BR112015029121B1 - PROTECTIVE SOLUTION TO PREVENT OR REDUCE BRAIN AND WHOLE BODY REPERFUSION - Google Patents

Abstract

PROTECTIVE SOLUTION TO PREVENT OR REDUCE REPERFUSION OF THE BRAIN AND WHOLE BODY. The present invention relates to a protective solution for preventing or reducing brain reperfusion injury containing magnesium ions and having an osmolarity of 350 to 600 mOsm/L, a pH value of 6.8 to 7.8 and albumin in an amount of 1 to 20% by weight.

Description

[0001] Sudden circulatory arrest is frequent with an incidence of 60/100,000 residents in the European Union. Cardiopulmonary resuscitation (CPR) including external chest massage, defibrillation, mechanical ventilation and drug therapy is the established mode of treatment. However despite increasing efforts in training for medical staff and potential bystanders, improved equipment and extensive interdisciplinary guidelines, CPR remains a difficult and somewhat improvised task. Furthermore, the outcome regarding survival and neurological recovery is not satisfying: mortality rates of >70% are reported in patients undergoing in-hospital CPR than much higher rates in out-of-hospital settings. Furthermore, brain injury is frequent in the rare case of survival.

[0002] Trummer et al., The Journal of Thoracic and Cardiovascular Surgery, May 2010, pp., 1325-1332 report successful resuscitation after prolonged periods of heart attack. According to this report it was possible to resuscitate in an animal model using pigs up to 15 minutes after the heart attack. In the experiments, extracorporeal blood circulation was established using Ringer's solution together with heparin as the initiating solution.

[0003] It is an object of the present invention to provide protective solutions that can be used in the process of resuscitation after a heart attack. It is desirable to keep damage in particular to the brain as low as possible. It has been observed in many cases where patients were resuscitated after a longer period after a heart attack that significant brain damage occurred. Such damage had the effect that people were severely compromised as far as the mind was concerned and such patients would not return to normal life.

[0004] Protective solutions are described here which can be used preferably in resuscitation procedures which are in more detail described for example in WO 2011/045011. Within a relatively short period of time after the heart attack, blood circulation has to be re-established. When heart attack occurs in a hospital, for example in the course of heart surgery treatments, the conditions are better manageable compared to cases where sudden heart attack occurs outside the hospital. There is usually a delay of time before doctors and bystanders can restore blood circulation. The organ that is most seriously compromised by a heart attack is the brain. The protective solutions described here prevent or reduce the damage that is caused by the resumption of blood circulation after a heart attack.

[0005] To this date, it is generally accepted that normal cerebral and myocardial function can only recover when conventional CPR is employed within the first 3 to 5 minutes after circulatory arrest. Therefore, since time is the most important factor in determining patient outcomes after CPR, every attempt is made to begin CPR as soon as possible after the heart attack to avoid post-resuscitation brain damage. However, from a pathophysiological point of view, circulatory arrest following heart attack can be interpreted as whole-body ischemia followed by reperfusion injury when the blood supply is restored. This phenomenon is known as an “ischemia reperfusion injury” in the tissue of certain organs. However these organs can be saved for very long intervals if the initial reperfusion after a significant ischemic discharge is controlled in terms of the reperfusion conditions (pressure, flow, temperature) and the composition of the reperfusate. Beneficial effects of this treatment regimen have been shown in cardiac muscle, skeletal muscle, liver, lung, and kidney tissue.

[0006] Assuming that a reperfusion injury of severe ischemia of the whole body and brain after circulatory arrest is the underlying cause of morbidity and mortality after CPR, special attention was paid to conditions of whole body reperfusion after CPR. The option to connect the patient via cannulation of arterial and venous vessels to an extracorporeal life support system (ECLS) with subsequent stabilization using cardiopulmonary bypass has been shown to be a useful tool to improve survival after CPR. Since restoration of circulation and a sufficient blood supply to organs, especially the brain, are key aspects concerning CPR, the use of ECLS during and after CPR potentially allows control of reperfusion conditions in terms of blood pressure, flow, and reperfusate. However, efficient conditions for whole-body reperfusion after normothermic heart attack have yet to be defined.

