WO1999004783A1 - Composition for protection from damage by ischemia - Google Patents

Abstract

The invention includes a method for protecting a tissue from damage due to ischemia. The method includes the step of administering to the tissue prior to the onset of ischemia an effective amount of a compound of formula (I). The method also includes inhibiting damage due to ischemia in a patient in need thereof by administering an effective amount of the compound above to the patient prior to the onset of ischemia.

Description

COMPOSITION FOR PROTECTION FROM DAMAGE BY ISCHEMIA

Background of the Invention

Tissue damage caused by ischemia is a serious problem encountered in stroke, myocardial infarct, transplantation, certain surgeries, and other conditions, diseases and procedures. It has been observed that some tissues, such as the heart, are rendered more tolerant to subsequent ischemia or injury during reperfiision by one or more brief episodes of ischemia through a process known as ischemic preconditioning. In the heart, preconditioning against ischemia results in protective effects such as reduction of infarct size, reduced stunning, and fewer arrhythmias. To this point, the mechanisms or agents that cause preconditioning are unclear.

There are numerous metabolic pathways and metabolites that are known to be involved in ischemia and reperfiision injury, and that may be implicated in preconditioning. For example, it has been reported that ischemic preconditioning activates protein kinase C (PKC). A role for PKC in ischemic preconditioning is supported by the observation that activators of PKC mimic ischemic preconditioning in protecting tissues and that inhibitors of PKC block the protective effects of ischemic preconditioning. Known PKC activators have a broad range of biological activities which make them unsuitable for therapy of ischemic tissue damage or reperfiision injury. For example, PKC activators reduce contractility of the heart and cause vasodilation, which can dramatically slow heartbeat and be fatal. In addition, many PKC activators are carcinogens. Furthermore, the usefulness of administering PKC activators is in doubt since the role of PKC in preconditioning remains controversial. Arachidonic acid is also released from phospholipid stores during ischemia or hypoxia in many tissues including the myocardium. Preconditioning results in increased amounts of the 12-lipoxygenase products in the tissue, and a lipoxygenase inhibitor reduces the protective effect of preconditioning. However, no 12- lipoxygenase product has been observed to have a protective effect against tissue damage due to ischemia or reperfiision injury.

Ischemia and reperfiision are significant causes of tissue injury and degradation in diseases or conditions such as stroke or myocardial infarct and in transplantation. A therapeutic method and composition that produces the beneficial effects of ischemic preconditioning presents a valuable addition to current therapies for tissue damage due to ischemia or reperfiision. Accordingly, there is a need for a method or composition that selectively provides beneficial effects of ischemic preconditioning.

Summary of the Invention

The present invention includes methods and compositions for protecting tissues and/or patients from damage due to ischemia or from reperfiision injury and that meet the needs described above. The method includes administering a 12- hydroperoxyeicosatetraenoic acid (12-HPETE), an active analog thereof, or a pharmaceutical composition including 12-HPETE or an active analog thereof to the patient or tissue. Preferably, the 12-HPETE or active analog is administered before the onset of ischemia. The tissue can be isolated, in a patient, or being prepared for transplantation. Tissues that can be treated or protected by the method of the invention include heart, brain, liver, lung, kidney, cornea, pancreas, stomach, bowel, and the like.

In the method of the invention, the patient can be determined to be susceptible to ischemia. Patients susceptible to ischemia include patients at risk for stroke, myocardial infarct, heart failure, an aneurysm, hemorrhage, organ transplant, organ donation, and the like. Indications that a patient is susceptible to an ischemic episode include a medical history including ischemic episodes, and indications such as angina, a transient ischemic episode, and the like. In addition, patients who are to undergo procedures such as transplantation, angioplasty, catheterization, cardiopulmonary bypass surgery, such as valve replacement, and the like are susceptible to ischemia.

Accordingly, in one embodiment the present invention includes a method for protecting a tissue from damage due to ischemia, including the step of administering to the tissue prior to the onset of ischemia an effective amount of a compound of the formula:

wherein: R, is an unsubstituted or substituted two carbon alkyl or alkene moiety or an alkyne moiety. R₂ is a two to ten carbon aryl or heteroaryl group or a five atom linear chain comprising substituted or unsubstituted carbon atoms, up to two heteroatoms, and up to two carbon-carbon double bonds, up to two carbon-carbon triple bonds, or one carbon-carbon double bond and one carbon-carbon triple bond. R₃ is a three to ten atom linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms. R₄ is a two to ten carbon linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms. Compounds of this formula include an optical or geometric isomer thereof, or a pharmaceutically acceptable salt thereof.

Accordingly, in a second embodiment the present invention includes a method for inhibiting damage due to ischemia in a patient in need thereof, including the step of administering to the patient prior to the onset of ischemia an effective amount of a compound of the formula:

R₄-R₂-C=C-C-CH₂-Rι-R₃

O

I

O I H wherein: R, is an unsubstituted or substituted two carbon alkyl or alkene moiety or an alkyne moiety. R₂ is a two to ten carbon aryl or heteroaryl group or a five atom linear chain comprising substituted or unsubstituted carbon atoms, up to two heteroatoms, and up to two carbon-carbon double bonds, up to two carbon-carbon triple bonds, or one carbon-carbon double bond and one carbon-carbon triple bond. R₃ is a three to ten atom linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms. R₄ is a two to ten carbon linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms. Compounds of this formula include an optical or geometric isomer thereof, or a pharmaceutically acceptable salt thereof. Brief Description of the Drawings

Figure 1 is a schematic presentation of the experimental protocol of the duration and timing of exposure to agents and time-course of ischemia and reperfiision.

Figure 2 is a bar graph showing recovery of left ventricular developed pressure (LVDP % of initial) measured at 20-minute reflow after 20 minutes of ischemia. Values are means ± S.E.M. *p<0.05 compared with untreated control hearts. Figure 3 is a bar graph showing recovery of left ventricular developed pressure (LVDP % of initial) measured at 20-minute reflow after 20 minutes of ischemia. Values are means ± S.E.M. *p<0.05 compared with untreated control hearts.

Detailed Description

Definitions

Abbreviations used in the Figures and throughout include:

DOG, 1,2-dioctanoyl-s/ϊ-glycerol;

PC, preconditioning; PC+baicalein, preconditioning in the presence of baicalein;

DOG+baicalein, perfiision with DOG in the presence of baicalein;

NAC, N-acetyl-cysteine;

DOG+NAC, perfiision with DOG in the presence of NAC;

I, 5 minutes of ischemia; R, 5 minutes of reflow;

12(S)-HPETE, 12-hydroperoxyeicosatetraenoic acid;

4-AP, 4-aminopyridine;

12(S)-HPETE+4-AP, 12(S)-HPETE perfiision in the presence of 4-aminoρyridine.

