WO2007038341A2 - Methods of organ protection - Google Patents

Abstract

Cyclocreatine can be contacted to a mammalian organ to treat or prevent, for example, ischemic injury.

Description

METHODS OFORGANPROTECTION

CLAIM OF PRIORITY

This application claims priority under 35 U. S. C. 119(e) to U.S. Provisional Application No. 60/719,580, filed September 23, 2005.

TECHNICAL FIELD

The present invention relates to methods for organ transplants.

BACKGROUND

A persistent consequence of even transient ischemia is a substantial decrease in adenosine triphosphate (ATP) levels. The lowering of ATP during ischemia is caused by anoxic inhibition of oxidative phosphorylation (i.e., conversion of adenosine diphosphate (ADP) to ATP) in the mitochondria, and accordingly excess ADP is accumulated. Investigations of cellular pathophysiology of ischemic injury in acute myocardial infarction have consistently shown that a significant part of tissue damage occurs during reperfusion, the period when blood flow is restored after an ischemic period of more than about ten minutes. Reperfusion injury or ischemic reperfusion injury (IRI) refers to the tissue damage inflicted on myochondrial tissue when blood flow is restored after ischemia. This injury is responsible for paradoxical organ death and tissue injury after termination of the reperfusion period. IRI can be shown in almost all organ systems. The mechanisms involved in IRI include a reduction in high energy phosphate (ATP) levels for several hours after tissue ischemia, inflammatory cell (neutrophils) mediated cellular and microvascular injuries, no reflow phenomena (inadequate reperfusion), microvascular dysfunction with platelet plugging and endothelial damage with inadequate tissue perfusion during the reperfusion period, and calcium overload mediated reperfusion injury.

SUMMARY

In one aspect, a method of protecting a mammalian organ from ischemic injury can include contacting cyclocreatine to the organ and harvesting the organ from the mammal. In another aspect, a method of protecting a mammalian organ from ischemic injury can include contacting an isolated mammalian organ with cyclocreatine.

In another aspect, a method of treating a mammalian recipient of an organ transplant from ischemic injury can include contacting cyclocreatine to the mammal. In yet another aspect, a method of protecting a mammalian organ from ischemic injury can include contacting cyclocreatine to the organ wherein the organ has been subjected to prolonged ischemic conditions.

Cyclocreatine can include a cyclocreatine precursor, prodrug, or salt. The organ can be isolated before contacting with cyclocreatine. The organ can be a heart, liver, kidney, or lung.

Contacting cyclocreatine to the organ can include treating the mammal with cyclocreatine. Contacting cyclocreatine to the organ can include delivering cyclocreatine to a mammal, delivering cyclocreatine to a mammal intravenously, intraperitoneally, or orally, isolating the organ before contacting with cyclocreatine, contacting with cyclocreatine before harvesting the organ, and incubating or exposing the organ to cyclocreatine after harvesting the organ. Exposing the organ to cyclocreatine can include perfusing the organ.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DETAILED DESCRIPTION

Ischemic injury can occur in many forms including myocardial stunning, apoptosis, and necrosis. Myocardial stunning refers to diminished contractility of ischemic but viable tissue following full restoration of blood flow. Approximately 20% to 80% of surgical patients experience myocardial stunning after cardiac surgery, such as when hearts were arrested during bypass surgery.

Apoptosis is programmed cell death characterized by cell shrinkage, membrane blebbing, and chromatin condensation. These apoptotic bodies are subsequently phagocytosed by surrounding cells or macrophages. Since cellular contents are not released, apoptosis does not stimulate an inflammatory response. Many agents that cause necrosis can also cause apoptosis, although usually at lower doses and over longer periods of time.

Necrosis occurs when a cell suffers lethal injury and is characterized by swelling, rupturing of the cell, release of cellular contents and consequently, inflammation. Necrosis can occur with myocardial infarction, for example. Ischemia associated with myocardial infarction can lead to further loss of oxidative phosphorylation in mitochondria and reduction in ATP production. Insufficient ATP can also result in failure of the ATP dependent sodium/potassium pumps and calcium pumps that normally maintain healthy levels of intracellular cell potassium, sodium and calcium. Without sufficient ATP regulation, excessive calcium levels can accumulate in the cell, resulting in increased glycolysis and acidosis, which damages lysosomal cell membranes and results in leakage of harmful lysosomal enzymes into the cytoplasm. In necrosis, the leaked lysosomal enzymes digest the cell contents resulting in nuclear changes and cell death. Organ transplantation is the process of transferring a relatively healthy tissue or organ from an organ donor to replace or augment a damaged or injured tissue or organ in an organ recipient. A donor can be a mammal that is brain dead. Organ transplantation is an important therapeutic option for patients with end-stage organ diseases. Although improved immunosuppressive strategies have dramatically increased the rate of short- term success of such transplants, the issue of organ damage upon organ isolation or harvest is a continuing problem.