[0007] While it is the goal in resuscitation to avoid severe damage to the brain likewise other essential organs should not be fatally affected. In resuscitation procedures, care has to be taken that essential organs such as in particular the heart, liver, lung and kidney are not damaged to such an extent that the patient cannot survive resuscitation. Since the best conditions for each organ may not be identical, sometimes the best compromise has to be selected that avoids severe brain damage on the one hand and irreversible damage to other essential organs like heart or lung on the other hand.

[0008] Within an extended series of animal experiments the relevance of these conditions was explored in an established porcine model. The results obtained with this allow a safe extrapolation to humans. A primary objective of this research is to provide controlled perfusion of the whole body with priority given to the demand of the most sensitive organ, the brain. As described above, reperfusion injury from ischemia, expressed as the extent of cerebral edema, should be limited as far as possible. In addition to defining the physical conditions of reperfusion (temperature, pressure/blood flow), the composition of the reperfusate is the objective of this description. In preferred modalities, hypocalcemia, hypermagnesemia and hyperosmolarity are relevant aspects to avoid edema in the reperfused tissue. The protective solutions described here are also referred to as priming solutions.

[0009] Without wishing to be bound by theory, the use of these elements in a modified reperfusate is based on the following considerations:

[00010] A key aspect of the initiator solution is hyperosmolarity. Osmosis occurs when a substance in solution crosses a membrane from an area of low concentration to an area of higher concentration to establish equilibrium. The concentration of dissolved particles in solution expressed as moles of solute per liter of solvent is referred to as "osmolarity". In human plasma, the concentration of dissolved particles is about 0.290 mol. Therefore, its osmolarity is 290 mOsm/L. The normal human plasma range normally extends from about 250 to 310 mOsm/L.

[00011] Water flows from an area of low osmolarity to an area of high osmolarity at a rate directly proportional to the (radiant) difference in osmolarity until equilibrium is reached. Solutions containing the same concentration of particles as blood are iso-osmotic (isotonic). In medicine, a 0.9% sodium chloride solution that is iso-osmotic with blood and the venous endothelium is often used. Solutions with a lower osmolarity (a lower concentration of resolved particles) are designated as hypotonic. Solutions with an osmolarity higher than that of normal saline (0.9% sodium chloride) are designated as hypertonic or hyperosmolaric.

[00012] The protective solutions of the present invention are hyperosmolaric and have an osmolarity ranging from 300 to 700 mOsm/L. A preferred range is 400 to 600 mOsm/L and particularly preferred is an osmolarity of 440 to 550 mOsm/L. All particles dissolved in solution contribute to osmolarity.

[00013] A preferred component for increasing the osmolarity of the protective solutions described here is albumin, preferably human albumin. Human albumin was chosen as a basic component of the initiator solution to generate a hyperosmolaric reperfusate. The comparable high molecular weight of albumin potentially reduces the transfer of these molecules into the extravasal space and potentially binds to intracellular fluid preventing cellular edema.

[00014] In addition to human albumin, the hyperosmotic properties of sugar alcohols such as mannitol can be used as a useful supplement to the starter solution. In other modalities, it is possible to use other substances in the same way to increase osmolarity. However, it has to be considered that some of such components likewise have side effects when used as infusion solutions. Mannitol has, for example, a diuretic effect. Other suitable components are sugars such as, for example, glucose. By selecting other components, unwanted side effects have to be avoided.

[00015] Another important aspect of the protective solutions described here is the increased content of magnesia ions in the solution. The high concentration of magnesia ions (Mg2+) is believed to have protective effects in particular with regard to preventing or reducing damage caused by reperfusion injury. Cytoprotective effects of hypermagnesemia have been described on respiration in isolated cardiac mitochondria. Furthermore, platelet aggregation may be decreased with potential effects on the “no-reflux” phenomenon which is another symptom of reperfusion injury. Therefore preferably magnesium is added to the starter solutions described here. Mg2+ ions can be introduced into the solution in the form of a suitable salt. A preferred component contributing to high Mg2+ concentration is magnesium citrate. In this preferred embodiment the citrate anion has other advantageous properties as described below. Other preferred sources of Mg2+ ions are magnesium sulfate or magnesium aspartate.