Ischemia

Ischemia can arise from numerous causes, conditions, and diseases. Typically, ischemia results from interruption of the blood supply to an organ or tissue. For example, occlusion of a blood vessel can prevent adequate oxygen from reaching a tissue by restricting the flow of oxygen-carrying blood to the tissue. This is the case in myocardial infarct, stroke, or ischemia of other tissues such as liver, lung, kidney, cornea, pancreas, stomach, bowel, and the like. Diseases, such as diabetes, which involve restricted circulation can also result in ischemia in certain tissues and organs. In addition, therapies including chemotherapies, such as with adriamycin, can cause damage to tissues that are similar to ischemic damage.

Certain subjects are more susceptible to ischemic episodes than others. Patients susceptible to ischemia include those at risk for stroke, myocardial infarct, heart failure, an aneurysm, hemorrhage, organ transplant, organ donation, and the like. Susceptible patients can be selected based on their medical history and specific indications. Such indications include angina, a transient ischemic episode, a medical history including ischemic episodes, and the like.

A tissue or organ can also be damaged by ischemia when the blood or oxygen supply to the tissue is reduced or cut off during catheterization, angioplasty, transplantation or surgery, such as a cardiopulmonary bypass surgery like valve replacement. In many surgeries, blood supply to an organ or tissue is reduced or stopped to allow manipulation of the tissue or organ without loss of blood or without blood interfering with the physician's manipulation of the organ. In transplantation, an organ being transplanted can be ischemic for many hours. In each instance of ischemia, additional tissue damage is caused by reperfiision injury, which occurs when the oxygen supply or blood flow to the tissue is re-established.

Interestingly, one or more brief periods of ischemia can render a tissue, such as the heart, more tolerant to subsequent episodes of ischemia and reperfiision through a process known as preconditioning. The protective effect of preconditioning can be produced by treating the tissue with an agent that induces some or all of the protective effects of preconditioning. For example, activators of protein kinase C can precondition tissues against damage from ischemia and reperfiision. Protein kinase C is involved in diverse metabolic pathways and activation of this enzyme can result in production of a wide variety of metabolites and physiological effects. Unexpectedly, an arachidonic acid metabolite from the 12-lipoxygenase pathway, 12- hydroperoxyeicosatetraenoic acid (12-HPETE) was found to induce a protective effect that mimics preconditioning in tissue that is later subject to ischemia; to protect against, reduce or inhibit tissue from damage due to ischemia; and to protect against, reduce or inhibit reperfiision injury.

12-HPETE

12-HPETE is an arachidonic acid metabolite produced by 12-lipoxygenase. This enzyme is stereospecific, producing 12(S)-HPETE. As used herein, 12-HPETE refers to racemic 12-HPETE, 12(R)-HPETE, and/or 12(S)-HPETE. Racemic 12- HPETE and 12(R)-HPETE share many activities with 12(S)-HPETE. Geometric isomers of 12-HPETE include cis and trans isomers of 12-HPETE. 12-HPETE can be isolated from natural sources or produced through chemical or enzymatic synthesis by methods known in the art. 12-HPETE can be represented by the compound of Formula 1 :

1

12-HPETE and analogs that share its activities can be represented by a compound of Formula 2:

RrR₂-C=C-C-CH₂-R_rR₃

O

I O

I H

2 Compounds of Formula 2 include optical and geometric isomers of these compounds. In a compound of Formula 2:

R_j is a two carbon alkyl, alkene or alkyne moiety, preferably alkene. When R_t is an alkyl or alkene moiety, the carbon atoms can bear small substituents, such as a hydroxyl, mercapto, lower alkyl group (such as methyl or ethyl), lower alkoxy (such as methoxy or ethoxy), lower hydroxyalkyl (such as -CH₂OH or -CH₂CH₂OH), acyl, allyl, a halogenated-lower alkyl group (e.g., a trifluoromethyl group), -C(O)-, - C(S)-, -C(O)H, =O, -C(O)OH, a halogen and the like. As used herein, lower refers to groups with about 3 or fewer carbon atoms.

R₂ is a two to ten carbon aryl or heteroaryl group or R₂ is a five atom linear chain comprising carbon atoms, up to two heteroatoms, and in any combination up to two carbon-carbon double and/or triple bonds. One or more carbon atoms of R₂ can be substituted with a variety of common small substituents, such as a hydroxyl, mercapto, lower alkyl group (such as methyl or ethyl), lower alkoxy (such as methoxy or ethoxy), lower hydroxyalkyl (such as -CH₂OH or -CH₂CH₂OH), acyl, allyl, a halogenated alkyl group (e.g., a trifluoromethyl group), -C(O)-, -C(S)-, - C(O)H, =O, -C(O)OH, a halogen and the like. R₂ is preferably a linear chain comprising five carbon atoms and two non-conjugated carbon-carbon double bonds, more preferably comprising five carbon atoms and two cis non-conjugated carbon- carbon double bonds. As used herein, heteroatom refers to oxygen, nitrogen, sulfur, and the like.

R₃ is a three to ten atom linear and/or cyclic group including carbon atoms and up to about three heteroatoms. One or more carbon atoms of R₃ can be substituted with a variety of common substituents, such as hydroxy, hydroxyalkyl (e.g., - CH₂OH), alkyl, cycloalkyl (with a 3-7 membered ring), carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, a halogenated alkyl group (e.g., a trifluoromethyl group), -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵ (where R⁵ is an alkyl, cycloalkyl or aryl group), acyloxy, -OSO₂R⁶ (where R⁶ is an alkyl, cycloalkyl or aryl group), -NR⁷R⁸ (where R⁷ and R⁸ are independently hydrogen, alkyl, cycloalkyl or aryl), and the like. These substituents may themselves be substituted with functional groups such as a hydroxy group, a carboxy group, an acetoxy group, or a halogen. A preferred R₃ is a substituted or unsubstituted hydrocarbon chain including one or more carbon-carbon double and/or triple bonds. More preferably R₃ is a 3-7 carbon saturated or unsaturated alkyl or heteroalkyl moiety or a 6-10 carbon aryl, heteroaryl, or substituted aryl moiety. More preferably R₃ is a n-pentyl moiety. R₄ is a two to ten carbon linear and/or cyclic group including carbon atoms and up to about three heteroatoms. One or more carbon atoms of R₄ can be substituted with a variety of common substituents, such as hydroxy, hydroxyalkyl (e.g., -CH₂OH), alkyl, cycloalkyl (with a 3-7 membered ring), carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, a halogenated alkyl group (e.g., a trifluoromethyl group), -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵ (where R⁵ is an alkyl, cycloalkyl or aryl group), acyloxy, -OSO₂R⁶ (where R⁶ is an alkyl, cycloalkyl or aryl group), -NR⁷R⁸ (where R⁷ and R⁸ are independently hydrogen, alkyl, cycloalkyl or aryl), and the like. These substituents may themselves be substituted with functional groups such as a hydroxy group, a carboxy group, an acetoxy group, or a halogen. A preferred R₄ is a linear, cyclic, or substituted hydrocarbon including one or more carbon-carbon double and/or triple bonds. More preferably R₄ is a 2-6 carbon saturated or unsaturated alkyl or heteroalkyl moiety or a 6-10 carbon aryl, heteroaryl, or substituted aryl moiety. Preferably R₄ has a terminal carboxyl group. More preferably R₄ is a n-butyric acid moiety.