Organ transplantation can include, for example, a heart transplant, liver transplant, lung transplant, or kidney transplant. There are at least two different surgical approaches to transplantation: the orthotopic and the heterotopic approach. The more common of the two procedures is the orthotopic approach, which includes replacing the recipient organ with the donor organ. After the donor organ is removed, it can be preserved, packed for transport, and it can be transplanted into the recipient within approximately four to five hours. In the case of a heart transplant, the recipient can receive general anesthesia and can be placed on a bypass machine to oxygenate the blood while the heart transplant is being performed. After the recipient's organ is removed, the donor organ can be prepared to fit and implantation can begin.

The second approach to transplantation is called heterotopic transplantation, which can be accomplished by leaving the recipient's organ in place and connecting the donor organ to the recipient organ. In a heart transplant, for example, the recipient's heart can remain in place and the donor heart can be connected to the right side of the chest. The procedure is rare compared to orthotopic transplantation, but it can be advantageous because the new heart can act as an assist device if complications occur. There are several steps to an organ transplant procedure. Procurement of a suitable graft or transplant is the first step. The requirements for matching donor and recipient include blood compatibility and organ size.

Donor management is a crucial issue when obtaining a suitable graft or transplant organ and complications can occur during after harvesting the organ. Naturally, the best way to avoid complications is to procure the organs as soon as possible and to minimize cellular damage during organ procurement and transport by contacting the organ with an appropriate agent or compound.

The procurement process can include multiple teams. One team can be responsible for harvesting an organ, such as a heart, a lung, a liver or a kidney. The sequence of graft removal can be dictated by the maximum allowable cold ischemia time, which can differ depending on the organ. The heart can have a maximum cold ischemia time of about eight hours and can be removed first. The lungs can have a maximum cold ischemia time of about six hours and can be removed with or after the heart. The liver can have a maximum cold ischemia time of about 24 hours and can be removed next. The kidneys can have a maximum cold ischemia time of about 48 hours and can be removed last. All organs can be rapidly cooled by perfusion and doused with a solution such as iced saline, for example, to arrest metabolism and clear blood from the organs. The age of the donor, mechanism of injury leading to brain death, gross appearance of the organ, the anatomy and how well the organ flushes are all important factors in judging the quality of the transplant. The sum of these factors can be weighed against the acuity of the recipient before a final decision can be made to use the transplant.

The first step for harvesting an organ for transplant is to cut open the donor's chest, abdomen, or other body part containing the organ. In a heart transplant operation, for example, a surgeon can saw through the breast bone and pull the ribs outward to reveal the heart. While other teams are working on other parts of the body, the heart team can clamp the different blood vessels leading into the heart and pump in a high potassium cardioplegic solution to stop the heart from beating and help preserve it during transportation. The surgeon can then sever the vessels and remove the heart from the body, placing it in a vessel with a preservative chemical. This bag can then be placed in a cooler filled with ice, which is rushed to the recipient's hospital. Meanwhile, the recipient can be fully anesthetized. The surgery can begin when or after the organ arrives. When the donated heart or other organ has arrived, the transplant team begins the procedure. First, they hook up an IV and inject an anticoagulant into the patient's bloodstream. As with the harvesting surgery, the team begins the surgery in the recipient by making an incision in the patient's chest, sawing through the breastbone and pulling back the ribs. The doctors can then hook up a heart-lung machine to the patient's body. The machine's plastic tubes are connected to blood vessels leading to and from the heart. Instead of being pumped to the lungs to get rid of carbon dioxide and pick up oxygen, blood returning to the heart is diverted to the machine. The machine drives the blood through a series of chambers to release carbon dioxide and pick up oxygen and then returns it into the body to be re-circulated. This enables a surgical team to remove the heart without disrupting respiration and circulation.

