WO1994026261A1 - Treating body parts susceptible to ischemia using creatine analogs - Google Patents

Abstract

Methods of using creatine analogs for treating body parts for ischemia are described. The methods are useful for both prophylactic and/or therapeutic treatments of ischemia. Methods of treating organs intended for transplantation also are described along with the creatine analog-containing compositions used in the forementioned methods.

Description

Treati ng body parts susceptibl e to i schemi a usi ng creati ne anal ogs.

Background of the Invention

A sudden lack of blood supply to a tissue is typically referred to as an ischemic episode or ischemia. Many tissues in the body are susceptible to damage or death following such a period of inadequate oxygen or nutrient supply. Examples of types of ischemia include myocardial ischemia (inadequate circulation of blood to the myocardium), cerebral ischemia or stroke (sudden reduction in cerebral blood supply), and ischemia of the spinal cord (sudden reduction of blood supply to the spinal cord). The frequency of cardiac surgery has increased with time as surgical procedures are continually being refined. Despite the use of cardioplegic solutions and deep hypothermia during cardiac surgery, patients are still at risk for injuries, e.g. protracted ventricular dysfunction or myocardial tissue damage, resulting from prolonged ischemic arrest. Cardioplegic solutions and/or surgical techniques have been varied in attempts to decrease the patient's risk for these injuries. For example, creatine phosphate has been added to cardioplegic solutions in attempts to provide protection to the myocardial tissue during cardiac surgery (Robinson et al., J. Thorac Cardiovasc Surg (1987) 93:415-27: Thelin et al. Thorac. Cardiovasc. Surgeon (1987) 35:137-142). Creatine phosphate, however, is rapidly hydrolyzed at the lower pH values that occur during ischemia which may limit its usefulness in cardioplegic solutions.

Cyclocreatine is a synthetic analog of creatine. The use of cyclocreatine for preserving and/or restoring the physiological functionality of an in vivo animal muscle tissue subject to ischemia was described in U.S. Patent No. 5,091,404 issued on February 25, 1992 to Salwa A. Elgebaly, the contents of which is expressly incorporated by reference herein. Elgebaly (a co-inventor of the present application) describes the administration of cyclocreatine in an amount effective for restoring post-ischemic physiological function of a muscle tissue to a substantially pre-ischemic level. Elgebaly specifically states that "administration may be initiated prior to surgery and optionally continued during and following the surgical procedure, although it is believed that only the pre-ischemic administration is fully effective" and that "use during full ischemia where no pre-ischemic dose has been administered is not effective". Summary of the Invention

The present invention provides methods of using creatine analogs for treating body parts (cardiac and non-cardiac related body parts) for ischemia. The creatine analogs of this invention are useful for both prophylactic and/or therapeutic treatments of ischemia. The present invention is based, at least in part, on the discovery that creatine analogs are useful for treating non-cardiac related body parts for ischemia, e.g. spinal cord, pancreas and kidney, and on the discovery that creatine analogs can provide prophylactic and/or therapeutic relief to body parts susceptible to ischemia. The present invention provides a method for treating a body part susceptible to ischemia by administering to a body part susceptible to ischemia an effective amount of a creatine analog such that the body part is protected against injury from ischemia or treated for ischemia.

The present invention further pertains to a method for treating a body part subjected to ischemia to restore functionality to the body part by administering a creatine analog post- ischemia such that functionality is restored to the body part. The present invention essentially provides a therapeutic use of a creatine analog for treating injuries resulting from ischemia and/or a prophylactic use of a creatine analog for preventing injury or protecting body parts from extensive injury due to ischemia.

The present invention even further pertains to compositions for treating, e.g. preventing or storing, organs intended for transplantation. The compositions of the present invention include an effective amount of the creatine analog in a pharmaceutically acceptable organ treatment solution, e.g. an organ preservation solution. The compositions optionally may include the organs being treated in the solutions, e.g. heart, kidney, or lung. The invention further includes methods for treating an organ intended for transplantation in a patient by contacting, e.g. infusing, an organ with the forementioned compositions. Other aspects of this invention include packaged creatine analogs and methods of using creatine analog for treating conditions or diseases which are associated with ischemia and/or wherein ischemia is at least one of the underlying causative factors, e.g. congestive heart failure. The packaged creatine analogs include instructions for using the creatine analog for treating ischemia or instructions for using the creatine analog as a component in a pharmaceutically acceptable organ treatment solution.

Brief Description of the Drawings

Figure 1 is a graph depicting the recovery of aortic flow of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH).

Figure 2 is a graph depicting the recovery of cardiac output of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH). Figure 3 is a graph depicting the recovery of stroke volume of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH).

Figure 4 is a graph depicting the recovery of stroke work of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH).

Figure 5 is a graph depicting the recovery of coronary flow of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH). Figure 6 is a bar graph depicting the increase in heart weight after both preservation and reperfusion of rat hearts treated with saline and then stored in University of Wisconsin solution (UW) versus rat hearts treated with CCrP and then stored in UW containing CCrP (HH).

Figure 7 is a graph depicting the recovery of aortic flow (percent of preischemic aortic flow) for CCrP and saline treated hearts after hypothermic ischemic arrest.

Figure 8 is a graph depicting the recovery of aortic flow (percent of preischemic aortic flow) for CCrP and saline treated rat hearts after normothermic ischemic arrest.

Figure 9 is a graph depicting the recovery of aortic flow (percent of preischemic aortic flow) for CCr and saline treated rat hearts after normothermic ischemic arrest. The CCr was administered two hours prior to arrest.

Figure 10 is a graph depicting the recovery of aortic flow (percent of preischemic aortic flow) for CCr and saline treated rat hearts after normothermic ischemic arrest. The CCr was administered thirty minutes prior to arrest.

Figure 11 is a graph depicting the dose-response for CCr for Example 3 below. Figure 12 describes the recovery of cardiac function in CCr treated dogs which underwent cold cardioplegic arrest for one hour using a cardiac index expressed as a percent of baseline.

Figure 13 is a graph depicting the recovery of cardiac function in CCr treated dogs which underwent cold cardioplegic arrest for one hour using myocardial segment shortening expressed as a percent of baseline.

Figure 14 is a graph depicting the recovery of cardiac function in CCr treated dogs which underwent cold cardioplegic arrest for three hours.

Figure 15 is a bar graph depicting the recovery of aortic flow (percent of preischemic aortic flow) for CCrP treated and control rat hearts which were arrested in vivo for seven minutes, nine minutes and ten minutes.

Figure 16 is a graph comparing the effects of CCrP versus CrP on the recovery of aortic flow (percent of preischemic aortic flow) of rat hearts following hypothermic ischemia arrest. Figure 17 is a graph comparing the effects of CCrP versus CrP on the recovery of aortic flow (percent of preischemic aortic flow) of rat hearts following normothermic ischemia arrest.

Figure 18 is a graph depicting the effect of CCrP on the recovery of aortic flow (percent of preischemic aortic flow) of rat hearts following normothermic ischemia.

Figure 19 is a graph depicting the effect of CCr on the recovery of aortic flows (percent of preischemic aortic flow) of rat hearts following normothermic ischemia.

Figures 20 and 21 are bar graphs depicting the dose-response for CCrP for Example below.

Detailed Description

The present invention pertains to a method for treating a body part susceptible to ischemia. The method involves the administration of an effective amount of a creatine analog to a body part susceptible to ischemia such that the body part is protected against injury from ischemia or treated for ischemia.

The language "treating a body part" is intended to cover both prophylactic and/or therapeutic treatments. The body part can be protected from damage or injury by the creatin analogs or treated therapeutically after incurring damage or injury. For example, the creatine analog can restore at least some functionality to a body part subjected to ischemia. The restoration of functionality does not have to be complete restoration to the preischemic level but rather restoration of the functionality to an extent which allows the body part to perform at least some of its intended function(s). The preferred restoration is to a substantially preischemic level, e.g. greater than 60 percent, more preferably greater than 70 percent.

The term body part is intended to include parts of a body which are susceptible to ischemia, i.e. parts of the body in communication with the body's blood supply. The body part can be directly involved in the ischemia, e.g. the heart in open heart surgery, or be indirectly effected by the ischemia, e.g. a satellite organ from which blood is being deprived to some extent during open heart surgery. The body preferably is a mammalian body such as humans, dogs, cats, horses, pigs, goats, rats and mice. Examples of body parts include cells, tissues, organs.

The term tissue is intended to include an aggregation of cells of characteristic form together with their intercellular matrix specialized for the performance of some limited function or functions. Examples of classes of tissues which are intended to be included are muscular, connective, epithelial, nervous and vascular. A cardiac tissue is an example of a type of muscular tissue.

The term organ is intended to include a multicellular structure made up of various tissues for the performance of a function. Examples of classes of organs include reproductive, respiratory, digestive, excretatory. urinary, sensory, and skeletal. Specific organs intended to be included in this invention include kidney, heart, pancreas, liver, gall bladder, brain, spleen and spinal cord.

