrated in Fig. 4, between control patients and patients who have received alpha-ketoglutarate is of a considerable clinical interest, since troponine-t can be regarded as a marker of the induced activity of proteolytic enzymes during the ischemic events. This suggests that alpha- keto glutarate not only is active as an energy substrate for the myocardium, but also is active in inhibiting the catabolism during the ischemia by reducing the degradation of contractile elements in the myocardial tissue and /or inhibit proteolytic enzymes.

It is evident that an alpha-ketoglutarate containing cardioplegic solution, according to the present invention, provides an improved myocardial protection during cardiac surgery and enhances the recovery of the heart, which is an important finding, since cardiac dysfunction is one of the major problems in cardiac surgery. The invention is therefore likely to have significant clinical effects.