Additionally, the heart-lung machine can be adjusted to warm or cool the blood. During the operation, it is set to cool all the blood that passes through it. This cools the rest the body, which helps protect the other organs during the operation. Typically, the machine will have an attachment to suction blood from the surgery area and send it directly back into the bloodstream. Capturing shed blood and returning to the patient's circulation can reduce the patient's loss of blood during surgery.

When the blood has been effectively diverted around the heart and lungs, the surgeons can remove the diseased heart or other organ by cutting it loose from the attached blood vessels. In a heart transplant, for example, surgeons can remove the back walls of the donor heart's atria and suture the donor heart to the remaining tissue of the old heart. Then, they can suture the blood vessels formerly leading to the diseased heart to the vessels leading out of the donor heart.

After the new heart is in place, the team can gradually warm up the blood flowing through the patient's body. As the body warms, the heart may start beating on its own. If it does not, the team can apply an electric shock. The team can allow the newly transplanted heart and the heart-lung machine to share the job of circulating blood for some time, giving the heart time to build strength. The team can wire the halves of the breast bone back together and stitches up the patient's chest using dissolving stitches, for example. The patient is connected to a ventilator and brought to the recovery room. Typically, the entire procedure can take about five hours. Transplant surgery methods can vary depending on the organ that is being harvested, the type of donor and recipient, and other factors, but in general, the method can be described by three phases: incision, removal, and reperfusion.

In a liver transplant, for example, the incision phase can begin with an abdominal incision. The actual incision can be a bilateral subcostal cut extending from the midclavicular line on the left to the midaxillary line on the right, with an upper midline extension and xiphoidectomy. The shape is similar to the Mercedes-Benz trademark and is commonly referred to as a "Mercedes incision". With a liver transplant, for example, after opening the abdomen, the ligamentous attachments are taken down and the hilar structures are dissected as close to the liver as possible. The extrahepatic biliary structures are divided followed by the branches of the hepatic artery. The hepatic artery is dissected back to the gastroduodenal artery, allowing skeletonization of the portal vein. Prior to dividing the portal vein, cannulas are placed in the femoral and axillary veins. These cannulas act as a bypass circuit which allows blood to be siphoned away from the lower half of the body and visceral venous outflow to be re-infused back to the right atrium. The portal vein can then be divided and a large cannula can be placed in the proximal end.

The removal phase, in an organ transplant usually involves clamping and isolation, hi a liver transplant, for example, the infrahepatic and suprahepatic venae cavae are clamped and the liver is removed. Hemostasis is obtained and the new liver is sewn into place. The suprahepatic vena cava is sutured first, followed by the infrahepatic cava. During the lower caval anastomosis, the liver is flushed through the portal vein to remove residual preservation solution, clot and foreign debris. With the caval anastomoses complete, the portal vein anastomosis is performed. The bypass cannula in the portal vein is removed, leaving only the femoral and axillary veins in the circuit. When the anastomosis is complete, the clamps are removed from the vena cava and the portal vein, initiating the reperfusion phase.

During the reperfusion phase, anastomosis, or the connection of tissues can be completed. Typically, an hour can elapse by the time the anastomosis, such as an artery and bile duct reconstruction, has been completed and there are biochemical signs of liver function. A liver biopsy can be taken and an intraoperative cholangiogram obtained if a duct-to-duct reconstruction was done. Drains can then be placed, the wound can be closed, and the patient can be transferred to the intensive care unit. With any type of transplantation technique, ischemic injury to the donated organ sustained either during harvest or thereafter can compromise the organ. A compromised transplant can include a transplanted organ that has undergone cellular damage (such as edema, lactic acidosis, or cell death), and may require retransplantation or other forms of intervention. Currently, the number of patients waiting for organ transplants, particularly heart transplants, exceeds the number of organ donors. Preserving a donated organ can help to ensure that donated organs result in successful transplants. Organ transplantation can require a lifelong commitment by the transplant team and the patient. A recipient must be willing and able to comply with a complex regimen which can include multiple expensive drugs, frequent follow-up visits and periodic biopsies. Since transplanted organs are at risk of ischemic injury from the moment of harvest, there is a need for an agent that protects organs after removal, reduces cellular damage, and maintains or preserves organ viability.

The critical changes that make injury irreversible and lethal in ischemia are related to the inability of the mitochondria to recover from injury, even after re-oxygenation, thereby producing permanent damage on cell membrane function and integrity.