The term "ischemia" is art-recognized and is intended to include conditions resulting from a reduction in or lack of oxygen or nutrients, e.g. blood supply, to a body part, e.g. cell, tissue, or organ. The reduction in oxygen and/or nutrients typically is sudden and/or prolonged resulting in damage or injury to the cells, tissue or organ. The damage or injury can be extensive resulting in the death of the cell, tissue or organ. Examples of types of ischemia include myocardial ischemia (inadequate circulation of blood to the myocardium), cerebral ischemia or stroke (sudden reduction in cerebral blood supply), and ischemia of the spinal cord (sudden reduction of blood supply to the spinal cord), ischemia of the kidney (sudden reduction of blood supply to the kidney), and ischemia of the pancreas (sudden reduction of blood supply to the pancreas).

The language "susceptible to ischemia" is intended to include those body parts which can be damaged or injured by a lack of oxygen or nutrients supplied to the body part, e.g. the heart is susceptible to ischemia because the heart can be damaged when the blood supply is suddenly reduced to the heart. The compounds of the present invention can have a cardio protective effect. The damage or injury is derived from the lack of oxygen or nutrient supply to the cells or tissues. The damage or injury can be morphological or physical damage or injury, e.g. cells can lose their integrity or tissues can become distorted or develop infarcts (areas of necrosis). The damage or injury can also be cell or tissue death and even further death of an organ. The damage or injury also can be damage or injury to the functional aspect of a body part, i.e. the body part's ability to perform its intended function. Morphological or physical injury can accompany the damage or injury to the functional aspect of the body part or the body part can appear normal but not be able to function normally. The term "administering" is intended to include routes of administration which allow the creatine analog to perform its intended function of protecting body parts against injury from ischemia or treating body parts for ischemia. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, etc.), oral, inhalation, transdermal, and rectal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the creatine analog can be coated with or in a material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The creatine analog can be administered alone or further can be co-administered with a pharmaceutically acceptable carrier, e.g. a cardioplegic solution. Further, the analogs can be administered as a mixture of creatine analogs which also can be in a pharmaceutically acceptable carrier. The creatine analogs even further can be coadministered with other different art-recognized reagents useful for treating ischemia as part of a combination therapy regime.

The language pharmaceutically acceptable carrier is intended to include substances capable of being coadministered with the creatine analog(s) and which allow the analog to perform its intended function of treating a body part susceptible to ischemia. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media and agent compatible with the creatine analog can be used within this invention.

The language "effective amount of a creatine analog" in the context of the methods of treating ischemia is that amount necessary or sufficient to prevent, protect, significantly reduce or eliminate the effects of ischemia on the body part. The effective amount can vary depending on such factors as the body part being treated, the type of ischemia and the extensiveness of the ischemia-associated symptoms. The regime of adminstration also can effect what constitutes an effective amount. For example, the effective amount of the creatine analog in this invention can be a 1% solution of the creatine analog in a pharmaceutical carrier up to the percent of saturation of the creatine analog in the particular pharmaceutical carrier. The creatine analog can be administered prior to an ischemic episode and/or post-ischemically. Further, several divided doses can be administered daily or sequentially or the dose can be infused continuously. Further, the dose can be proportionally reduced as indicated by the exigencies of the therapeutic or prophylactic situation.

Creatine (also known as N-(amidinomethyl-N-methylglycine, methylglycosyamine or N-methyl-guanidino acetic acid) is a well-known substance (see The Merck Index Ninth Edition, No. 2556 (1976)) and its formula is as follows:

NH,

V +

NH-

N CH,

CH,

O^'

O

Creatine is present in the muscular tissue, brain and other organs of many vertebrates and the naturally occurring product commercially is extracted from meat. The terms creatine or creatine analog are intended to include both the isolated naturally occurring form, if available, and the chemically synthesized form. Creatine presently is commercially available and further may be chemically synthesized using conventional techniques such as by heating cyanamide with sarcosine (Strecher Jahresber. Chem. (1868), 686; cf. Volhard Z. Chem. 5,318 (1869); Paulmann, Arch. Pharm. 232, 638 (1894); Bergmann et al. 7. Physiol. Chem. 173, 80 (1928); and King J. Chem. Soc. (1930), 2374).

The language "creatine analog" is intended to include compounds which are structurally similar to creatine and/or compounds which are art-recognized as being analogs of creatine. The creatine analogs of this invention are those analogs which are useful for treating body parts susceptible to ischemia. The term creatine analog also is intended to include pharmaceutically acceptable salts of the analog. Creatine analogs have previously been described in copending application Serial No. 07/061,677, entitled Methods of Treating Body Parts Susceptible to Ischemia Using Creatine Analogs, filed May 14, 1993; copending application Serial No. 08/009,638 entitled Creatine Phosphate, Creatine Phosphate Analogs and Uses Therefore filed on January 27, 1993; copending application Serial No. 07/812,561 entitled Creatine Analogs Having Antiviral Activity filed on December 20, 1991; and copending application Serial No. 07/610,418 entitled Method of Inhibiting Transformation of Cells in Which Purine Metabolic Enzyme Activity is Elevated filed on November 7, 1990. The entire contents of each of the copending applications are expressly incorporated by reference herein along with their published foreign counterparts and all of the creatine analogs along with their methods of synthesis discussed in the forementioned applications are intended to be part of this invention unless specifically stated otherwise. The preferred creatine analogs of this invention are those encompassed by formula (I) set forth below:

NR₄R₅

NR₂R₃

R .

Y.

wherein A is selected from the group consisting of N or CH; Z is selected from the group consisting of

B, B_

wherein B1-B4 are each independently selected from hydrogen and OX4 and X1-X4 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and pharmaceutically acceptable salts;

Y\ and Y2 are each independently selected from the group consisting of a direct bond, alkylene, alkenylene, alkynylene and alkoxylene;

R] is selected from the group consisting of hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, and alkoxyl; and R2 - R5, if present, are each independently selected from the group consisting of hydrogen, a phosphorus containing moiety, alkyl, alkenyl, alkynyl, alkoxy and haloalkyl, wherein A may form a ring structure with one of the nitrogens in the amidino moiety or with Y2.

A preferred subgenus of the above formula (I) are compounds encompassed by formula (II) set forth below wherein the variables are as defined above in formula (I).

The alkylene, alkenylene, alkynylene, alkyl, alkenyl and alkynyl groups (hereinafter hydrocarbon groups) may have straight or branched chains. The unsaturated groups may have a single site of unsaturation or a plurality of sites of unsaturation. The hydrocarbon groups preferably have up to about ten carbons, more preferably up to about six carbons, and most preferably up to about three carbons. A hydrocarbon group having three carbon atoms or less is considered to be a lower hydrocarbon group. For example, an alkyl group having three carbon atoms or less is a lower alkyl. Examples of lower hydrocarbon groups which may be used in the present invention include methyl, methylene, ethyl, ethylene, ethenyl. ethenylene, ethynl, ethynylene, propyl, propylene, propenyl, propenylene, propynyl, and propynylene. Examples of higher hydrocarbon groups (from four to about ten carbons) include butyl, t-butyl, butylene, butenyl, butenylene, and butynyl, butynylene, nonyl, nonylene, nonenyl, nonenylene, nonynyl, and nonynylene. The alkoxy, haloalkyl, alkoxyene, and haloalkylene groups (hereinafter substituted hydrocarbon groups) are alkyl or alkylene groups substituted with one or more oxygen or halogen atoms. The alkoxy and haloalkyl groups also may be straight or branched chain and preferably are made up of up to about ten atoms (including carbon, oxygen or halogen), preferably up to about six atoms, and most preferably up to about three atoms. The term halogen is art-recognized and includes chlorine, fluorine, bromine, and iodine. Examples of substituted hydrocarbon groups which are useful within this invention are similar to the examples of the hydrocarbon groups set forth above except for the incorporation of oxygen(s) or halogen(s) into the groups.

The term pharmaceutically acceptable salt (as a possibility for "X" is formula (I) and as it pertains to creatine analog salts) is intended to include pharmaceutically acceptable salts capable of being solvated under physiological conditions. Examples of such salts include sodium, e.g. disodium, potassium, e.g. dipotassium, and hemisulfate. The term further is intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, i.e. alkyl esters, e.g. methyl, ethyl and propyl esters. For purposes of this invention, the amidino moiety of formula I is depicted below:

NR₄R_£

NR 2,R^3

The nitrogens in this moiety can form a ring structure with A or with X2- The ring can be a hydrocarbon ring or a hetero ring containing atoms such as O, N or S. The ring structure further can be a single ring or alternatively can be a fused ring system. The preferred ring structures are single rings having five, six or seven ring members and most prefereably five membered rings such as those present in cyclocreatine- or carbocreatine-like compounds. The creatine analogs of this invention preferably possess inherent characteristics enhancing their ability to perform their intended function of treating body parts for ischemia. For example, the creatine analogs preferably have a solubility which allows them to be delivered to the body part in a pharmaceutically acceptable formulation. A saturated solution is not considered to be a pharmaceutically acceptable formulation. The creatine analogs can be selected based on their ability to act as a substrate for creatine kinase. Creatine analogs which are useful in this invention are listed in Table 1 below. TABLE 1

Prephosphagens

Phosphagens

The analogs of creatine can be purchased or alternatively can be synthesized using conventional techniques. For example, creatine can be used as the starting material for synthesizing at least some of the analogs encompassed by formula I. Appropriate synthesis reagents, e.g. alkylating, alkenylating or alkynylating agents can be used to attach the respective groups to target sites, e.g. a nitrogen in the guanidino moiety. Appropriate protecting groups can be employed to prevent reaction at undesired sites in the molecules.