Examples of suitable aryl groups include aromatic hydrocarbons such as a phenyl group, a biphenyl group, or a naphthyl group, as well as aromatic heterocyclic groups such as a pyridine group, a purine group, a pyrimidine group, or a furan group. The aromatic group may optionally be substituted with one or more substituents such as fluorine, chlorine, alkyl groups having from 1 to 10 carbon atoms (e.g., methyl or ethyl), alkoxy groups having from 1 to 10 carbon atoms (e.g., methoxy or ethoxy), alkoxyalkyl groups having from 1 to 10 carbon atoms and one or more oxygen atoms, or amido groups having from 1 to 10 carbon atoms, such as acetamido. The substituent(s) may also be a fluorinated alkyl group having from 1 to 10 carbon atoms (e.g., trifluoromethyl) or a fluorinated alkoxy groups having from 1 to 10 carbon atoms (e.g., trifluoromethoxy). Preferably the aromatic groups are a phenyl group or a naphthyl group which are either unsubstituted or substituted with fluorine, a lower alkyl group (having from 1 to 6 carbon atoms), a lower alkoxy group (having from 1 to 6 carbon atoms), or trifluoromethyl.

The compounds of Formula 2 include pharmaceutically acceptable salts of the active compound be it 12-HPETE, an active analog thereof, or another compound of Formula 2. When the active compound includes a carboxyl group, the carboxyl group can be present as a salt such as a salt with a alkali or alkaline earth metal (e.g. sodium, potassium, calcium, magnesium), an amine or amino acid (e.g. glycine). Pharmaceutically acceptable salts include salts of any potentially charged group in the compound, such as a salt of any amine and acid addition salts. Acid addition salts may be made from mineral acids, such as HC1, phosphoric acid, sulfuric acid, and the like, or from organic acids such as gluconic acid, acetic acid, and the like. Compounds of Formula 2 include compounds referred to herein as active analogs of 12-HPETE. Active analogs of 12-HPETE share the activity of 12- HPETE in protecting tissues against damage due to ischemia or reperfiision injury and are structural analogs of 12-HPETE. Preferred active analogs of 12-HPETE include homologues of 12-HPETE, optical isomers of 12-HPETE, geometric isomers of 12-HPETE, analogs of 12-HPETE in which one or more double bonds is saturated and/or one or more double bonds is replaced by a triple bond, and the like.

Active analogs of 12-HPETE also include fatty acid hydroperoxides produced by the action of 12-lipoxygenase on substrates other than arachidonic acid. For example, 12-lipoxygenase will accept unsaturated fatty acids such as linoleic acid, 9,12-octadecadienoic acid; linoelaidic acid, t,t-9,12-octadecadienoic acid; a- linolenic acid, 9,12,15-octadecatrienoic acid; -linolenic acid, 6,9,12- octadecatrienoic acid; dihomo- ^-linolenic acid, 8,1 1,14-eicosatrienoic acid; timnodonic acid, 5,8,11,14,17-eicosapentaenoic acid; and the like as substrates. Other typical 12-lipoxygenase substrates that form active analogs 12-HPETE include homologues of arachidonic acid, cis and trans isomers of arachidonic acid, analogs of arachidonic acid in which one double bond is saturated, and analogs of arachidonic acid in which one double bond is replaced by an acetylenic bond, and the like. Such fatty acid peroxides share biological activities with 12-HPETE.

12-HPETE is commercially available, can be made enzymatically using 12- lipoxygenase, or can be chemically synthesized. Methods for stereospecific chemical synthesis of 12-HPETE are reported in Corey et al. J. Am. Chem. Soc. 102(4)1433-5 (1980) and Nagata et al. Tetrahedron Lett. 30(21):2817-2820 (1989). Methods for making compounds of Formula 2 are known in the art. For example, such compounds can be made by oxidation of an unsaturated acid precursor with an oxidizing agent, e.g. permanganate, hydrogen peroxide, oxygen or other known oxidizing agents. Pharmaceutical Compositions

12-HPETE can be used in pharmaceutical compositions for treatment of conditions and diseases that give rise to ischemia. Patient treatment using the method of the present invention involves administering therapeutic amounts of the 12- HPETE composition. In the context of the present invention, the terms "treat" and "therapy" and the like refer to alleviate, slow the progression, prophylaxis, attenuation or cure of existing disease. The pharmaceutical compositions of the present invention include 12-HPETE in effective unit dosage form and a pharmaceutically acceptable carrier. As used herein, the term "effective amount", "effective unit dosage" or "effective unit dose" is denoted to mean a predetermined amount that provides protection against damage due to ischemia or reperfiision, or that results in preconditioning against ischemia or reperfiision injury.

Pharmaceutically acceptable carriers are materials useful for the purpose of administering the medicament, which are preferably non-toxic, and can be solid, liquid, or gaseous materials, which are otherwise inert and medically acceptable and are compatible with the active ingredients. Suitable pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences," 15th Ed.; Mack Publishing Co., Easton (1975); see, e.g., pp. 1405-1412 and pp. 1461-1487. Such compositions will, in general, contain an effective amount of the active compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the host.

A 12-HPETE composition may be formulated with conventional pharmaceutically acceptable parental vehicles for administration by injection. These vehicles comprise substances which are essentially nontoxic and nontherapeutic such as water, saline, Ringer's solution, dextrose solution, Hank's solution, a perfiision solution, and the like. It is to be understood that 12-HPETE formulations may also include small amounts of adjuvants such as buffers and preservatives to maintain isotonicity, physiological and pH stability. The compositions according to the invention can be presented in unit dose form in ampoules or in multi-dose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free buffer saline, before use. The present compositions can also be in the form of encapsulated liposomes.

As indicated by the above formulation, the 12-HPETE can be administered parenterally. 12-HPETE can also be delivered or administered topically, by transdermal patches, intravenously, intraarterially, intraperitoneally, in aerosol form, orally, for example in tablets, capsules, enterically coated capsules, and the like, or in drops, among other methods. When the 12-HPETE is administered intravenously, it can be delivered as a bolus or on a continuous basis. 12-HPETE can be administered intrarterially through a catheter, such as during angioplasty.

The dose of the 12-HPETE formulation to be administered will depend upon the patient and the patient's medical history, and the severity of the disease process. However, the dose should be sufficient to effectively precondition against ischemia or reperfiision injury, or to effectively prevent, reduce, or inhibit damage due to ischemia or reperfiision. Dosages for adult humans envisioned by the present invention and considered to be therapeutically effective will range from between about 0.01 mg/kg and about 330 mg/kg and preferably between about 0.1 mg/kg and about 40 mg/kg. These doses may be repeated up to several times per day. In addition, lower and higher doses may be more appropriate depending on the individual patient and the disease or condition to be treated.