Therefore, the balance between cell death by apoptosis and necrosis depends not only upon the intensity of the injury but also the level of available intracellular ATP. A lack of ATP can cause a switch of the mode of cell death from apoptosis to necrosis.

Since ischemic injury can result from a lack of ATP, an agent that enhances ATP production can protect against ischemic injury. Conventionally, adenosine, a precursor of AMP, ADP, and ATP, has been used as an agent to increase cellular synthesis of ATP. However, adenosine has several disadvantages: it has a half-life of less than 5-10 seconds and therefore requires injections every few minutes, which can be costly an inconvenient. Moreover, adenosine must be given intravenously by a health care professional, which limits its applications, especially in emergency situations. Futhermore, adenosine can be difficult to tolerate for some patients, with adverse events occurring in approximately 60% of the population of those treated with adenosine. Therefore, while there is preclinical and clinical evidence that adenosine is a cardioprotective agent, its clinical use remains limited because it is relatively unstable and has limited modes of administration. Thus, there is a need for an organ-protective agent that is easy not limited to intravenous administration, well-tolerated, stable, safe, and functional.

The creatine analog cyclocreatine (including cyclocreatine, cyclocreatine phosphate, pharmaceutically acceptable salts, precursors and prodrugs thereof), are more stable, well tolerated, do not require intravenous administration, and can be contacted to a mammalian organ, for example by treating the mammal donor or recipient, perfusing the organ, or administering the compound (orally or intravenously) to the mammal. When contacted to a mammalian organ prior to ischemia, cyclocreatine can delay ATP depletion during ischemia and can restore organ function in models of warm and hypothermic cardioplegic cardiac arrest, regional warm ischemia, and global warm ischemia (such as models of cardiac arrest). Cyclocreatine can have beneficial effects when administered before or after ischemia occurs. Cyclocreatine can be synthesized from inexpensive starting materials in a stable form. The synthesis can be performed on a large scale, such as more than 100 grams, more than 1 kilogram, or 10 kilograms or more. Creatine is 2- (amidino-methyl-amino)acetic acid. Cyclocreatine is l-carboxymethyl-2- iminoimidazolidine. Synthesis of cyclocreatine and its analogues is described, for example in U.S. Pat. App. No. 60/640,061, which is incorporated by reference in its entirety. Cyclocreatine can be administered as a pharmaceutically acceptable salt. A pharmaceutically acceptable salt refers to a salt prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include salts with one or more of the following cations: aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts.

Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropyulamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compound is basic, salts may be prepared from pharmaceutically acceptable acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic (besylic), benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids. Cyclocreatine can be administered as a prodrug or a precursor which can undergo chemical conversion by metabolic processes before becoming an active pharmacological agent. The prodrug can be designed to improve the delivery and absorption of the drug through targeting to cellular transporters. See BS Vig, PJ Lorenzi, et al, Amino acid ester prodrugs of floxuridine: synthesis and effects of structure, stereochemistry, and site of esterification on the rate of hydrolysis. Pharm Res. Sep; 20(9):1381-8 (2003) and U.S. Pat. No. 6,669,954, both of which are incorporated by reference in its entirety. It can be desirable to protect a functional group during preparation of cyclocreatine. For example, an amino group can be protecting with a protecting group to prevent undesired reactions of the amino group. A protecting group is a suitable chemical group which may be attached to a functional group of a molecule, then removed at a later stage to reveal the intact functional group and molecule. Examples of suitable protecting groups for various functional groups are described in Theodora W. Greene, Peter G. M. Wuts: Protective Groups in Organic Synthesis, 3^rd ed. Wiley Interscience, 1999; L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); L. Paquette, ed. Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995); each of which is incorporated by reference in its entirety.