If the creatine analog contains a ring structure, i.e. one of the nitrogens in the amidino moiety forms a ring with "A" or "Y2", then the analog can be synthesized in a manner analogous to that described for cyclocreatine (Wang, T., J. Org. Chem. 12:3591-3594 (1974)). The various "R", "X" groups can be introduced before or after the ring is formed and "Y" group can be introduced before the ring is formed.

Many creatine analogs have been previously synthesized and described (Rowley et al. J. Am. Chem. Soc. 93:5542-5551 (1971): McLaughlin et al. J. Biol. Chem. 247:4382- 4388 (1972); Nguyen, A.C.K., "Synthesis and enzyme studies using creatine analogs", Thesis, Dept. of Pharmaceutical Chemistry, Univ. Calif., San Francisco (1983); Lowe e_ aL, J. Biol. Chem. 225:3944-3951 (1980): Roberts et al.. J. Biol. Chem. 260:13502-13508 (1985); Roberts et al.. Arch. Biochem. Biophvs. 220:563-571 (1983); and Griffiths et aL, J. Biol. Chem. 251:2049-2054 (1976)). The creatine analogs of this invention can be synthesized chemically or enzymatically. The chemical conversion of the prephosphagens (see Table 1) to the respective phosphagens can be done in the same manner as that descibed by Annesley et al. (Biochem. Biophys. Res. Commun. (1977) 74:185-190). Disodium salts, e.g. disodium salts, of the creatine analogs can be prepared as describe in forementioned copending application Serial No. 08/009,638 filed on January 27, 1993. The contents of all of the forementioned references are expressly incorporated by reference. Further to the forementioned references, Kaddurah-Daouk et aL (WO92/08456) also provide citations for the synthesis of a plurality of creatine analogs (see Examples 2 and 3 including Table 4). The contents of the entire Kaddurah-Daouk et al. published patent application including the contents of any references cited therein also are expressly incorporated by reference.

Transplantation of organs is now considered to be a definitive treatment for patients with end stage liver, kidney, heart and pancreas disease. There is thus a great deal of interest in improving ex vivo storage of cadaveric organs and thus the viability of organ transplants. The two most commonly used methods for organ treatment, e.g. preservation, are hypothermic storage and continuous pulsatile perfusion. With hypothermic storage,the organs are rapidly cooled immediately after removal from the cadaver donor using a combination of external cooling and a short period of perfusion. The hypothermic storage method is a preferred method due to its practicality and the ease of transportation of the organs. Continuous pulsatile perfusion involves hypothermic pulsatile perfusion after flushing with a chilled electrolyte solution. The present invention also pertains to compositions and methods for treating an organ intended for transplantation. The composition contains an amount of a creatine analog effective to treat the organ intended for treatment (or a satellite organ) and a pharmaceutically acceptable organ treatment solution. The methods involve contacting an organ with the composition. For example, the organ can be infused with the compositions of this invention. The language "treating an organ" is intended to include contact of the organ by the creatine analog either directly or indirectly, e.g. via a solution. This language is intended to include storage, both short-term or prolonged storage, and preservation. The treatment can also be of the organ in vivo prior to removal for transplantation. The language "effective amount" in the context of the organ treatment compositions is intended to include those amounts which have a beneficial effect on an organ being transplanted or a satellite organ. The beneficial effect does not have to be an improvement of the condition of the organ and can be maintenance of the organ above a selected acceptable level allowing it to be functional after transplantation. The effective amount can vary depending on the organ being treated.

The language "pharmaceutically acceptable organ treatment solution" is intended to include solutions used in organ transplantation procedures, e.g. organ preservation solutiosn. A number of organ preservation solutions have been develped with a view to extended organ preservation time. Ringer's lactate isotonic saline solutions have been used as extracellular flushing solutions and have been reported to allow for safe renal preservation for shor periods of time. i.e. up to four hours. Storage for longer periods of time may result in severe histologic ischemic damage and subsequent non-function of the organs (U.S. Patent No. 4,920,004).

An intracellular electrolyte solution devleoped by Collins et al., (Lancet 2:1219, 1969), has been reported to offer several advantages for hypothermic storage. Modified Collins' solutions also have been developed for use in hypothermic storage. For example, Euro-collins solution is similar to Collins solution with, the exception that it does not contain magnesium.

Other organ preservation solutions which have been developed include Sacks' solution (S\ and S2) which have high intracellular ion concentration and osmotic pressure (Sacks, S.A., Lancet 1 :1024, 1973). These solutions have been reported to provide improved transplantation results after storage of kidneys for up to 72 hours. Protective additives such as ATP-MgCl2, AMP-MgCl2 and inosine have also been included in preservation/flush solutions. (Siegel, N.J. et al., Am. J. Phvsiol. 245:530, 1983; Stromski, M.E. et al., Am. J. Phvsiol. 250:834, 1986; Sumpio, B.E. et al., Am. J. Phvsiol. 247:1046; Stromski, M.E. et al, Am. J. Phvsiol. 250:834, 1986), Belzer et al., (Transpl. Proc. 16:161, 1984).

U.S. Patent No. 4,920,004 discloses a hyperosmotic intracellular flush and storage solution that is reported to combine the alient features of Belzer' s ATP-MgCl2 perfusate and commonly used Collins' C-2 Flush solutions. Mannito is substituted in place of dextrose in Collins C-2 solution and adenosine and magnesium are added to the solution to improve the preservation properties of the flush solution.

U.S. Patent Nos. 4,798,824 and 4,873,230 disclose solutions for the preservation of organs (particularly kidneys) prior to implantation, containing a specific synthetic hydroxyethyl starch in place of serum albumin. In particular, U.S. Patent No. 4,798,824 discloses a solution including 5% hydroxyethyl starch having a molecular weight of from about 200,000 to about 300,000 and wherein the hydroxyethyl starch is substantally free of ethylene glycol, ethylene, chlorohydrin, sodium chloride and acetone. U.S. Patent No. 4,873,230 discloses a solution containing hydroxyethyl starch having a molecular weight of from about 150,000 to about 350,000 daltons, a degree of substitution of from about 0.4 to about 0.7 and being substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone.

U.S. Patent No. 4,879,283, discloses a solution for the preservation of organs which contains lactobionate and raffinose and has a solution osmolality of about 320 mOsm/L, K+ of 120 mM and Na+ of 20 mM. The solution also contains a synthetic hydroxyethyl starch and other components such as glutathione and adenosine. The solution disclosed in U.S. Patent No. 4,879,283 is commonly known as the University of Wisconsin solution or UW solution. The solution has been reported for preserving the liver (Jamieson, N.V. et al., Transplantation 46:517, 1988), and pancreas (Wahlberg, J.A., Transplantation. 43:5, 1987).and heart (Wicomb, W.N., Transplantation 47:733, 1988; and Swanson, D.K., J. Heart Transplant 7:456. 1988).

This invention also pertains to methods for treating a subject for an ischemia- associated disease or condition by administering to the subject an effective amount of a creatine analog. The ischemia-associated disease or condition includes diseases or conditions in which ischemia is at least one of the underlying causative factors. Examples of ischemia- associated disease or conditions include congestive organ failure, e.g. congestive heart failure, angina, etc.

The term "subject" is intended to include mammals susceptible to ischemia. The subject can have or just be susceptible to an ischemia-associated disease or condition at the time of treatment. Examples of subjects include humans, dogs, cats, pigs, cows, horses, rats, and mice.

The language "effective amount of creatine analog" in the context of these methods is that amount necessary or sufficient to significantly reduce or eliminate symptoms associated with the ischemic-associated disease or condition. This amount can vary depending on such factors as, the particular disease or condition, the weight of the subject and severity of the symptoms.

The following invention is further illustrated by the following examples which should in no way be construed as being further limiting. The contents of all references, pending patent applications and published patent applications, cited throughout this application are hereby incorporated by reference. The following methodology described in the Materials and Methods section was used throughout the examples set forth below. It should be understood tht the animal models used throughout the examples are accepted animal models and that demonstration of efficiacy in these animal models is predictive of efficiacy in humans.