The present invention is also drawn to methods of treating ischemia or preconditioning against ischemia using the present pharmaceutical compositions. Typically, the compositions will be administered to a patient (human or other animal, including mammals such as, but not limited to, cats, horses and cattle and avian species) in need thereof, in an effective amount to reduce or prevent damage from ischemia or to precondition against ischemia. The present compositions can be given either orally, intravenously, intramuscularly or topically.

The composition can be administered to an isolated tissue or organ, or to a patient. When administered to a patient, the composition can be administered systemically, or directly to an organ or tissue, for example through a catheter to the blood stream entering the tissue or organ. For example, the composition can be administered to the blood stream entering the heart, entering a particular portion of the heart, or another tissue prior to catheterization, angioplasty or cardiopulmonary bypass surgery. Alternatively, the composition can be administered via injection directly into the tissue or organ, or into other fluids or solutions that enter the organ, such as lymph or iv solutions. When a tissue or organ is to be transplanted, or is being transplanted, the composition can be administered to the donor while the donor is alive, to the donor or tissue before the organ or tissue is excised, or to the excised organ or tissue, for example in a perfiision solution.

The present invention also encompasses a kit including the present pharmaceutical compositions and to be used with the methods of the present invention. The kit can contain a vial which contains a 12-HPETE and suitable carriers, either in dried or liquid form. The kit further includes instructions in the form of a label on the vial and/or in the form of an insert included in a container in which the vial is packaged, for the use and administration of the compounds. The instructions can also be printed on the container in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow a worker in the field to administer the drug. It is anticipated that a worker in the field encompasses any doctor, nurse, or technician who might administer the drug.

Animal Models of Ischemia The effect of ischemia on the heart can be assessed in a rat model described in Murphy et al., Circ. Res. 76: 3, 457-467 (1995) and Chen, et al., J. Mol. Cell Cardiol. 28, 871-880 (1996) (the disclosures of which are incorporated herein by reference). Such a model can also be adapted for use with hearts of other species, such as rabbit (Chen et al, J. Biol. Chem. 271 :18, 7398-7403 (1996) (the disclosure of which is incorporated herein by reference). The animals are anesthetized and the hearts rapidly excised and the aorta cannulated. Retrograde perfiision is used to introduce perfiision solutions with and without a test agent, such as 12-HPETE. Ischemia is created by stopping perfiision. Contractility of the heart is measured by a balloon that is inserted into the left ventricle, and also connected to a pressure transducer, at desired times during the test. Those of skill in the art know that such general models for ischemia can be used in other animals such as rodents, primates, and the like and other tissues such as kidney, lung, muscle, liver, brain, and the like. An in vivo model for myocardial infarct has been developed in the rat (see Richard et al., Cardiovascular Res., 27, 2016-2021 (1993) for a general description of the model). This ischemia model involves reversible occlusion of a left coronary artery using a loop of suture to constrict the artery. This results in regions of ischemia in the heart. Loosening the loop results in reperfiision. This model can be employed to simulate myocardial ischemia in humans and other larger animals.

A model for transient focal cerebral ischemia has been developed in the rat (see Matsuo et al, Stroke, 25, 1469-1475 (1994) for a general description of the model). This ischemia model involves reversible occlusion of the middle cerebral artery ("MCA") through the intravascular insertion of a nylon thread. The insertion of the thread blocks blood flow to the middle cerebral artery and results in area of regional ischemia within the brain. Neutrophil infiltration and cerebral edema formation develop in the infarcted cortex after MCA occlusion. The neutrophil infiltration has been implicated in the pathogenesis of ischemia-reperfusion injury. The ischemia model is employed to simulate ischemic injury in humans, such as the injury observed with cerebrovascular occlusive disease (cerebral infarction), myocardial infarctions, pulmonary infarctions, intestinal infarctions, renal ischemia- reperfusion injury, peripheral vascular occlusive disease, and the like.

The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

Examples Methods

Perfiision of Hearts

All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publications No. 8523, revised 1985). Male Sprague-Dawley rats (170-350 g) were anaesthetized with intraperitoneal pentobarbitone (~25 mg). The animals were heparinized (200 units intravenously), and the heart was rapidly excised and the aorta cannulated. Retrograde perfiision was begun under constant pressure (90 cm H₂O). The nonrecirculating perfusate was a Krebs-Henseleit buffer containing (in mM): NaCl 120, KC1 4.78, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.25, NaHCO₃ 25, and glucose 11. The buffer was maintained at pH 7.4 by bubbling with a mixture of 95% O₂/5% CO₂, and at a temperature of 37°C. To monitor contractility, a latex balloon connected to a Statham pressure transducer was inserted into the left ventricle. The balloon was inflated to give an end-diastolic pressure of 5-10 cm H₂O. Global ischemia was created by cross- clamping the perfusate inflow line. To minimize "no-reflow" at the end of the ischemic period, the balloon was collapsed when the heart was reperfused. After a few minutes of reperfiision, the balloon was reinflated to an end diastolic pressure of 5-10 cm H₂O to assess recovery of a contractile function.

Rabbits and rabbit hearts were handled, perfused, and monitored by similar methods suitable for rabbits and known to those of skill in the art.

Biochemical Assay for 12(S)-HETE

12(S)-HETE was extracted by the method described in "Relationship between lysophospholipid accumulation and plasma membrane injury during total in vitro ischemia in dog heart" by C. Steenbergen and R. Jennings. J. Mol. Cell. Cardiol. 1984; 16:605-621. During the treatment protocol hearts were freeze- clamped using tongs precooled with liquid nitrogen at the time indicated by the solid arrow in Figure 1. The protocol was otherwise the same as used for measuring hemodynamic parameters.

Frozen hearts were placed in liquid nitrogen and stored in liquid nitrogen until extracted. Frozen hearts were placed in a homogenizing tube containing 2 ml chloroform, 4 ml methanol, and 0.8 ml pH 7.4 imidazole buffer containing 10 mM imidazole (Sigma), 10 mM EGTA (ethyleneglycol-bis-(b-aminoethyl ether) N, N'- tetraacetic acid, (Sigma), and 100 mM KC1. For evaluations of the extraction recovery, ¹⁴C-palmitate was added in selected extractions. The heart homogenate was kept ice cold at all times. Butylated hydroxytoluene (BHT, 0.005%) was added to the chloroform as an antioxidant. The tissue was homogenized with a Polytron (PT20) in several short bursts over a period of several minutes. Following the first homogenization, an additional 2 ml chloroform was added and the tissue was rehomogenized. An additional 2 ml of the imidazole buffer was then added, and the tissue was rehomogenized for a third time in several short bursts over several minutes. The homogenate was spun at 900 g for 10 minutes. The chloroform layer was removed. The tissue residue and aqueous phase was re-extracted by addition of 3 ml chloroform and 0.5 ml methanol, followed by homogenization and centrifugation as described above. This was repeated an additional time so that the tissue was extracted a total of three times. The chloroform layers from these three extractions were combined. The chloroform phase was dried under nitrogen gas and stored at -70°C until assayed. The extraction recovery measured by C-palmitate was -100%.