Cyclocreatine can be an effective agent to protect against ischemic injury for the following reasons. First, unlike adenosine whose half life is less than 5 to 10 seconds and requires repeat injections every few minutes, cyclocreatine is relatively stable, even when compared to the naturally occurring creatine phosphate, and showed prolonged restoration of organ function after one bolus injection before ischemia. Second, in terms of ease of administration, unlike adenosine, which is typically given intravenously by a health professional while the patient is hooked up to a heart monitor, cyclocreatine can be orally administered (i.e., in tablet, pill, suspension or other form), by patients without the help of a physician or health care provider. Cyclocreatine can also be administered intravenously to the donor, recipient, or both, or as an infusion before surgery such as a transplant surgery. Third, unlike adenosine which has been associated with adverse events in approximately 40 to 60 percent of patients, cyclocreatine is well tolerated. Finally, while adenosine has been shown to protect mainly ventricular areas, cyclocreatine can protect both atrial and ventricular areas. A newly recognized adenosine receptor, the A3 subset, is expressed on cardiac ventricular cells and its activation protects the ventricular heart cells against injury during a subsequent exposure to ischemia. Interestingly, cardiac atrial cells lack native A3 receptors and therefore exhibit a shorter duration of cardioprotection than do ventricular cells. Surprisingly, neutrophils accumulate to a greater extent in the atria compared to the ventricular in dog hearts that underwent cardioplegic arrest for 1 hour followed by reperfusion. See Elgebaly SA, et al., "Evidence of cardiac inflammation after open heart surgery," Ann. Thome. Surg 57:35- 41, 1994, which is incorporated by reference in its entirety. This observation further supports that atrial tissues were less protected during surgery. Unlike lack of adenosine protection in the atrium, cyclocreatine can provide equal protection to the atrium and ventricular areas. Furthermore, the administration of cyclocreatine prior to surgery can result in a reduction in the current rates of postoperative stunning and infarction, and represent a significant advance in the field of ischemic protection.

Administering cyclocreatine to organ donors, for example, heart donors, achieved a number of surprising results that enhanced organ viability and demonstrated, for example, decreased ischemic injury. For example, when dog hearts (cyclocreatine-treated hearts and control hearts) were subjected in vivo to 1 hour of warm global ischemia, isolated, and then subjected to 4 hours of continuous perfusion, the ATP stores in the heart of the CCr-treated dog were threefold higher than in the control hearts. The results indicate that cardiac function is also restored by cyclocreatine after 4 hours of reperfusion.

As an example, cyclocreatine can maintain and improved the viability of a donor organ. For example, cyclocreatine was injected intravenously to a dog one hour before inducing warm ischemia. Ex vivo hearts that were subjected to one hour ischemia and four hours of continuous reperfusion with cyclocreatine were assessed for viability by measuring systolic recovery. The results indicate that as ATP levels increased (in nM/mg protein), the level in systolic recovery likewise increased. Thus, cyclocreatine affects the amount of ATP synthesized, which bears a direct relationship to systolic recovery. Specifically, the cyclocreatine-treated heart continued to beat for 9 minutes after inducing warm ischemia, while the control heart stopped beating after 2 minutes. Cyclocreatine can be used as a protective agent in tissues and organs where reduced ATP levels result in ischemic injury, such as the heart, the kidney (see Qingqing Wei, et al., Am. J. Physiol. Renal Physiol. Dec. 16, 2003 (abstract), the liver (see Janssen H, Eur. Surg. Res. 2004; 36(l):26-32), and the lung (see Williams M, Biochem Pharmacol. 2000 May 15; 59(10):l 173-85). Cyclocreatine can be administered to an ischemic organ. Cyclocreatine can be administered orally or intravenously, for example, into a brain dead donor before harvesting the heart. The heart can then be perfused with cyclocreatine phosphate until they are transplanted into recipients. This can significantly prolong the viability of the hearts and improve the heart's function in the recipient.

Cyclocreatine can also be administered to ischemic hearts of donors whose hearts have stopped beating for a substantial period of time, such as four minutes or more, for example. The ischemic hearts can be removed and contacted or perfused with cyclocreatine to increase heart viability and increase chance of recovery of the recipient. Cyclocreatine can be administered to organ transplant recipients to preserve and maintain the function of the transplanted organ. Cyclocreatine can also be administered to patients undergoing heart transplants, before, during, and after surgery. To avoid potential future complications, surgical patients can be maintained on an oral dose of cyclocreatine after surgery.