Example 1 - A Transplant Model

This example demonstrates the effect of cyclocreatine phosphate on the recovery of rat hearts after prolonged cold storage which is intended to be a model for the circumstances surrounding an organ transplant. The Material and Methods section provides information which is applicable throughout all of the examples.

Materials and Methods

Experimental preparation

Male Sprague-Dawley rats weighing 300-325 gm were injected intravenously with 1 ml saline (n=6) or cyclocreatine phosphate (CCrP) (200 mg/1 ml saline) (n=6). After two hours, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/rat) and heparinized intravenously (2 mg/rat). The excised hearts were rapidly submerged in ice-cold Krebs-Henseleit bicarbonate buffer (KHB-119 mM NaCl, 25 mM NaHCO₃, 4.6 mM KCL, 1.2 mM KH₂PO₄, 1.2 mM MgSO 2.5 mM CaCl , and 11 mM glucose) and weighed. The KHB buffer was passed through a 5 um porosity filter before use. The hearts were then attached to a Langendorff perfusion apparatus via the aorta and perfused at a pressure of 45 cm H2O with cold (4°C) University of Wisconsin (UW) solution (The Dupont Merck Pharmaceutical Co., Wilmington, Delaware). The perfusion apparatus was water-jacked for temperature control. Each heart received 10 ml of UW solution. Control saline hearts were then immersed in 40 ml ice-cold UW solution. CCrP treated hearts were immersed in 40 ml ice-cold UW solution containing 100 mg CCrP (Hartford Hospital solution (hereinafter HH.)).

After 6 hours cold storage (4°C), hearts were weighed, mounted on the perfusion apparatus and perfused through the aortic root at a pressure of 100 cm H2O with KHB buffer at 37°C equilibrated with 95% oxygen and 5% carbon dioxide. The pulmonary artery was incised to ensure free drainage of coronary venous effluent. The Langendorff perfusion was conducted for fifteen minutes, during which the left atrium was cannulated (hereinafter Langendorff mode). Hearts were then converted to the working mode by initiating left atrial perfusion at a pressure of 18 cm H2O and allowing the left ventricle to eject into a recirculating aortic column against the afterload of 95 cm H2O (hereinafter working heart mode). During the thirty-minute working mode, aortic flow was measured (every five minutes) by a flowmeter in the aortic column. Similarly, coronary flow was measured by timed (sixty seconds) volumetric collections of the effluent from the right side of the heart. The aortic pressure was monitored via the side arm of the aortic cannula with a pressure transducer (Hewlett Packard, USA). The heart rate was monitored and recorded from EKG. Cardiac output was derived from the sum of aortic and coronary flow rates. Stroke volume was obtained by dividing cardiac output by heart rate, while stroke work was calculated by multiplying stroke volume by systolic pressure. After thirty minutes of reperfusion in the working heart mode, hearts were weighed and biopsy samples for electron microscopy analysis were taken from the transmural tissue. The remaining hearts were placed in liquid nitrogen and kept frozen at -70°C to determine myocardial levels of CCrP.

Enzyme Leakage

Creatine kinase and lactate were assayed in coronary effluent samples collected after thirty minutes of reperfusion (Arch. Neurol. (1961) 4:520; Analyst (1972) 97:142) using commercially available kits (Sigma Chemical Co., St. Louis, MO).

Myocardial Levels of CCrP

A modified colorimetric assay to measure myocardial levels of cyclocreatine and its phosphorylated metabolite cyclocreatine phosphate was used (Walker 1976, J. Biol. Chem. (1976) 251 :2049-2054). Frozen hearts (UW control and HH treated groups) were weighed then quickly minced with a scalpel and placed into a four ml test tube containing three ml of 0.4 M perchloric acid. The mixture was homogenized with a chilled Tekmar tissue homogenizer for two minutes on a medium-high setting. The sample was then vortexed and split into two eppendorf tubes. Each was centrifuged at 14K rpm for two minutes at -20°C and the supernate was drawn off into clean eppendorf tubes. One sample was placed in a 65° C waterbath for forty-five minutes to measure the total amount of cyclocreatine and CCrP, while the other was kept on ice for forty-five minutes to measure the free cyclocreatine. Each sample was neutralized with 0.4 ml of 4M KOH and again centrifuged at 14K rpm. The supernatant (0.5 ml) was assayed colorimetrically for free and total cyclocreatine by adding 0.25 ml of a 1% aqueous solution of NA3 (mM Fe(CN)5NH3) and 1 ml of 30 mM Na2CO3. Cuvettes containing the later mixture were incubated at room temperature for thirty minutes and were then read at 605 nM using a spectrophotometer.

The amount of free cyclocreatine present in the chilled heart samples was calculated by correcting for the dilution factors and reported as umol/gm of tissue. The phosphorylated cyclocreatine concentrations were obtained by substracting the free cyclocreatine (i.e., chilled heart samples) from the total values (i.e., heated heart samples).

Electron Microscopy

At the end of thirty minutes reperfusion, biopsy samples were taken from the transmural tissue of the left ventricles of HH rats (n=6) and UW rats (n=6). Samples were immediately fixed in 4% buffered glutaraldehyde and processed for electron microscopy analysis (SAE).

Ultrastructural analysis was performed blindly. Parameters evaluated were based on morphologic changes associated with reversible versus irreversible injury, including: nuclear chromatin clumping, nuclear margination, mitochondrial swelling, mitochondrial amorphous matrix densities, disrupted mitochondrial eristae, decreased glycogen, and ruptured sarcolemmal membranes.

Statistical Analyses All results were expressed as mean ± standard error of the mean. HH group (n=6) was compared to UW group (n=6) using the t-test. A difference was considered statistically significant if the P value was less than 0.05.

All animals received humane care in compliance with the "principles of "Laboratory

Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institute of Health (NIH publication No. 80-23, revised 1978).

Results

Cardiac function The nonischemic aerobic control aortic flow and cardiac output were 54.8 ± 3.1 ml/min and 69.9 ± 2.4 ml/min, respectively. As shown in Figure 1, the HH group (hearts pretreated with CCrP then stored in UW containing CCrP) showed significantly better recovery of aortic flow compared to control UW group (saline treated hearts then stored in UW). After thirty minutes reperfusion, HH hearts showed aortic flow values of 28 ± 1.9 ml/min (52% baseline), while control hearts showed recovery of 13.3 ± 2.9 ml/min (25%) (P <0.01). Cardiac output recovery (Figure 2) was 51 ± 3.3 ml/min (73% baseline) for HH group and 32 ± 4 ml/min (46%) (P<0.01) for UW rats. Similar patterns of recovery were observed for stroke volume (Figure 3) and stroke work (Figure 4). However, the recovery of coronary flow was significantly different between the two groups only during the first ten minutes and the last thirty minutes of reperfusion (Figure 5). The recovery of heart rate and aortic pressure was essentially the same among the two groups.

Heart Weights

As shown in Figure 6, the increase in heart weights after the initial six hours of cold storage was significantly higher in UW rats (i.e., 0.3 ± 0.03 gm) compared to HH hearts (i.e., 0.17 ± 0.04 gm) (P<0.05). After thirty minutes reperfusion UW hearts showed an increase of 0.53 ± 0.09 gm, while HH hearts showed an increase of 0.36 ± 0.08 gm. Enzyme leakage

No significant leakage of creatine kinase was detected after thirty minutes reperfusion in both groups. The lactate leakage was similar in HH group (0.54 ± 0.1 mmol/L) and UW rats (0.44 ± 0.1 mmol/L).

Myocardial levels of CCrP

After thirty minutes of reperfusion, HH hearts showed a total accumulation of cyclocreatine and cyclocreatine phosphate of a range of 0.16-0.34 u mol/gm of tissue with a mean value of 0.256 ± 0.03. The percent phosphorylated was ranged from 78%-90% with a mean value of 82% ± 2.11. UW hearts showed no detectable levels of both compounds.

Discussion

Hearts of rats pretreated with cyclocreatine phosphate then incubated in UW solution containing cyclocreatine phosphate (HH solution) showed statistically significant better recovery of cardiac function after six hours of cold storage compared to control hearts. The improved recovery was observed during the first ten minutes of reperfusion and continued throughout the remaining twenty minutes. Cyclocreatine phosphate treatment also resulted in statistically significant reduction in the increase in heart weights after prolonged preservation. The reduction in tissue edema may be associated with the observed restoration of function. Although drug treatment improved function recovery, there was no detectable difference between the two groups in regard to myocardial injury measured by the release of creatine kinase and lactate as enzymatic indicators, or analyzed ultrastructurally by electron microscopy. This further supports the need to assess myocardial protection using multiple independent indexes of tissue function. After thirty minutes reperfusion, treated hearts showed residual accumulation of cyclocreatine and its phosphorylated metabolite CCrP of 0.256 ± 0.03 u mol/gm of tissue. Cyclocreatine phosphate constituted 82% of the total amount.