The extracted 12(S)-HETE was quantitated using an enzyme immunoassay kit (PerSeptive Diagnostics, Catalog No. 8-6812).

Example 1 — Activation of Protein Kinase C Induces the Protective Effect of Preconditioning

In various cells and tissues activation of protein kinase C is involved in diverse aspects of metabolism including activation of arachidonic acid metabolism and preconditioning against ischemic injury. The protein kinase C activator 1,2- dioctanoyl-sπ-glycerol (DOG) was used to stimulate rat hearts and the effect on ischemic injury to the hearts was examined.

Both control and experimental groups were subjected to a 20 minute equilibration period, a treatment period, a 20 minute period of global normothermic ischemia, and a 20 minute reperfiision period. The procedures used on the control and experimental groups are shown schematically in Figure 1. Group I (control 1 , n=l 1) received the control treatment. During the treatment period, hearts were perfused with phosphate-free Krebs-Henseleit buffer for 25 minutes. Group II (DOG, n=8) hearts were treated with DOG. During the treatment period hearts were perfused with Krebs-Henseleit buffer for 10 minutes, and then 3μM DOG was perfused for 10 minutes followed by a 5-minute washout period. Group IV (PC, n=5) hearts were subjected to preconditioning. In group IV, hearts were preconditioned with four cycles of 5 minutes of ischemia (I) each separated by 5 minutes of reflow (R). During perfiision left ventricular developed pressure (LVDP) was monitored continuously and the recovery of LVDP measured at 20 minutes of reflow was reported as a percent of the preischemic LVDP, prior to preconditioning or drug administration, as the index of the protective effect.

Effects of Interventions on Hemodynamics Before Ischemia

Table 1 compares the hemodynamics of control and treatment groups. The PKC activator DOG showed no significant differences in LVDP, heart rate, or coronary flow rate at the end of the control period. DOG did not significantly modify heart rate or coronary flow. Preconditioning with 4 cycles of 5 minutes of ischemia and 5 minutes of reflow resulted in a decline in LVDP to 72% of the initial value at the end of the fourth reflow, immediately before 20 minutes of sustained ischemia.

Results shown in Figure 2A demonstrate that PKC activation by DOG improved postischemic LVDP recovery (as % of initial) at 20 minutes of reperfiision after 20 minutes of ischemia (77 ±7% versus 48 ±4% in control untreated hearts, p<0.05), which was not significantly different than that in the preconditioned hearts (90+2%, p=0.725, PC vs DOG).

There were no significant differences in ATP content at the end of 20 minutes of ischemia among the experimental groups in this and the following

Examples. Upon reperfiision, ATP recovered to 20-40% of initial in all groups in all Examples. Recovery of coronary flow during reperfiision was nearly complete (75- 95% of the initial value) and was not significantly different among the groups among any of the Examples, indicating that the differential functional recovery on reflow was not due to differences in tissue perfiision.

Example 2 — Activation of Protein Kinase C Stimulates Production of 12

Lipoxygenase Products

12-Lipoxygenase products were measured to determine whether the protective effects of protein kinase C activation involved arachidonic acid metabolism. Production of the stable 12-lipoxygenase end-product 12(S)-HETE was monitored as described hereinabove under Methods. Control, DOG treated, and preconditioned groups (Groups I, II and IV) were treated generally according to the protocol described in Example 1 but 12(S)-HETE was determined. In group I (control, n=5), 12-HETE was determined after 25 minutes of control perfiision. In group II (DOG, n=5), 12-HETE was determined at the end of 10 minutes of DOG perfiision. In group IV (PC, n=5), 12-HETE was determined at the end of the second 5 minute period of ischemia.

Results

The results shown in Table 2 demonstrate that hearts treated with DOG have an increased content of 12(S)-HETE as compared with untreated control hearts (10.3+2.0 vs 4.0±0.8 ng/gww, p<0.05), and a content similar to that in preconditioned hearts (8.9± 1.2 ng/gww, p>0.05).

Example 3 — A 12-Lipoxygenase Inhibitor Prevents Activation of Protein Kinase C From Inducing the Protective Effect of Preconditioning

The effect of a 12-lipoxygenase inhibitor on hemodynamic parameters and the production of 12-HETE was measured to determine whether a 12-lipoxygenase product was involved in the protective effect of preconditioning. The 12- lipoxygenase inhibitor used was baicalein. For determining hemodynamic parameters control, DOG treated, and preconditioned groups (Groups I, II, and IV) were treated according to the protocol described in Example 1. Baicalein treated groups (Groups III, V, and VII) were treated generally according to the protocol of Example 1, with the following differences. In group III (DOG+baicalein, n=5), 10 μM baicalein was perfused for 25 minutes beginning 10 minutes prior to the addition of DOG, throughout the 10 minutes with DOG, and during the 5-minute DOG-washout period, similar to group II except for the baicalein. Group V (PC+baicalein, n=5) was identical to group IV except that 10 μM baicalein was added to the perfusate 10 minutes before preconditioning and was present throughout the preconditioning protocol. In group VII (Baicalein, n=4), 10 μM baicalein was added to the perfusate for 25 minutes. For determining production of 12-HETE, control, DOG treated, and preconditioned groups (Groups I, II, and IV) were treated according to the protocol described in Example 2. -Baicalein treated groups (Groups III, V, and VII) were treated generally according to the protocol of Example 2, with the following differences. In group III (DOG+baicalein, n=5), 12-HETE was determined at the end of 10 minutes of DOG perfiision in the presence of baicalein. In group IV (PC, n=5), 12-HETE was determined at the end of the second five minute period of ischemia. In group V (PC+baicalein, n=5), 12-HETE was determined at the end of the second five minute period of ischemia.

Results The results of Table 1 demonstrate that the decrease in LVDP in hearts preconditioned in the presence of baicalein were similar to those with preconditioning alone. This improvement, the decrease in LVDP, was attenuated when the hearts were perfused with DOG in the presence of the 12-LO inhibitor, baicalein (42+1-9%, p<0.05 compared with DOG alone). A similar effect was observed in the hearts preconditioned in the presence of baicalein (50+/-6%, p<0.05 compared to preconditioned alone), while baicalein by itself had no significant effect on postischemic functional recovery (41+/-6%, p>0.05 compared to control).

The effects on LVDP were paralleled by the effects on levels of 12-HETE, which are shown in Table 2. The increases in 12(S)-HETE observed with preconditioning or activation of protein kinase C were attenuated by the baicalein treatment protocol (5.7+/- 1.0 ng/gww in DOG+baicalein treated hearts, 4.6+/- 1.3 ng/gww in PC+baicalein treated hearts, neither value is significantly different than control). Baicalein treatment by itself did not affect the 12(S)-HETE content (mean value of 5.2 ng/gww from 2 hearts).