Cyclocreatine can also be administered orally (i.e. by tablet, pill, suspension, or other form). The administration of cyclocreatine preferably is carried out by the most convenient or direct route available. Thus, injection of the material, typically within a fluid carrier such as a sterile saline solution, can be employed, with parenteral administration (e.g., intravenous injection) being preferred when the treatment involves the cardiovascular system. Oral administration can also be employed. Such solutions typically have an essentially neutral pH, such as a conventionally employed saline solution. Of course, other appropriate means of administration can be used depending upon the particular tissue of concern and the vehicle used for its administration. See, for example, U.S. Pat. No. 5,091,404, which is incorporated by reference in its entirety. In most instances, sterile saline solutions containing substantially more than one percent and typically more than three percent by weight of cyclocreatine have been effective within one half hour of surgery, although somewhat lower percent levels of cyclocreatine may be effective when administered within one to two hours prior to surgery. Where a saturated solution containing about five percent by weight cyclocreatine at room temperature is employed, it has been found to be consistently effective within about thirty minutes and less. Of course, the concentration level at saturation will vary with temperature. The dosage administered may be as low as about 2 grams per 70 kilograms of body weight but typically is greater than about 6 g/70 kg. Excellent results have been achieved at dosage levels of about 8-12 g/70 kg of body weight. The cyclocreatine employed can be synthesized according to a known procedure as set forth by Griffiths et al. in J. Bio. Chem. 1976, Vol. 251, pages 2049-2054, incorporated by reference in its entirety. Various methods of administering cyclocreatine are described, for example, in Pat. No. 5,091,404, which is incorporated by reference in its entirety. Cyclocreatine treatment can increase organ viability despite prolonged ischemia.

Prolonged ischemia refers to ischemic conditions for approximately 20 minutes, 40 minutes, 1 hour, two hours, or more than two hours. Cyclocreatine-treated dogs were able to sustain substantially longer periods of ischemia compared to control hearts. Because cyclocreatine phosphate is a long-acting phosphagen, it can help to sustain ATP levels longer during ischemia compared to controls containing creatine phosphate (CrP) as the sole phosphagen. Studies showed that cyclocreatine phosphate possesses a substantially less negative Gibbs standard free energy of hydrolysis than creatine phosphate and, therefore, it can continue to buffer thermodynamically the adenylate system at the lower pH values and lower cytosolic phosphorylation potentials that occur during the latter stages of ischemia, conditions in which CrP is no longer effective (see, for example, Griffiths, G.R. and Walker J.B., J. Biol. Chem. 251 : 2049-2054, 1976, which is incorporated by reference in its entirety).

Furthermore, because an organ such as the heart relies almost exclusively on mitochondrial oxidative phosphorylation for high-energy phosphate production, a decrease in oxygen delivery below a critical limit-due to pathological block of adequate blood supply, asphyxia, poisoning, or experimental and surgical intervention, will change cardiac energy metabolism. Research showed that contractile performance in- vivo decreases precipitously and ceases when 75% of CrP is depleted, but only when 20% of ATP is depleted. See, for example, Gudbjarnason S, et al., J. MoL Cell Cardiol. 1 :325- 339, 1970, which is incorporated by reference in its entirety. Administering Cyclocreatine to Isolated Hearts

Isolated ischemic rabbit hearts treated with cyclocreatine maintained high levels of ATP and CrP compared to control hearts treated with saline or creatine, which lost more than 95% of ATP and CrP (see, for example, Elgebaly SA, et al., Am. J. Pathol. 137:1233-1241, 1990, which is incorporated by reference in its entirety). Preservation of ATP would likely explain the significant reduction of cardiac-derived chemotactic factor released by cyclocreatine treated hearts and the high release of the chemotactic factor by controls (see Elgebaly SA, et al., Surg. Forum 41 :274-278, 1991; and U.S. Patent No.

5,091,404, each of which is incorporated by reference in its entirety). In the intact canine model of coronary artery occlusion followed by reperfusion, cyclocreatine-treated hearts maintained 85% of the ATP (loss of 15%) and 97% of the CrP (loss of 3%) of normal non-ischemic levels (see Elgebaly SA, et al. J. Pharmacol. Exp. Therap. 266(3): 1670- 1677, 1993, which is incorporated by reference in its entirety). Control saline treated hearts, on the other hand, maintained 66% of the ATP (loss of 34%) and 18% of the CrP (loss of 83%), thereby impacting contractile performance of the heart. See, for example, Gudbjarnason S, et al., J. MoI. Cell Cardiol. 1:325-339, 1970, which is incorporated by reference in its entirety. A number of studies have also established that the decline in ATP associated with ischemia could have many adverse consequences, including loss of ionic gradients, resulting in a calcium overload and activation of endogenous phospholipases or proteases. Catabolites of lipid degradation may act as a detergent and damage cell membranes, leading to edema. Adenosine nucleotides and bases accumulate and might be a major source of free radicals via the xanthine oxidase reaction (see Reimer, K.A., et al, J. MoI Cell Cardiol. 21 : 1255-1239, 1989, which is incorporated by reference in its entirety).