Example 2 - The Use of Cyclocreatine Phosphate In A Bypass Model This example demonstrates myocardial protection by cyclocreatine phosphate by showing improved cardiac function after cold and warm ischemic cardioplegic arrest. This example is intended to be a bypass model. The term bypass includes all situations in which the heart is arrested and the body's blood circulation is bypassing the heart, e.g. revascularization of the heart, valve, etc. Both cold and warm ischemic cardioplegic arrest are included in this example because some surgeons reduce the temperature via carioplegic solution for surgical procedures while others do not use techniques which alter the temperature. The administration of cyclocreatine phosphate (CCrP) prior to ischemia enhances the recovery of rat hearts during reperfusion. Rats (n=6 per group) were injected intravenously with 1 ml saline or CCrP (200mg/ml saline). After one hour, hearts were excised and perfused in the Langendorff mode for five minutes and then in the working heart mode for twenty minutes (i.e., preischemic function). Hypothermic arrest was induced by infusing an initial dose of cold St. Thomas solution (15 ml at 22°C), followed by subsequent doses every thirty minutes for 2.5 hours. Normothermic arrest was induced by infusing warm St. Thomas solution (15 ml at 37°C) once and then keeping the hearts at 37°C for forty minutes. Following arrest, the hearts were reperfused in the Langendorff mode for fifteen minutes and then in the working heart mode for thirty minutes. As shown in Figure 7, hearts pretreated with CCrP showed significantly better restoration of aortic flow (i.e., 60-75% preischemia)(mean ± S.E.) after 2.5 hours of cold arrest compared to saline hearts (i.e., 27- 44%)(P<0.001). In normothermically arrested hearts (Figure 8), CCrP treatment also resulted in better recovery of aortic flow (i.e., 21-60%) compared to that of saline hearts (i.e., 14- 37%)(P<0.05). Similar patterns of recovery of cardiac output, stroke volume, and stroke work were observed. This improved postischemic recovery demonstrates that cyclocreatine phosphate can provide a cardioprotective effect against cold and warm ischemic arrest.

Example 3 - The Use of Cyclocreatine In Bypass Models

The term "bypass" is is as defined in Example 2 above. The purity and physical characteristics of cyclocreatine were verified using high performance liquid chromatography and nuclear magnetic resonance. The isolated rat heart model was used to determine the cardioprotective effect of cyclocreatine under controlled pre and afterload conditions. Using the standard Langendorff-working heart model, aortic flow and coronary flow were measured before cardioplegic arrest (forty minutes at 37°C) and during reperfusion for thirty minutes. Cyclocreatine (600 mg/kg) was administered either two hours or thirty minutes before removal of the hearts. The cardioprotective effect of cyclocreatine also was determined at two lower doses (i.e., 300 mg/kg and 150 mg/kg) administered thirty minutes before sacrifice. Control rats were injected with saline at similar time points.

The canine model of cardiopulmonary bypass was used to determine the cardioprotective effect of cyclocreatine in vivo. Cyclocreatine (500 mg/kg) was injected intravenously one hour before experiment. Control dogs were injected with saline. Dogs underwent cold cardioplegic arrest for one hour, perfused for forty five minutes and then weaned off bypass and monitored for four hours. In another study, hearts were arrested for three hours, perfused for forty five minutes and weaned off bypass for four hours. The effect of cyclocreatine on normal myocardial contractility, heart rate and arterial blood pressure was also measured to determine whether cyclocreatine has inotropic or chronotropic effects on the heart.

All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institute of Health (NIH published 80-23, revised 1978). Isolated Rat Heart Model

Male Sprague-Dawley rats weighing 300-325 gm were injected intravenously with saline (control) or cyclocreatine (CCr). Rats were divided into three groups:

Group I: Rats were injected with saline (n = 6), or CCr (600 mg/kg) (n = 6) for two hours before sacrifice. CCr treated rats receiving 180 mg/rat were injected twice with a 4% preparation of saturated drug at physiologic temperature two hours (90 mg/2.25 ml) and one hour (90 mg/2.25 ml) before sacrifice. Control rats were injected similarly twice with saline; namely two hours (2.25 ml) and one hour (2.25 ml) before sacrifice.

Group II: Rats were injected with saline (n = 6) or CCr (600 mg/kg) (n = 6) for thirty minutes before sacrifice. CCr treated rats (180 mg/rat) were injected with a 4% preparation of saturated drug at physiologic temperature (180 mg/4.5 ml) thirty minutes before sacrifice. Control rats were similarly injected with saline; namely thirty minutes (4.50 ml) before sacrifice.

Group III: Rats were injected with saline (n = 6) or CCr at 600 mg/kg (n = 6), 300 mg/kg (n = 6), and 150 mg/kg (n = 6) for thirty minutes before sacrifice. CCr treated rats (600 mg/kg) were injected twice with 2.25 ml of 4% preparation of saturated drug at physiologic temperature (90 mg/2.25 ml) thirty minutes and fifteen minutes before sacrifice. Rats receiving 300 mg/kg CCr were injected with 2.25 ml of 4% preparation of saturated drug at physiologic temperature (45 mg/2.25 ml) thirty min before sacrifice. Rats receiving 150 mg/kg CCr were injected with 2.25 ml of 4% preparation of saturated drug at physiologic temperature (22.5 mg/2.25 ml) thirty minutes before sacrifice. Control rats were injected with 2.25 ml saline thirty minutes and fifteen minutes before sacrifice.

After two hours or thirty minutes, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/rat) and heparinized intravenously (2 mg/rat). Hearts were rapidly excised and submerged in ice-cold Kreb-Henseleit bicarbonate buffer (KHB:119 mM NaCl, 25 mM NaHCO₃, 4.6 mM KCL, 1.2 mM KHPO₄, 1.2 mM MgSO₄2.5 mM CaCl₂, and 11 mM Glucose). Hearts were mounted on the perfusion apparatus and perfused through the aortic root at a pressure of 100 cm H₂O with KHB buffer at 37°C equilibrated with 95% oxygen and 5% carbon dioxide. The KHB buffer was passed through a 5 um porosity filter before use. The pulmonary artery was incised to ensure free drainage of coronary venous effluent. The Langendorff perfusion was conducted for a five minute washout and equilibration period, during which the left atrium was cannulated. Hearts were then converted to the working mode by initiating the left atrial perfusion at a pressure of 18 cm H₂O and allowing the left ventricle to eject perfusate into a recirculating aortic column against the afterload of 95 cm H₂O. At the end of twenty minutes working mode, aortic flow was measured by a flowmeter in the aortic column. Similarly, coronary flow was measured by timed volumetric collections of the effluent from the right side of the heart. Cardiac output was derived from the sum of aortic and coronary flow rates. After recording the preischemic baseline values of cardiac output, normothermic arrest was induced by infusing 15 ml of warm St. Thomas solution (sodium chloride 110.0 mmol/L; potassium chloride 16.0 mmol/L; magnesium chloride 16.0 mmol/L; calcium chloride 1.2 mmol/L; sodium bicarbonate 10.0 mmol/L adjusted to a pH of 7.8) at 37°C. Hearts were then kept at 37°C for forty minutes. After forty minutes, hearts were reperfused in the Langendorff mode with KHB buffer for fifteen minutes. Hearts were then converted to the working mode for an additional thirty minutes of reperfusion in which the recovery of function was measured every five minutes.

Intact Canine Heart Model

Surgical Techniques: Twenty two adult preconditioned dogs (20 to 25 kg) were used in this demonstration. The dogs were anesthetized with sodium pentobarbital (30 mg/kg) and maintained on a respirator supplied with 100% oxygen. Femoral arterial pressure was displayed on a Honeywell multichannel monitor (AR - 6 Simultrace Recorder). A heating pad was placed under each dog to keep its body temperature at 37°C before and after cardiac arrest. The heart was exposed in a pericardial cradle through a median sternotomy.

Anticoagulation was accomplished with intravenous heparin (2,500 U/kg). Cannulas were placed in the ascending aorta, inferior vena cava, and superior vena cava. A pediatric bubble oxygenator (Bentley Temptrol Oxygenator; American Bentley, Irvine, CA) was used. The perfusion pump was primed with Ringer's solution (900 ± 30 ml), and the dogs were then placed on cardiopulmonary bypass (CPB) (Travenol Laboratories, Morton Grove, IL). The azygos vein was occluded and the vena cava snared during the cross-clamp period (i.e., one hour or three hours). Hearts were defibrillated (10 to 30 mj) ten minutes after release of the aortic cross-clamp, as needed. The cold cardioplegic solution (4°C) (Roberts et al. (1983) Arch Biochem Biophys 220: 563-71) consisting of one liter of plasmalyte and containing 30 ml of 50% dextrose, 24 mEq of sodium bicarbonate, 20 mEq of potassium chloride, 20 IV of soluble insulin, and 1 mEq of calcium chloride was infused (250 ml every twenty minutes) during the aortic cross-clamp period. Topical ice cooling was applied. The myocardial temperature (apical left ventricle) was kept below 20°C and the body temperature (rectal) between 25°C and 28°C. Peripheral blood samples were drawn before, during, and after CPB to monitor the arterial pH, carbon dioxide tension, oxygen tension, hematocrit, and the potassium and calcium ion levels. The pulmonary artery wedge pressure and the blood pressure were maintained during reperfusion to that of the baseline values by applying the infusion- venting technique. None of the dogs received blood transfusions.