[00016] Since ischemia causes an insufficiency of the cell membrane-based and energy-dependent Na+/Ca2+ antiporter, the concentration of calcium in the cytosol is increased excessively with subsequent accumulation of fluid within the cell. This increase in fluid is synonymous with an edema of the cell ultimately leading to malfunction and potentially terminal cell failure. Therefore the reduced supply of calcium to the cell limits this effect. Reduction of the calcium content is achieved by adding magnesium citrate or sodium citrate to the starter solution. Alternatively, other chelating agents, such as for example 2,3-dimercapto-1-propane sulfonic acid (DMPS), alpha lipoic acid (ALA) or ethylenediamine tetraacetic acid or methylamine can be used. Selection of the proper chelating agent depends on the other components of the solution. Chelating agents that bind Ca2+ better than Mg2+ are particularly preferred.

[00017] Another preferred component of the protective solution is lidocaine (2-diethylamino-N-(2,6-dimethylphenyl)acetamide). Lidocaine is available for example under the trade name Xylocaine and is well known as a locally acting anesthetic and antiarrhythmic agent.

[00018] In a preferred embodiment, the protective solution comprises a high dosage of Lidocaine. Dosages range from 1 to 20 mg/kg of body weight, more preferred 5 to 15 mg of Lidocaine per kg of body weight of the patient being treated, and particularly preferred around 10 mg/kg of body weight is added to the initiating solution. The amount of lidocaine present in the protective solution depends somewhat on the body weight of the patient being treated.

[00019] Normally, the protective solution has a concentration of 0.05-1.0 g per l solution. In more preferred embodiments, the lidocaine content ranges from 0.1-0.7g per L solution. Even more preferred is an amount of 0.3-0.7 g per L solution. Since the patient's body weight in an emergency case is not known the Lidocaine concentration can be calculated on an average body weight of eg 70 kg. Lidocaine causes a blockade of voltage-gated sodium channels ultimately leading to a stabilization of the cell membrane. This effect is beneficial in myocardial cells and neurons. Therefore Lidocaine is a preferred component of the initiator solution.

[00020] Another preferred adjunct to the initiator solution is heparin. Heparin is used to provide sufficient anticoagulation, which is necessary to conduct cardiopulmonary bypass. Since many patients are not heparinized at the time of CPR or their coagulation status is unclear, adding this drug early is conceivable.

[00021] In a preferred embodiment, the protective solution for preventing or reducing reperfusion injury to the brain has an osmolarity of 350 to 600 mOsm/L, a pH value of 6.8 to 7.8, preferably 7.4 to 7.6. The physiological pH value of the human body is about pH 7.4. In a particularly preferred embodiment of the present invention the protective solutions described herein have a somewhat lower pH value. It is therefore preferred to use a pH value of around 7.0 to 7.4 which lowers the pH value in the body.

[00022] For maintaining the pH value, the protective solutions described here may also contain a buffering agent to allow an alkalotic pH of the solution at certain times of the perfusion process to neutralize cellular acidosis. A suitable buffering agent can be a phosphate, hydrogen phosphate system or a bicarbonate buffering system. Such a buffer can be administered at a dosage of 0.1 mmol kg body weight and hour up to 3 mmol kg body weight and hour. The buffering agent concentration comprises all ions that can contribute to the buffering effect. In a phosphate buffering solution all phosphate ions (PO43-, HPO42-, H2PO4- and H3PO4) are taken into account when calculating the concentration of the buffering agent.

[00023] One of the components that mainly contribute to the hyperosmolarity of the protective solution is albumin for which human serum albumin is preferably used. Albumin is added to the protective solution in an amount of 1-20%, preferably 215% and most preferably 5-10% by weight based on solution. Albumin is normally available as a solution with a high concentration of albumin, for example, 20% human albumin in solution. Such a solution with a high concentration of albumin is used for the preparation of a protective solution. Although albumin from other sources can just as well be used it is preferred to use human serum albumin as this material is available in large quantities. The human serum albumin to be used must be specifically prepared for human application. This means it must be free of contamination, viruses or other unwanted traces of toxic components.