Administering Cyclocreatine Prior to Ischemia

Cyclocreatine can be effective when administered prior to the induction of ischemia. Long-term feeding of rats and chickens (up to 3 weeks) with 1% cyclocreatine significantly delayed the reduction of myocardial ATP, exhaustion of high-energy phosphates, and onset of rigor tension during cardiac ischemia. Upon reperfusion, the number of hearts recovering mechanical function was significantly higher in cyclocreatine treated rats compared to controls (see Roberts, JJ. and Walker, J.B., Am. J. Physiol. 243: H911-H916, 1982, which is incorporated by reference in its entirety).

Short-term administration (30-120 min) of cyclocreatine can be as effective in protecting an organ such as the heart from ischemic injury as long-term administration for up to 21 days. Intravenous injection in dogs, rabbits, and rats of cyclocreatine 30-120 min prior to the induction of ischemia reduced the cardiac production of Nourin-1 and the accumulation of neutrophils into the myocardium during reperfusion (see, for example, Elgebaly SA, et al, Am. J. Pathol 137:1233-1241, 1990; and Elgebaly SA, et al, J. Pharmacol. Exp. Therap. 266(3):1670-1677, 1993, each of which is incorporated by reference in its entirety).

Cyclocreatine and cyclocreatine phosphate promoted significant restoration of organ function and preservation of ATP and CrP. For example, in intact canine models of myocardial ischemia followed by reperfusion, as well as isolated rat heart working models, drug administration prior to ischemia protected the hearts against warm and hypothermic ischemia, in the presence and absence of cardioplegic arrest, and when ischemia was induced for 40 min, 2.5 hours, and 6 hours. See, for example, Allam ME, et al, Surg. Forum XLL246-249, 1990; Elgebaly SA, et al, Transplantation 57(1) 1-6, 1994; and Houser SL, et al, J. MoI. Cell Cardiol. 27:1065-1073, 1995, each of which is incorporated by reference in its entirety. Below is a regimen for the administration of cyclocreatine phosphate as an organ- protective agent aimed at interfering with the injury process before ischemia, during ischemia, and after ischemia.

Cyclocreatine can be administered immediately after documentation of ischemia or ischemic injury, and before surgery. Since apoptosis, which occurs due to drop in ATP, has been demonstrated to occur at the border zone of infarction and even in sites remote from the region of ischemia, the advantages of the early administration of cyclocreatine phosphate before establishing perfusion is that the drag will protect the myocardium adjacent to infarction and according reduce future incidences of congestive heart failure.

Thus, administration of cyclocreatine and cyclocreatine phosphate can protect organs including myocardial tissue against ischemic injury and can restore cardiac function in models of acute myocardial infarction, global cardiac arrest, coronary bypass surgery, and heart transplant. In a heart transplant rat model, cyclocreatine phosphate did not only improve the recovery of function during reperfusion after 6 hours of cold storage, but also significantly reduced the increase in heart weight compared to control untreated hearts. Since the breakdown of ATP is the immediate source of energy for contraction, and that contractile performance decreases precipitously and ceases when only 20% of ATP is depleted, the reported cyclocreatine preservation of over 85% of ATP (loss of only 15%) in ischemic myocardium is likely the major contributor to the observed restoration of post-ischemic myocardial contractility (see Allam ME, et al, Surg. Forum XLI:246-249, 1990, which is incorporated by reference in its entirety).

In a dose-response study, cyclocreatine exerted a strong cardioprotective effect at 600 mg/kg and 300 mg/kg. No effect was observed, however, at 150 mg/lcg. Cyclocreatine phosphate also exerted strong cardioprotective effect at 1000 mg/lcg, 667 mg/kg, and 484 mg/kg, but not at 300 mg/kg. In a molar equivalent basis, CrP was effective at 510 mg/kg when injected intravenously and at 10 mM when placed in the perfusate.