Example 4 -- 12-Hydroxyeicosatetraenoic Acid (12-HETE) Does Not Induce the

Protective Effect of Preconditioning A separate group of animals was used to investigate the protective effects of direct perfiision of exogenously added 12(S)-HETE and 12(S)-HPETE. Because 12(S)-HETE and its peroxide analog 12(S)-HPETE are labile, these compounds were added to a 1 OX stock perfused by the following variation of the procedure described under Methods. These compounds were perfused at 1/10 the coronary flow rate and added directly above the aorta via PE tubing connected to a Harvard pump. Preliminary experiments were performed at various concentrations of 12(S)- HETE, up to 3 μM. Based on these results, 3 μM was studied in detail (n=8). Concurrently a control group (n=7) was treated identically to those in group I and hemodynamic parameters were measured.

Results

Pretreatment with 12(S)-HETE did not significantly affect contractility or coronary flow. In addition, perfiision with up to 3 μM 12(S)-HETE resulted in no significant improvement in postischemic recovery of LVDP. This finding suggests that the active metabolite is not a downstream product of the 12-LO pathway or a remote effect of its metabolites. This indicates that the active 12-lipoxygenase product is 12(S)-HPETE.

Example 5 — 12-Hydroperoxyeicosatetraenoic Acid (12-HPETE) Induces the

Protective Effect of Preconditioning

12-HPETE was perfused into hearts using the protocol described in Example 4. Several preliminary studies were performed to determine if a concentration of 12(S)-HPETE could be found that would have an effect on recovery of function after 20 minutes of global ischemia. These studies showed that 0.4 μM was an effective concentration and this was used for subsequent studies.

Four groups of hearts were studied using the protocol described above and illustrated in Fig. 1. The differences from the standard protocol i _*n the 12-HPETE treatment are summarized as follows: A second control group (control 2, n=5) was treated identically to the first control group, group I. In group VIII (4-AP, n=4), 1 mM 4-AP was added to the perfusate for 20 minutes. In group IX (12(S)-HPETE, n=4), the hearts were perfused for 5 minutes with Krebs-Henseleit buffer followed by perfiision with 0.4 μM 12(S)-HPETE for 10 minutes followed by a 5-minute washout period. In group X (12(S)-HPETE+4-AP, n=4), 1 mM 4-AP was perfused for 20 minutes beginning 5 minutes prior to the addition of 12(S)-HPETE, throughout the 10 minutes with 12(S)-HPETE, and during the 5 minute 12(S)- HPETE-washout period. In another series of experiments, hearts were perfused for 5 minutes with Krebs-Henseleit buffer followed by perfiision with 12(S)-HPETE for 10 minutes followed by a 5 -minute washout period.

Results Pretreatment with 12(S)-HPETE and/or 4- AP did not significantly affect contractility or coronary flow. As shown in figure 4, pretreatment of hearts with 12(S)-HPETE resulted in a significant reduction in postischemic contractile dysfunction. Hearts treated with of 12(S)-HPETE recovered 82 ±7% of their preischemic function compared to 38 ±7% for untreated hearts. Thus, 12(S)-HPETE results in recovery from postischemic LVDP to levels comparable to those observed with preconditioning. The protective effects of 12(S)-HPETE are specific for this compound since addition of up to 3 μM 12(S)-HETE resulted in no significant improvement in postischemic recovery of LVDP. Furthermore, this finding suggests that the active metabolite is not a downstream product of the 12-LO pathway. We further tested if the protective effect of 12(S)-HPETE could be blocked by the potassium channel inhibitor 4-aminopyridine (4-AP). As shown in Figure 4, we found that the improvement in functional recovery observed with the addition of 12(S)-HPETE (82+/-7% vs 38+/-4% in controls, p<0.05) was lost when hearts were perfused with 12(S)-HPETE plus 4-AP (56+/-3.6%, p<0.05 vs 12(S)-HPETE alone and p>0.05 compared to control). 4-AP treatment by itself did not affect postischemic LVDP recovery.

Discussion

An equivalent protective effect on postischemic contractile dysfunction was seen with perfiision of 12(S)-HPETE and perfiision of DOG. Protective effects were not seen with arachidonic acid, suggesting that 12-LO activation is necessary for the effect, or with 12(S)-HETE, suggesting that the effect is not due to a downstream metabolite of the 12-LO pathway. Furthermore, the protection afforded by 12(S)- HPETE was blocked by the K+-channel blocker, 4-aminopyridine, suggesting that 12(S)-HPETE mediated protection might involve activation of the K+-channel. These results indicate that the 12-lipoxygenase pathway of arachidonic acid metabolism induces protection of the tissue from damage during periods of ischemia. Improved post-ischemic functional recovery induced by DOG was blocked by 12-lipoxygenase inhibition, which was confirmed by direct measurement of 12(S)-HETE production. Furthermore, the protective effect was also attained by direct administration of 12(S)-HPETE, but not by administration of 12(S)-HETE. Thus the data indicate that 12(S)-HPETE is the active metabolite in the 12-LO pathway that is responsible for the protective effect. Although not limiting to the present invention, it is believed that ischemic preconditioning in rat heart leads to PKC activation, which stimulates the 12-LO pathway of arachidonic acid metabolism, which generates 12(S)-HPETE, which confers a protective effect against postischemic contractile dysfunction.

Table 1. Left ventricle developed pressure (LVDP), heart rate (HR) and coronary flow rate (flow) during the pre-ischemic periods. end control period (end treatment)!

Groups LVDP (mmHG) HR (bpm) flow2 % LVDP % HR % flow

I (control 1) 117±4 299±6 9.9±0.6 101%±4 94%±6 85%±2.7

II (DOG) 132±8 313±6 9.7+0.6 92%±5 98%±4 90%±6.5

III (DOG+baicalein) 104± 12 298±9 10.1 ±0.6 83%±6 96%±6 83%±3.4

IV (PC) 135±6 295±4 10.2+0.6 72%±2* 92%±2 90%± 1.2

V (PC+baicalein) 137+ 16 304±9 9.7±0.4 75%±2* 94%±2 88%±2.1

VI (DOG+NAC) 133+ 11 318+6 8.8±0.4 92%±6 92%± 1 113%±7.5*

VII (baicalein) 99± 11 317± 17 8.9±0.4 87%+9 96%±3 81%±3.6

I (control 2) 102±3 340± 11 10.9±0.8 97%±3 96%±4 95%±4.1

VIII (4-AP) 115+3 303 ±34 10.9±0.4 89%±4 92%±6 92%±2.2

IX (12(S)-HPETE) 91 +7 319± 19 10.3±0.6 92%±4 98%±6 98%±2.1

X (12(S)-HPETE+4-AP 101 ±6 354± 14 10.6±0.6 97%+ 3 88%± 1 93%± 1.6

Abbreviations: DOG, 1,2-dioctanoyl-sΗ-glycerol; NAC, N-acetyl-cysteine; PC, preconditioning; DOG+baicalein, perfusion with DOG in the presence of baicalein; DOG+NAC, perfusion with DOG in the presence of NAC; PC+baicalein, preconditioning in the presence of baicalein; 12(S)-HPETE, 12-hydroperoxyeicosatetraenoic acid, 4-AP, 4-aminopyridine; 12(S)-HPETE+4-AP, perfiision of 12(S)-HPETE in the presence of 4-AP; bpm, beats per minute. Values are means ± S.E.M. *p<0.05 compared with initial value.