[00024] In another embodiment, the protective solution likewise contains another sugar alcohol. A preferred sugar alcohol is mannitol. However, it is also possible to use erythritol.

[00025] It is an essential aspect of the present invention that the protective solution contains a high content of magnesium ions. The magnesium concentration ranges from 0.1-15 mmol/L, preferably from 1.0-8.0 and more preferred from 1.5-4.5 mmol/L.

[00026] Another aspect of the protective solution is that the solution should be essentially free of calcium ions. To remove calcium contained within blood vessels, the protective solution contains a chelating agent. A preferred chelating agent is citrate. Citrate may be present in an amount of 0.1-20 mmol/L, preferably in an amount of 1.0-5.0 mmol/L. It is possible in a particularly preferred embodiment for the protective solution to be prepared using magnesium citrate to obtain a high magnesium concentration and to introduce the citrate anions into the solution without counter ions that could disrupt the effect of the protective solution.

[00027] In another preferred embodiment the protective solution contains a substantial amount of heparin. Preferably, the heparin concentration ranges from 5000 to 50,000 IU per L of solution, more preferred is a range of 15,000-40,000 IU per L of solution, and particularly preferred is a range of 20,000-30,000 IU per L of solution.

[00028] An important aspect of the present invention is the viscosity of the protective solutions described herein. In resuscitation procedures where protective solutions (initiator solutions) are used, the solution and the mixture of the solution and the patient's blood have to be pumped through the patient's body and the machine used. The viscosity of the solution therefore plays an essential role. Mixed solution viscosities are difficult to describe precisely. Furthermore, the viscosity is dependent on the temperature of the solution. It is essential that the protective solution described here has a viscosity of less than 3 mPas at 37°C and less than 5 mPas at 32°C. When protective solutions are mixed with human blood, the viscosity of the protective solution should be less than 4mPas at 37°C and less than 6mPas at 32°C. Viscosity is determined by methods well known to the person skilled in the art. A very common method for measuring viscosity is a Brookfield viscometer. Alternatively, however, viscosity can likewise be measured by a capillary viscometer or rotational viscometer. For the measurement of blood plasma a specialized capillary viscometer (hardness viscometer) was recommended. Other methods are for example described in Haidekker et al., Am J Physiol Heart Circ Physiol, 2002, H1609-H1614).

[00029] The protective solutions described here are preferably used for the prevention or reduction of brain reperfusion injury that potentially occurs after cardiopulmonary resuscitation. The term cardiopulmonary resuscitation comprises events that may occur in the hospital, for example heart surgery, or that may occur spontaneously in daily life and likewise outside a hospital, for example after myocardial infarction or spontaneous ventricular fibrillation.

[00030] In a particularly preferred embodiment, the protective solution for reducing brain reperfusion injury is used in a device which is described in detail in WO 2011/04011.

[00031] A preferred protective solution contains lidocaine in an amount of 0.05-1.0 g per L solution, a sugar alcohol in an amount of 1.0-50 g/L, whereby the sugar alcohol is preferably mannitol, magnesium which is present in a concentration of 1.0 to 15 mmol/L, 5000 to 50,000 IU of heparin / L solution, and citrate ions in an amount of 20 to 100 mmol/l solution.

[00032] The protective solution is preferably used in the prevention or reduction of brain reperfusion injury after cardiopulmonary resuscitation.

[00033] In another preferred embodiment, the protective solution is used in preventing or reducing reperfusion injury to the brain after cardiopulmonary resuscitation with a potassium-containing solution of 0.1-25.0 mmol per L solution. In some embodiments, it may be preferable to have a concentration of greater than 8 mmol/L of potassium in the solution. In such embodiments, the potassium concentration is 8 to 25 mmol of potassium per L of solution. The effect of high potassium concentration is that cardiac defibrillation can be avoided or performed less frequently. Adding potassium to the electrical solution and consecutive heart muscle activity is reduced to almost zero. The advantage thereof is that the substrate and energy consumption process of ventricular fibrillation can be terminated. This means that after replenishing heartbeat substrates can be better started. Furthermore, the risk of displacement of inserted cannulas during potential defibrillation is decreased.