Cyclocreatine Administration A total of twenty two adult dogs were used in this demonstration. Cyclocreatine (500 mg/kg) was prepared in saline and administered one hour before each test. A 3% solution (360 ml) was injected over a one hour period (60 ml every ten minutes). Control dogs received saline at similar time points. Dogs were divided into three groups:

Group IN: Control (n = 6) and cyclocreatine (n = 6) treated dogs underwent cardiac arrest for one hour, then hearts were reperfused on bypass for forty five minutes and weaned off bypass for an additional four hours. Postischemic cardiac indexes (cardiac output divided by body surface area, American Edwards Laboratories, Santa Anna, CA) were measured during off bypass and compared to baseline values recorded before arrest. Three readings of cardiac output were taken at each time point.

Group V: Control (n = 3) and cyclocreatine (n = 3) treated dogs underwent cardiac arrest for one hour followed by a forty five minute reperfusion on bypass then hearts were weaned off bypass for an additional two hours. Segmental contractility measured during bypass and off bypass were calculated as percent of baseline values recorded before arrest. Segmental myocardial contractility was measured using one pair of piezo ultrasonic crystals implanted in a circumferential plane (1 cm apart) in the left ventricle apex (Theroux et al. (1974) Circ Res 35:896-908). Changes in the distance between the crystals were recorded every fifteen minutes before and after aortic cross-clamping. Three readings of segmental contractility were taken at each time point.

Group VI: Control (n = 2) and cyclocreatine (n = 2) treated dogs underwent cardiac arrest for three hours followed by reperfusion on bypass for forty five minutes then off bypass for four hours. Cardiac function was measured as cardiac index.

To determine whether cyclocreatine has inotropic or chronotropic effects on the dog hearts (n = 3-6), baseline values of contractility, heart rate, and arterial blood pressure (monitored with a carotid artery catheter) were obtained before and after the administration of cyclocreatine for one hour. Statistical Analysis

All data were expressed as mean value ± standard error of the mean. The Student's t- test was used to calculate the p value (p < 0.05 was considered to be significant).

Isolated Rat Hearts

As shown in Figure 9, hearts of rats which received cyclocreatine (600 mg/kg) for two hours (Group I) before removal and cardioplegic arrest for forty minutes at 37°C showed significantly better recovery of cardiac output at the end of thirty minutes of reperfusion compared to saline hearts (i.e., 69.7 ± 3.54% CCr of baseline values vs. 56.6 ± 2.39% saline, p < 0.05). When the drug was administered thirty minutes instead of two hours (Group II) prior to the removal of hearts, the recovery of cardiac output continued to significantly be better in cyclocreatine hearts (i.e., 75.96 ± 5.34%) compared to saline hearts (i.e., 58.13 ± 1.93%, p < 0.05) (Figure 10). A dose response study was, therefore, performed using the thirty minute preinjection time. The cardioprotective effect of cyclocreatine at a 600 mg/kg dose was compared to lower doses of 300 mg/kg and 150 mg/kg (Group III). As shown in Figure 11 , no significant difference in the cardioprotective effect was observed in rats receiving cyclocreatine at a dose of 600 mg/kg (i.e., 75.96 ± 5.34%) or 300 mg/kg (i.e., 78.85 ± 2.74%). However, when the dose was reduced to 150 mg/kg, the drug did not show any cardioprotective effect during reperfusion (i.e., 47.51 ± 9.27%, p < 0.05 vs. 600 mg/kg and 300 mg/kg) under these conditions in this particular demonstration. Control rats showed a range of cardiac output recovery of 56.6-58.13%.

Intact Canine Hearts

Figure 12 and Figure 13 describe the recovery of cardiac function in dogs which underwent cold cardioplegic arrest for one hour. After four hours of reperfusion off bypass, cyclocreatine treated hearts (Group IV) showed significantly better recovery of cardiac index (i.e., 87.70 ± 7.55% of baseline values) compared to control canine hearts (i.e., 51.50 ± 5.8% - p < 0.001. (Figure 12). Throughout the four hours of reperfusion, cyclocreatine hearts maintained their cardiac index of an average of 92.46 ± 3.04%, while control hearts showed an initial recovery of 80.11 ± 1.236% after thirty minutes which then declined to 51.50 ± 5.8% by four hours. To determine the effect of cyclocreatine on segmental contractility, six additional dogs were used (Group V). As shown in Figure 13, cyclocreatine treated hearts exhibited better segmental myocardial contractility at the end of two hours reperfusion (i.e., 115 ± 25% of baseline values) compared to control hearts (i.e., 68 ± 10%), supporting the cardiac index data. The hyperfunction seen shortly after hearts were weaned off CPB (Figure 13) may be associated with hypermetabolism due to the release of endogenous catecholamines (Silverman et al. (1988) Cardiovascular Surgery: State of the Art Events: Vol. 2, No. 2, Philadelphia: Hanley and Belfus Inc. 181-195). In another study, the aortic cross-clamp time was extended to three hours (Group VI). Although cyclocreatine treated hearts (Figure 14) continued to show improved recovery of cardiac index after being off bypass for three hours (i.e., 91 ± 8.79% of baseline values), control hearts ceased beating after two hours. The effect of cyclocreatine on normal heart function also was investigated. Baseline values of segmental myocardial contractility, heart rate, and arterial blood pressure were recorded prior to the administration of cyclocreatine, then after sixty minutes. Results indicated no changes in all parameters recorded before and after cyclocreatine administration (Table 2), suggesting that the drug does not have inotropic or chronotropic effects on the heart.

Table 2. Effects of cyclocreatine on heart rate, arterial blood pressure, and segmental contractility.

Before Cyclocreatine After Cyclocreatine

Heart rate (b/m) 124 ± 13 120 ± 3

Arterial blood pressure (mmHg) 125 ± 5 121 ± 9

Segmental contractility (cm) 2.55 ± 0.33 2.8 ± 0.27

This example demonstrates a cardioprotective effect of cyclocreatine against ischemic injury in isolated and intact models of cardioplegic cardiac arrest. Using isolated rat hearts (Langendorff-working heart model), cyclocreatine treated hearts showed significantly better recovery of cardiac output after normothermic ischemic arrest compared to saline hearts. The drug was effective at 600 mg/kg and 300 mg/kg. Furthermore, administering cyclocreatine for thirty minutes or two hours prior to removal of the heart was effective in protecting the hearts against ischemic injury. Having established the capability of cyclocreatine to enhance the recovery of cardiac function in a controlled model of fixed pre and afterload pressure, the demonstration was repeated using intact canine hearts. A prompt recovery of cardiac function was also seen in cyclocreatine treated hearts. Although the majority of cyclocreatine hearts resumed spontaneous sinus rhythm shortly after release of the aortic cross-clamp (i.e., one to two min), control hearts required defibrillation within the first ten minutes. After one hour of cold cardioplegic arrest, cyclocreatine hearts resumed normal function of cardiac output and segmental contractility. Control hearts, however, showed significantly reduced function during reperfusion. When the aortic cross-clamp time was extended to three hours, cyclocreatine treated hearts continued to exert normal recovery during reperfusion, while control hearts ceased beating. When cyclocreatine was tested on normal canine hearts, it had no effect on segmental contractility, heart rate, or arterial blood pressure, which excluded a possible inotropic or chronotropic effect of the drug.

Example 4 - A Model Demonstrating the Protective Effect of Creatine Analogs Against Injuries from Warm Ischemia Without the Use of a Protective

Solution

Cyclocreatine phosphate (CCrP) significantly improves recovery of rat heart function following hypothermic and normothermic cardioplegic arrest. The present study demonstrates the cardioprotective effect of CCrP during warm ischema without cardioplegic solution. Rats (n=6 per group) were injected intravenously with 1 ml saline or CCrP (150 mg/1 ml saline). After one hour, hearts were arrested in vivo for seven minutes, nine minutes and ten minutes by cross clamping the aorta. Excised hearts were then placed on the Langendorff reperfusion working heart apparatus to measure function. As shown in Figure 15, improved recovery of aortic flow after twenty minutes of reperfusion was observed in CCrP hearts for all groups (7 min = 75%, 9 min = 55%, 10 min = 45%) compared to their respective saline controls (7 m = 50%, 9 min = 25%, 10 min = 18%) (P<0.05). Similar improvement in cardiac output was also observed. These results demonstrate a cardioprotective effect of cyclocreatine phosphate against warm ischemic injury.