¹ % of the initial value

² flow rate = ml g wet weight of heart

Table 2. Tissue 12(S)-HETE content measured by the EIA-Kit

Groups 12(S)-HETE (ng/gww)

I (control) 4.0±0.8

II (DOG) 10.3 ±2.0* III (DOG+baicalein) 5.7 ± 1.0

IV (PC) 8.9± 1.2*

V (PC+baicalein) 4.6± 1.3

VI (DOG+NAC) 5.2±0.5

Abbreviations: DOG, 1,2-dioctanoyl-sn-glycerol; NAC, N-acetyl-cysteine; PC, preconditioning; DOG+baicalein, perfusion with DOG in the presence of baicalein; DOG+NAC, perfusion with DOG in the presence of NAC; PC+baicalein, preconditioning in the presence of baicalein. Values are means ± S.E.M. * p<0.05 compared to control value.

Example 6 - Rabbit Studies of Preconditioning

Rabbits were subjected to studies similar to those described in the Examples above. Rabbit studies were conducted by the methods described above but adapted to the larger species using adaptations known to those of skill in the art. Factors investigated included the improvement in postischemic functional recovery caused by preconditioning and preventing this improvement by administration of a 12- lipoxygenase inhibitor. Each study was conducted on a group of animals large enough to provide statistically significant results.

Results

Preconditioning improved postischemic LVDP recovery at 30 minutes of reflow after 30 minutes of ischemia The preconditioned rabbit heart recovered 67±3% but the control group recovered only 50±3% (p<0.05). This improvement was eliminated by administration of the 12-lipoxygenase inhibitor baicalein. The baicalein treated and preconditioned rabbit hearts recovered only 46+4% of the LVDP compared to 50±3% recovery for the control group (p<0.05).

Preconditioning resulted in an increase in 12(S)-HETE levels. The preconditioned rabbit tissue produced 54.3+8.1 ng/gww (gram wet weight) but the control tissue produced only 24.0±2.9 ng/gww (p<0.05). The increase in 12(S)- HETE was attenuated by administration of baicalein. The baicalein treated and preconditioned rabbit hearts produced only 25.0+4.3 ng/gww compared to 24.0±2.9 ng/gww produced by control hearts (p<0.05).

Example 7 — An In Vivo Model of Myocardial Ischemia

Animal Preparation

The investigation conforms with the Guide for the cure and use of laboratory animals published by the US National Institutes of Health (NIH publication No. 85- 23, revised 1985). The study is performed on male rats which are anesthetized. A midline incision is made in the neck and a tracheotomy performed. The rats are mechanically ventilated and the respiratory rate and tidal volume are adjusted to maintain arterial blood gases within the normal range. Body temperature is maintained at 37 °C with heating as necessary. The right jugular vein is cannulated for injection of drugs and of India ink for the delineation of area at risk. The left carotid artery is cannulated, and a small catheter is inserted in the artery to measure arterial blood pressure. An electrocardiogram is obtained with standard limb electrodes. Heart rate and arterial pressure are monitored.

A left thoracotomy is performed and the heart exposed. A suture is passed around the proximal left coronary artery, and the ends are passed through a small plastic tube to form a snare. The artery is occluded by pulling the snare, which is kept in place by means of a homeostatic clamp. Myocardial ischemia is confirmed by visual cyanosis. Reperfiision is induced by releasing the snare.

Experimental protocol Rats are assigned to groups. Group 1 rats (control group) are subjected to coronary occlusion followed by reperfiision. Group 2 rats (treatment group) are subjected to the same duration of ischemia and reperfiision, but receive 12-HPETE or an active analog thereof as an intravenous infusion before ischemia. Group 3 rats (preconditioning group) are preconditioned with three cycles of five-minute coronary occlusion each followed by five minutes reperfiision. At the end of the last five-minute reperfiision period, the rats are subjected to the same ischemia reperfiision cycle as in groups 1 and 2. Group 4 rats (preconditioning + treatment group) are subjected to the same protocol as in group 3, but receive 12-HPETE or an active analog thereof as an intravenous infusion before preconditioning.

Conclusion The protective effects of preconditioning occur in rabbits as well as rats indicating that this are a general effects.

Measurements of arrhythmias, areas at risk, and infarct size

The occurrence of ventricular tachycardia and fibrilation (reversible and irreversible) during each episode of ischemia and reperfiision is detected on the electrocardiogram (ECG) and the blood pressure tracing. Ventricular tachycardia is detectable on the ECG signal, whereas fibrillation is detectable both on the ECG and as a complete absence of arterial pressure.

At the end of the reperfiision, the artery is briefly reoccluded and Indian ink is injected slowly into the jugular catheter, to delineate the area at risk of infarction. The heart is excised, the right ventricle and the atria are dissected away and the remaining left ventricle is frozen. The frozen ventricle is then sliced from apex to base into sections. The slices are immersed in 1% triphenyltetrazolium chloride (TTC) in phosphate buffer for 20 minutes at 37 °C, to delineate the infarcted tissue. The sections are then fixed in phosphate buffered formalin. After fixation, each section is weighed and placed under a microscope and the area (mm²) of non- ischemic (Indian ink stained) viable (TTC positive), and infarcted (TTC negative) tissue are determined on each section. The surface of each slice is digitized, and total surface area and the infarcted surface area are calculated. The total and infarcted volume is calculated for each slice by multiplying the surface area by the slice thickness. Infarct and area at risk weights are calculated knowing the individual weight of each section. Area at risk size is expressed as a percentage of left ventricular volume, and the infarct size is expressed as a percentage of left ventricular volume and as a percentage of the area at risk. Example 8 — A Rat Transient Focal Cerebral Ischemia Model

Methods Animals

Sprague-Dawley (SD) rats weighing 270-350g, are divided into groups for the following treatments: (1) induction of ischemia only, (2) administration of 12- HPETE, (3) administration of vehicle only. 12-HPETE is administered intravenously as a saline solution at a desired dose prior to induction of ischemia, just after reperfiision, and after reperfiision. Neurological status during recovery is assessed and the animals are sacrificed after surgery. The brains are then removed and processed for histology to determine infarction size according to the procedure described below.

The animal procedures are carried out under complete, general anesthesia. The left femoral vein was cannulated for administration of 12-HPETE. An incision is made in the midline of the neck and the left carotid bifurcation is exposed. The common carotid artery is then occluded, and the branches of the external carotid artery are dissected and divided. The internal carotid artery is followed rostrally, and the pterygopalatine branch is identified and divided. An occluder, typically a suture, is then advanced from the external carotid artery into the lumen of the internal carotid artery until the origin of the middle cerebral artery is blocked. Reperfiision is accomplished by withdrawal of the occluder.