[00034] In another embodiment, the protective solution may contain norepinephrine at a concentration of 0.05 μg - 0.5 μg of norepinephrine per kg of the patient to be treated. Since the solutions are prepared in advance, the final concentration in the protective solution can vary from about 1 to 100 mg of norepinephrine per L of solution.

[00035] Ciclosporin attenuates mitochondrial permeability transition pore opening and stabilizes the inner mitochondrial membrane in ischemic cardiomyocytes. Thus cyclosporine A is a preferred component of the protective solution. In another embodiment, the protective solution can contain a concentration of 1.0-17.5 mg of cyclosporin A per kg of patient to be treated. More preferred is a range of 2.5-15.0 per kg of patient and particularly preferred is a range of 4.0 -12.0 cyclosporine A per kg of patient. The solution may therefore contain from 50 mg to 1300 mg of Ciclosporin per L of solution, preferably from 250 mg to 850 mg of Ciclosporin per L of protective solution.

[00036] An especially preferred starter solution is prepared using: Human Albumin 20% 500 mL Mannitol 20% 250 mL Sodium-Citric 3.13% 250 mL Xylocaine 2% 25 mL Magnesium 10% 20 mL Heparin 15000 IE

[00037] Laboratory analysis reveals the following chemical composition of a particularly preferred starter solution.

[00038] Although it is desired to keep the calcium content as low as possible the final solution may contain some calcium which is normally brought into the solution as an unwanted impurity. Since human albumin is obtained from blood it may happen that traces of calcium are introduced into the solution as an impurity. The calcium content should, however, be lower than 0.5 mmol/L.

[00039] This priming solution is used to moisten, flush and de-air the extracorporeal circuit before the patient is connected to a device which is described for example in WO 2011/045011. After connection to the patient and initiation of blood pumping, the initiator solution is mixed with the blood returning from the patient and reinfused into the patient through the arterial line. Dependent on the blood return analysis, the reinfused blood is modified. Part of this modification could be the addition of drugs through a dosing system that is part of our ECLS. The dosing system consists of a main line that is continuously stimulated with priming solution or any other intravenous crystalloid or colloid solution. Drugs can be added via separate side lines to the main line depending on the specific patient requirement. Therefore one can describe a dynamic drug composition at the end of the main line dosing system. Example 1

[00040] The protective solution was tested in an animal model. For the experiments a pig model was used which is described in detail in Trummer et al., Journal of Thoracic and Cardiovascular Surgery (2010), p. 1325 ff, which we explicitly refer to. In the experiments, pigs having a weight of about 55 kg were anesthetized with propofol. Anesthesia and muscle paralysis was maintained with fentanyl. Two incisions were made for vascular access. During the experiment, systemic blood pressure was monitored, heart attack was induced by ventricular fibrillation, and heart attack was maintained for 20 minutes. After 20 minutes resuscitation was started using different initiator solutions. After treatment the animals were monitored for seven days. After that, euthanasia was performed. The brain was removed immediately thereafter and stored in formalin solution for histological examination.

[00041] The neurological status of the test animals was assessed before anesthesia and every 24 hours after cardiopulmonary resuscitation. The neurological examination consisted of five test aspects under which they are summarized: A. Central nervous function (0-100 points): pupil size (0-10); eye position (0-10); light, eyelid, and corneal reflex (each 0-10); ciliospinal and oculocephalic reflex (each 0-10); hearing reflex and gag (each 0-10); carinal reflex (0-10) B. Breathing (0-100 points): normal (0), hyperventilation (25), abnormal (50), absent (100) C. Sensory motor function (0-100 points): extension reflex (0-25), motor response to pain (0-25), positioning (0-25), muscle tone (0-25) E. Level of consciousness (0-100 points): normal (0), vague (30), delirium (45), lethargy (60), coma (100) F. Behavior (0-100 points): drinking, chewing, sitting, and standing (each 0-15); walking (0-40)

[00042] The total count is the sum of all sections (0, normal; 500, brain dead). Numbers in parentheses indicate counts for each parameter.

[00043] Pigs were divided into four groups where one control group and three test groups were used.