In another study using isolated rabbit hearts, intravenous administration of cyclocreatine (600 mg/kg) for 2 hours prior to removing and perfusing hearts in- vitro for additional two hours, did not induce tissue damage. After 2 hours reperfusion, control hearts showed patches of eosinophilic degeneration of myocardial fiber cytoplasm characteristic of early ischemia in myocardium. Patches of contraction bands associated with ischemia were also evident. Cyclocreatine-treated hearts, on the other hand, showed only occasional small foci of contraction bands and no significant eosinophilic changes. Similar results were obtained when using the intact canine model of coronary occlusion followed by reperfusion for two hours. The administration of cyclocreatine showed marked reduction in cell damage compared to control hearts. As described above, the reduction in myocardial cell injury in cyclocreatine-treated hearts was associated with significant restoration of cardiac function further confirming that cyclocreatine is not toxic to heart tissues.

Other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

I . A method of protecting a mammalian organ from ischemic injury comprising contacting cyclocreatine to the organ and harvesting the organ from the mammal.

5 2. The method of claim 1 , wherein the organ is isolated before contacting with cyclocreatine.

3. The method of claim 1 , wherein the organ is a heart, liver, kidney, or lung.

4. The method of claim 3, wherein the organ is a heart.

5. The method of claim 1 , wherein contacting cyclocreatine to the organ includes o treating the mammal with cyclocreatine.

6. The method of claim 1 , wherein the cyclocreatine includes a cyclocreatine precursor, prodrug, or salt.

7. The method of claim 1, wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal.

5 8. The method of claim 1 , wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal orally.

9. The method of claim 1 , wherein contacting cyclocreatine occurs before harvesting the organ.

10. The method of claim 1, wherein contacting includes exposing the organ to 0 cyclocreatine after harvesting the organ from the mammal.

I 1. A method of protecting a mammalian organ from ischemic injury comprising contacting an isolated mammalian organ with cyclocreatine.

12. The method of claim 11, wherein the organ is isolated before contacting with cyclocreatine.

5 13. The method of claim 11 , wherein the organ is a heart, liver, kidney, or lung.

14. The method of claim 13, wherein the organ is a heart.

15. The method of claim 11, wherein contacting cyclocreatine to the organ includes treating the mammal with cyclocreatine.

16. The method of claim 11, wherein the cyclocreatine includes a cyclocreatine precursor, prodrug, or salt.

17. The method of claim 11, wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal.

18. The method of claim 11 , wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal orally.

19. The method of claim 11 , wherein contacting cyclocreatine occurs before harvesting the organ.

20. The method of claim 11 , wherein contacting includes exposing the organ to cyclocreatine after harvesting the organ from the mammal.

21. A method of treating a mammalian recipient of an organ transplant from ischemic injury comprising contacting cyclocreatine to the mammal.

22. The method of claim 21, wherein the organ is isolated before contacting with cyclocreatine.

23. The method of claim 21, wherein the organ is a heart, liver, kidney, or lung.

24. The method of claim 21, wherein the organ is a heart.

25. The method of claim 21, wherein contacting cyclocreatine to the organ includes treating the mammal with cyclocreatine.

26. The method of claim 21, wherein the cyclocreatine includes a cyclocreatine precursor, prodrug, or salt.

27. The method of claim 21, wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal.

28. The method of claim 21, wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal orally.

29. The method of claim 21, wherein contacting cyclocreatine occurs before harvesting the organ.

30. The method of claim 21 , wherein contacting includes exposing the organ to cyclocreatine after harvesting the organ from the mammal.

31. A method of protecting a mammalian organ from ischemic injury comprising contacting cyclocreatine to the organ wherein the organ has been subjected to prolonged ischemic conditions.

32. The method of claim 31 , wherein the organ is isolated before contacting with cyclocreatine.

33. The method of claim 31, wherein the organ is a heart, liver, kidney, or lung.

34. The method of claim 33, wherein the organ is a heart.

35. The method of claim 31, wherein contacting cyclocreatine to the organ includes treating the mammal with cyclocreatine.

36. The method of claim 31, wherein the cyclocreatine includes a cyclocreatine precursor, prodrug, or salt.

37. The method of claim 31, wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal.

38. The method of claim 31 , wherein contacting cyclocreatine includes delivering cyclocreatine to a mammal orally.

39. The method of claim 31, wherein contacting cyclocreatine occurs before harvesting the organ.

40. The method of claim 31, wherein contacting includes exposing the organ to cyclocreatine after harvesting the organ from the mammal.

1. The method of claim 31 , wherein the organ has been subjected to approximately two hours of ischemic conditions.