Example 5 - A Comparison of the Effects of Cyclocreatine Phosphate Versus Creatine Phosphate on the Recovery of Rat Heart Function

The hypothermic and normothermic arrest models were as described in Example 2 above. Both cyclocreatine phosphate (CCrP) and creatine phosphate (CrP) each contained in St. Thomas solution enhanced recovery of rat heart function after normothermic arrest (see Figure 16). The cardioprotective effect of CrP in hypothermically arrested rat hearts (2.5 hours at 22°C) also was compared to that of CCrP (see Figure 17). Using a Langendorff working rat heart model, CrP (2.55 mg/ml, n=6) in cold St. Thomas solution showed no improvement of postischemic function over that of control hearts (n=6) after thirty minutes of reperfusion (25% vs 30% preischemic aortic flow, respectively). Furthermore, following intravenous injection (n=6 per group) of 1 ml saline, CrP (152 mg/lml saline) or CCrP (200 mg/ml saline) one hour prior to experiment, better recovery of aortic flow was seen in CCrP hearts (72%) compared to that of saline (43%) (PO.001) or CrP hearts (22%) (P<0.0001).

Example 6 - A Comparison of the Effects of Cyclocreatine Phosphate Versus Cyclocreatine on the Recovery of Rat Heart Function

The normothermic arrest model was conducted as described in Example 2 above. Equivalent amounts of CCrP and CCr were delivered intravenously thirty minues prior to ischemic arrest and one hour prior to ischemic arrest. As shown in Figures 18 and 19, the cardioprotective effect of CCrP was greater than that of CCr after normothermic arrest. After thirty minutes of reperfusion following normothermic arrest, the percent of preischemia aortic flow was 62.85 ± 5.52 for CCr and 73.64 ± 5.15 for CCrP. More significantly, the CCrP was significantly better than CCr when the comparison is done based on percent of the control (saline solution). The recovery of aortic flow (base on % of saline control) was approximately 205 percent for CCrP and approximately 138 percent for CCr for the one hour pre-ischemic injection. Figures 20 and 21 are dose-response graphs for the present example.

Example 7 - The Effect of Post-Ischemic Administration of a Creatine

The present example demonstrates that the administration of cyclocreatine post- ischemia will exert a beneficial, e.g. cardioprotective, effect against reperfusion injury.

Twelve pigs underwent occlusion of the LAD for forty minutes followed by reperfusion for six hours. Immediately after the release of the occlusion, an intravenous injection of saline (n=6) or a 3% solution of CCr (500 mg/kg) (n=6) was administered followed by infusion of a 1% CCr (250 mg/kg) or saline. After a six hour reperfusion, cardia index values of CCr hearts (102.06 ± 5.72% baseline) were significantly better than that of control hearts (58.33 ± 5.41%) (P < 0.001). The results demonstrate a cardioprotective effect of CCr when administered post-ischemia.

Example 8 - The Use of Creatine Analogs for Protecting the Spinal Cord from Ischemic Injury

The preservation of spinal cord function is a major problem in surgery on the descending thoracic or the thoracoabdominal aorta. Aortic cross clamping reduces perfusion of kidneys, splanchnic organs and the spinal cord. Ischemic injury associated with the spinal cord includes paraparesis or paraplegia. The present example demonstrates that cyclocreatine protects the spinal cord against ischemic injury. A pig model involving thoracic aortic cross-clamping for forty minutes was used. Cyclocreatine (1% soluble solution) was administered daily for three days (six to eight hours infusion) and on the day of surgery. Four doses of cyclocreatine were used (40 gm 23 kg pig, 30 gm, 20 gm and 10 gm). Control pigs were similarly infused with saline. The end point of each test was the presence or absence of paraplegia evaluated twenty four hours after surgery.

Surgical Procedure

Yorkshire pigs of either sex, weighing 20-25 kg, were divided into three groups. CCr treated pigs received daily intravenous infusion of 1000 ml 1 % CCr solution (in saline) for four days (Group I, n=5; total 40 gm), three days (Group II, n=5; total 30 gm), or two days (Group III, n=2; total 40 gm and n=3; total 30 gm), including the day of surgery. Control pigs received an equal volume of saline for four days (Group I, n=6). three days (Group II, n=6), or two days (Group III, n=5). Pigs were anesthetized with telazol (200 mg) muscularly and maintained on an inhalation of 2% forane. A venous catheter was inserted into the left external jugular vein of each pig for the perfusion of normal saline (control) or 1% CCr (treated), which was dripped daily through the venous catheter for six to eight hours. On the day of surgery, pigs were anesthetized as described above, then intubated and ventilated. Optimal oxygenation was assured by regular blood gas analyses. Through two left thoracotomy (5th and 9th intercostal space), the descending aorta was exposed. An arterial catheter was inserted into the aorta (proximal to clamp) to monitor the proximal arterial pressure. Electrocardiography was also monitored. Heparin (2000 Units) was injected intravenously before cross-clamping. The descending aorta was clamped proximally (1 cm distal to the subclavian artery) and distally (2 cm above the diaphragm). The period of occlusion was thirty minutes. The proximal arterial pressure was reduced to pre-operative values with esmolol (5-20 mg intravenously once) during the period of occlusion. After thirty minutes, the distal clamp was removed first and then the proximal clamp. Sodium bicarbonate was administered to balance the expected acidosis after unclamping. The arterial catheter was then removed. A #16 chest tube was placed through a separate puncture (10th intercostal space). A Heimlich valve was connected to the chest tube, then the chest incisions were closed. Body temperature was continuously measured with a rectal thermometer during the operation and was kept relatively constant between 35-37° C with the aid of a heating blanket.

Neurologic assessment was recorded at emersion from anesthesia and at twenty four hours post-operation and graded according to Tarlov: 0-no movement of hind limbs; 1- perceptible movement of hind limbs; 2-hind limbs movement good unable to stand; 3-able to stand and walk; 4-complete recovery. Paralysis was defined as grade 0, paretic defined at grade 1, 2 and 3, and normal as grade 4. The pigs were sacrificed postoperatively after twenty four hours. Entire spinal cord and cerebral cortex were taken for pharmacological and morphological analyses.

Statistical method. For the descriptive data analysis, contingency table analysis using chi square statistics were used.

A. Cyclocreatine

This example demonstrates that the administration of cyclocreatine (CCr) prior to thoracic aorta cross-clamping provides protection against ischemic injury and improves the recovery of motor function. For this demonsration, thirty three pigs (Group I and II) weighing 23-25 kg were used. Eleven pigs, however, died due to surgical complications, malignant hyperthermia, or fluid overload. CCr treated pigs received daily intravenous infusion (i.e., one liter over six to eight hours) of 1% CCr/0.9% saline solution for four days (total 40 gm, n=5) or three days (30 gm, n=5), including the day of surgery. Control pigs received an equal volume of saline for four days (n=6) or three days (n=6). All animals underwent thirty minutes of proximal (just distal to the left subclavian artery) and distal just above the diaphragm) aortic cross-clamping. Animals were evaluated for neurologic function twenty four hours postoperatively. Overall results were that ten out often (100%) cyclocreatine treated animals were able to stand and walk (Tarlov scale 3 or 4), while only three out of the twelve control pigs (25%) were able to stand.

The neuroprotective effect of cyclocreatine (40 gm and 30 gm) when administered over two day period (Group III) also was tested. For the 40 gm/23 kg group (n=2), one liter (30 gm) of 3% cyclocreatine was infused (six to eight hours) one day prior to the day of surgery as well as 330 ml (10 gm) was given at the day of surgery. Control pigs (n=2) received similar volume of saline. For the 30 gm/23 kg group (n=3), one liter (20 gm) of 2% cyclocreatine was infused one day prior to surgery followed by 500 ml (10 gm) at the day of surgery. Control pigs (n=3) received saline. No paralysis was observed in control and cyclocreatine treated pigs.

Using the Chi square test, the P value was calculated for saline pigs and pigs that received cyclocreatine as 40 gm or 30 gm total dose for four days (CCr =5, saline n=5), three days (CCr n=5, saline n=6), or two days (CCr n=5, saline n=5). As described in Table 3, significant difference (P=0.004) was shown between cyclocreatine and control pigs regardless to days of prior drug administration.

Table 3: Comparison between cyclocreatine and saline treated pigs for recovery of motor function.

Number

Tarlov

Scale

P = 0.004 (Chi square test)

These results demonstrate that cyclocreatine administration for one to three days prior to cross-clamping of the descending thoracic aorta provides protection against the development of paraplegia.