[00044] For the control group as initiator solution a Ringer's solution was used. Animals were treated as described in Trummer et al. (Journal of Thoracic and Cardiovascular Surgery, 2010, pp 1325-1332). The time period between heart attack and cardiopulmonary resuscitation was 15 minutes in the control group.

[00045] To demonstrate that the time period between heart attack and resuscitation can be extended by the protective solutions described here, the time interval between heart attack and resuscitation was 20 minutes for test groups 1-3. The 5 minute difference is extraordinarily important as very often, despite better organisations, more time is required before the resuscitation action can start.

[00046] As solutions according to the invention three test solutions 1, 2 and 3 were used.

[00047] Test Solution 1 had an osmolarity of 440 mosm/L. It was a solution containing in addition to mannitol, lidocaine as well as human albumin. The animals were treated hypothermically aiming a body temperature of 32 C° for 30 minutes.

[00048] Test solution 2 had an osmolarity of 550 mosm/L. It contained albumin, human mannitol, lidocaine, high concentrations of magnesium and citric sodium. The animals were treated hypothermically aiming a body temperature of 32 C° for 30 minutes.

[00049] Test Solution 3 had an osmolarity of 550 mosm/L. It contained human albumin, mannitol, lidocaine, high concentrations of magnesium and sodium-citric. Furthermore, the concentration of natrium was decreased. The animals were treated under normal thermal conditions. No attempt was made to control temperature in this group. The body temperature of the pigs remained at ~36 C°.

[00050] Test results are provided in the following Table:

[00051] The experiments clearly show that the protective solutions described here do not substantially reduce a) mortality in these animals and b) brain damage and allow recovery after an incredibly long period of time between heart attack and cardiopulmonary resuscitation (20 minutes). Neurological recovery was quantified with the above-mentioned Score system (NDS). “Good recovery” includes full awareness, standing, walking, eating and drinking from animals.

[00052] The comparative example clearly shows that the test solutions described here allow for a prolongation of the time period between heart attack and the start of resuscitation procedures. The results of neurological deficit core suffered in the control after 15 minutes were well comparable to the test solution. The important difference, however, is the time difference between 15 minutes and 20 minutes. Furthermore, experience shows that hypothermic treatment also improves results.

[00053] With control solutions it was likewise possible to avoid brain damage in several cases. The longer the period of time is after which animals used in experiments have not suffered severe neurological damage, the better such solutions can be used in humans to prevent brain damage in cases of cardiogenic shock, severe circulatory failure or cardiopulmonary resuscitation.

Claims (11)

1. Protective solution to prevent or reduce brain reperfusion injury, characterized in that it contains: magnesium ions, has an osmolarity of 350 to 600 mOsm/L, a pH value of 7.5 to 7.8, and albumin, in an amount of 1 to 20% by weight. 2. Protective solution, according to claim 1, characterized by the fact that the albumin is human serum albumin. 3. Protective solution, according to claim 1 or 2, characterized in that it contains lidocaine in an amount of 0.05 to 1.0 g per L of solution. 4. Protective solution, according to any one of claims 1 to 3, characterized in that it contains a sugar alcohol in an amount of 1.0 to 50 g/L. 5. Protective solution, according to claim 4, characterized by the fact that the sugar alcohol is mannitol. 6. Protective solution, according to any one of claims 1 to 5, characterized by the fact that magnesium is present in a concentration of 0.1 to 15 mmol/L. 7. Protective solution, according to any one of claims 1 to 6, characterized in that it contains 10,000 to 50,000 IU of heparin per L of solution. 8. Protective solution, according to any one of claims 1 to 7, characterized in that it contains citrate ions in a concentration of 20 to 100 mmol/L of solution. 9. Protective solution, according to any one of claims 1 to 8, characterized in that it contains Ciclosporin in an amount of 50 mg to 1300 mg per L of protective solution. 10. Protective solution according to any one of claims 1 to 9, characterized in that it is for use in preventing or reducing brain reperfusion injury after cardiopulmonary resuscitation. 11. Protective solution, according to any one of claims 1 to 9, characterized in that it is for use in preventing or reducing brain reperfusion injury after cardiopulmonary resuscitation, which is used together with a solution containing 0.1 to 25.0 mmol of potassium per L of solution.