B. Creatine phosphate

The neuroprotective effect of creatine phosphate also was demonstrated. For this demonstration, four pigs received daily intravenous infusion of saline (i.e., one liter over six to eight hours, n=2) or creatine phosphate (42 gm 23 kg pig, n=2 ~ molar equivalent of the creatine moiety in creatine phosphate to creatine at 40 gm dose) for four days, including the day of surgery. Control (n=2) and creatine phosphate (n=2) treated pigs then underwent thoracic aorta cross-clamping for forty minutes. Results indicated that pigs receiving creatine phosphate were able to stand with Tarlov scale of 4, while control pigs were paralyzed. The neuroprotective effect of creatine phosphate (42 gm/23 kg pig) (n=2) when administered over two days only (i.e., 500 ml [30 gm] on day 1 and 166 ml [12 gm] on surgery day) also was tested. Control pigs (n=4) received similar volume of saline. None of the creatine phosphate pigs (n=2) showed any motor movement (Tarlov scale of zero). For control pigs, three out of four developed paraplegia and one pig was able to stand with Tarlov scale of 4. These results show that creatine phosphate administration three days prior to cross- clamping of the descending thoracic aorta provides protection against the development of paraplegia.

Example 9 - The Use of a Creatine Analog for Protecting the Kidney from Ischemic Injury

Prevention of a warm ischemic injury in renal transplantation would expand the potential donor pool by facilitating the procurement of organs subjected to longer periods of normothermic ischemia, i.e. following cardiac arrest of the donor.

CCrP was administered to rats prior to being subjected to forty-five minutes of warm ischemia. The warm ischemia was produced by cross-clamping the main artery to the kidney. The control rats (saline treated (n=6); 1 ml intravenously, one hour prior to ischemia) were compared to CCrP rats (n=2; 200 mg./ml of saline, intravenously one hour prior to ischemia) and the parameter used for comparison was the creatinine level in the blood of the rats. The creatinine level is an appropriate parameter for measuring the extent of ischemic injury because one of the kidneys functions is to clear creatinine from the blood. The control rats had creatinine levels ranging from 7 to 9 units in their blood and the CCrP rats had creatinine levels of 1.0 and 6.0 in their blood. These results show that CCrP had a protective effect on the kidney from ischemic injury in these rats.

Example 10 - The Use of Creatine Analog for Protecting the Pancreas from Ischemic Injury

Prevention of warm ischemic injury to the pancreas would enable more pancreases to be available for transplantation and would reduce the ischemic injury to the pancreas in other situations, e.g. surgery. The islet cells were removed from both control and CCrP treated rats and were subjected to fifteen minutes of warm ischemia (moderate damage). The islet cell size is an appropriate parameter to measure the extent of ischemic injury of the pancreas because larger islet cells are healthy and produce more insulin than smaller islet cells. The islet cell size of the control rats (saline treated (n=2); 1.0 ml. intravenously, one hour prior to ischemia) was compared to the islet cell size of the CCrP treated rats (300 mg/ml. saline, intravenously one hour prior to ischemia) and the results are set forth in Table 4 below.

TABLE 4

Pooled Samples CCrP Treated Control

Islet Cells (# of Large Islet Cells) (# of Large Islet Cells)

1 (n=3) 1265 707

2 572 910

3 (n=3) 1058 822

4 1025 847 980±146 893±79

EQUIVALENT

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for treating a body part susceptible to ischemia, comprising administering to a body part susceptible to ischemia an effective amount of a creatine analog such that the body part is protected against injury from ischemia or treated for ischemia, provided that the creatine analog is not creatine phosphate or cyclocreatine when the creatine analog is administered prior to ischemia.

2. The method of claim 1 wherein the creatine analog has a formula as follows:

NR4R5 +

C ^"— NR₂R₃

Y,

wherein A is selected from the group consisting of N or CH; Z is selected from the group consisting of

B B,

wherein B1-B4 are each independently selected from the group of hydrogen and -OX4 and X1-X4 are each independently selected from the group consisting of hydrogen, alkyl. alkenyl, alkynyl and pharmaceutically acceptable salts; Y\ and Y2 are each independently selected from the group consisting of a direct bond, alkylene, alkenylene, alkynylene and alkoxylene;

K\ is selected from the group consisting of hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, and alkoxyl; and

R2 - R5 are each independently selected from the group consisting of hydrogen, a phosphorus containing moiety, alkyl, alkenyl, alkynyl, alkoxy and haloalkyl, wherein A may form a ring structure with one of the nitrogens in the amidino moiety or with Y2.

3. The method of claim 2 wherein one of R2- 5 is a phosphorus containing moiety.

4. The method of claim 3 wherein one of R2-R5 is selected from the group consisting of

, and

B, B

wherein B1-B4 and X4 are as defined above.

5. The method of claim 1 wherein the creatine analog is cyclocreatine phosphate.

6. The method of claim 1 wherein the creatine analog is selected from the group of creatine analogs set forth in Table 1.

The method of claim 2 wherein A forms a ring structure with one of the nitrogens in the amidino moiety of the creatine analog.

8. The method of claim 7 wherein the ring structure is a five membered hetero ring structure.

9. The method of claim 2 wherein A forms a ring structure with Y2 in the creatine analog.

10. The method of claim 9 wherein the ring structure is a four-membered hetero ring structure.

11. The method of claim 9 wherein the ring structure is a five-membered hetero ring structure.

12. The method of claim 1 wherein the body part is tissue.

13. The method of claim 1 wherein the body part is an organ.

14. The method of claim 12 wherein the tissue is selected from the group consisting of muscle tissue, connective tissue, epithelial tissue and nervous tissue.

15. The method of claim 14 wherein the tissue is muscle tissue.

16. The method of claim 15 wherein the tissue is cardiac tissue.

17. The method of claim 13 wherein the organ is selected from the group consisting of reproductive organs, respiratory organs, digestive organs, excretatory organs, urinary organs, sensory organs and skeletal muscle organs.

18. The method of claim 13 wherein the organ is selected from the group consisting of kidney, heart, pancreas, liver, gall bladder, brain, spleen and spinal cord.

19. The method of claim 18 wherein the organ is the spinal cord.

20. The method of claim 1 wherein the creatine analog is administered prior to ischemia.

21. The method of claim 1 wherein the creatine analog is administered post ischemia.

22. A method for treating a body part susceptible to ischemia, comprising administering to a body part susceptible to ischemia an effective amount of a creatine analog such that the body part is protected against injury from ischemia or treated for ischemia, the body part being tissue selected from the group consisting of skeletal, nervous and epithelial.

23. A method for treating a body part susceptible to ischemia, comprising administering to a body part susceptible to ischemia an effective amount of a creatine analog such that the body part is protected against injury from ischemia or treated for ischemia, the body part being an organ selected from the group consisting of kidney, gall bladder, liver, pancreas and spinal cord.

24. The method of claim 23 wherein the body part is spinal cord.

25. A method for treating a body part susceptible to ischemia, comprising administering to a body part susceptible to ischemia an effective amount of a disodium trihydrate salt of a creatine analog such that the body part is protected against injury from ischemia or treated for ischemia.

26. A method for treating a body part subjected to ischemia to restore functionality to the body part, comprising: administering a creatine analog post-ischemia such that functionality is restored to the body part.

27. A composition for treating an organ intended for transplantation comprising an effective amount of a creatine analog in a pharmaceutically acceptable organ treatment solution.

28. The composition of claim 27 further comprising an organ.

29. A composition of claim 27 wherein the creatine analog has a formula as follows:

NR₄R₅

Y -

Y.

wherein A is selected from the group consisting of N or CH; Z is selected from the group consisting of O B, B,

— C — 0X₁ — ^NH0X2 , — SO₃X3 , — P =0, and — P

B, B.

wherein B1-B4 are each independently selected from the group of hydrogen and -OX4 and X1-X4 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and pharmaceutically acceptable salts;

Y\ and Y2 are each independently selected from the group consisting of a direct bond, alkylene, alkenylene, alkynylene and alkoxylene;

R\ is selected from the group consisting of hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, and alkoxyl; and

R2 - R5 are each independently selected from the group consisting of hydrogen, a phosphorus containing moiety, alkyl, alkenyl, alkynyl, alkoxy and haloalkyl, wherein A may form a ring structure with one of the nitrogens in the amidino moiety or with Y2.

30. The composition of claim 27 wherein the creatine analog is selected from the group set forth in Table 1.

31. The composition of claim 29 wherein the creatine analog contains phosphorus.

32. The composition of claim 31 wherein the creatine analog is creatine phosphate or cyclocreatine phosphate.

33. The composition of claim 32 wherein the creatine analog is cyclocreatine phosphate.

34. A method for preserving and storing an organ intended for transplantation in a patient comprising contacting an organ with a composition comprising an effective amount of a creatine analog in a pharmaceutically acceptable organ treatment solution.

35. The method of claim 34 wherein the organ is selected from the group consisting of kidney, heart, pancreas, liver and lung.

36. The method of claim 35 wherein the organ is a heart.

37. In a composition used in organ transplant procedures, the improvement comprising, the inclusion of a creatine analog in the composition.

38. In a method for transplanting an organ, the improvement comprising, contacting the organ with an effective amount of a creatine analog.

39. A method for treating a subject for an ischemia-associated disease or condition, comprising: administering an effective amount of a creatine analog to a subject susceptible to an ischemia-associated disease or condition or having an ischemia-associated disease or condition.