A neurological examination (as described in Zea Longa et al., Stroke, 20, 84- 91 (1989)), is performed 12, 24 and 48 hours after occlusion. The following standard scoring scale is used in the neurological examination: 0, normal, 1 , failure to extend the left forepaw; 2, circling to the left; 3, falling to the left; and 4, does not spontaneously exhibit a consciousness disturbance.

Measurement of infarct size

Ischemic animals are anesthetized after reperfiision. The brains are removed from the animals and kept at -70 °C. Each frozen brain is cut into coronal blocks. The brain slices are incubated in 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) and placed in 10% formalin for 48 hours. The unstained regions correspond well to regions of histopathological infarction. The surface of each slice is digitized, and total surface area and the infarcted surface area are calculated. The total and infarcted volume is calculated for each slice by multiplying the surface area by the slice thickness.

Example 9 - Preparation and Purification of 12-HPETE

12-HPETE can be prepared by methods known in the art, and is also available commercially. Methods for stereospecific chemical synthesis of 12- HPETE are reported in Corey et al. J. Am. Chem. Soc. 102(4)1433-5 (1980) and Nagata et al. Tetrahedron Lett. 30(21):2817-2820 (1989). Alternatively, 12- lipoxygenase, which can be obtained commercially or isolated from tissues such as heart by methods known in the art, can be used for enzymatic synthesis of 12- HPETE. Arachidonic acid is incubated with 12-lipoxygenase near neutral pH until the desired conversion to 12-HPETE is obtained. Alternatively, 12-HPETE can be chemically synthesized by methods known in the art. Numerous methods are known in the art for purification and isolation of 12-HPETE. For example, 12-HPETE can be separated from other arachidonic acid metabolites by reversed-phase HPLC (for example, C-18) using gradients of water, acetonitrile, acetic acid, and/or methanol. 12-HPETE can be separated from 12-HETE and other arachidonic acid metabolites by normal-phase HPLC.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

Claims

WE CLAIM:

1. A method for protecting a tissue from damage due to ischemia, comprising the step of: administering to the tissue prior to the onset of ischemia an effective amount of a compound of the formula:

R₄-R₂-C=C-C-CH₂-RrR₃

O

I O

I H wherein: R, is an unsubstituted or substituted two carbon alkyl or alkene moiety or an alkyne moiety; R₂ is a two to ten carbon aryl or heteroaryl group or a five atom linear chain comprising substituted or unsubstituted carbon atoms, up to two heteroatoms, and up to two carbon-carbon double bonds, up to two carbon- carbon triple bonds, or one carbon-carbon double bond and one carbon-carbon triple bond; R₃ is a three to ten atom linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms; and R₄ is a two to ten carbon linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms; or an optical or geometric isomer thereof, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1 , wherein R, is an alkyl or alkene moiety substituted with a hydroxyl, mercapto, lower alkyl, lower alkoxy, lower hydroxyalkyl, acyl, allyl, halogenated alkyl, -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, or a halogen.

3. The method of claim 1 , wherein R_j is an unsubstituted alkene.

4. The method of claim 1 , wherein a carbon in the two to ten carbon aryl or heteroaryl group or in the linear chain of R₂ is substituted with a hydroxyl, mercapto, lower alkyl, lower alkoxy, lower hydroxyalkyl, acyl, allyl, halogenated alkyl, -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, or a halogen.

5. The method of claim 1 , wherein R₂ is a linear chain of 5 unsubstituted carbon atoms with two non-conjugated carbon-carbon double bonds.

6. The method of claim 5, wherein the two double bonds are cis.

7. The method of claim 1 , wherein a carbon in the linear or cyclic chain of R₃ is substituted with a hydroxy, hydroxyalkyl, alkyl, cycloalkyl, carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, halogenated alkyl, -C(O)-, - C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵, acyloxy, -OSO₂R⁶, or -NR⁷R⁸.

8. The method of claim 1 , wherein the chain of R₃ is a substituted or unsubstituted hydrocarbon chain including one or more carbon-carbon double and/or triple bonds.

9. The method of claim 8, wherein R₃ is a three to seven carbon saturated or unsaturated alkyl or heteroalkyl moiety or a six to ten carbon aryl, heteroaryl, or substituted aryl moiety.

10. The method of claim 9, wherein R₃ is a n-pentyl moiety.

11. The method of claim 1 , wherein a carbon in the linear or cyclic chain of R₄ is substituted with a hydroxy, hydroxyalkyl, alkyl, cycloalkyl, carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, halogenated alkyl, -C(O)-, - C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵, acyloxy, -OSO₂R⁶, or -NR⁷R⁸.

12. The method of claim 1 , wherein the chain of R₄ is a substituted or unsubstituted hydrocarbon chain including one or more carbon-carbon double and/or triple bonds.

13. The method of claim 12, wherein R₄ is a two to six carbon saturated or unsaturated alkyl or heteroalkyl moiety or a six to ten carbon aryl, heteroaryl, or substituted aryl moiety.

14. The method of claim 13, wherein R₄ has a terminal carboxyl group.

15. The method of claim 14, wherein R₄ is a n-butyric acid moiety.

16. The method of claim 1 , wherein the compound is 12- hydroperoxyeicosatetraenoic acid (12-HPETE), an optical isomer of 12-HPETE, or a geometric isomer of 12-HPETE.

17. The method of claim 16, wherein the compound is 12(R,S)- HPETE.

18. The method of claim 17, wherein the compound is 12(S)-

HPETE.

19. The method of claim 1 , wherein the tissue is an isolated tissue.

20. The method of claim 1 , wherein the tissue is in a patient.

21. The method of claim 1 , wherein the tissue is being or is to be transplanted.

22. The method of claim 1 , wherein the tissue is heart, brain, liver, lung, kidney, cornea, pancreas, stomach, or bowel.

23. The method of claim 11, wherein the tissue is heart or brain.

24. The method of claim 1 , wherein the compound is administered after occurrence of angina or a transient ischemic episode.

25. The method of claim 1 , wherein the compound is administered before transplantation, angioplasty, or cardiopulmonary bypass surgery.

26. The method of claim 1 , wherein the administration is to a patient.

27. The method of claim 26, wherein the administration is directly to the tissue.

28. A method for inhibiting damage due to ischemia in a patient in need thereof, comprising the step of: administering to the patient prior to the onset of ischemia an effective amount of a compound of the formula:

wherein: R_t is an unsubstituted or substituted two carbon alkyl or alkene moiety or an alkyne moiety; R₂ is a two to ten carbon aryl or heteroaryl group or a five atom linear chain comprising substituted or unsubstituted carbon atoms, up to two heteroatoms, and up to two carbon-carbon double bonds, up to two carbon- carbon triple bonds, or one carbon-carbon double bond and one carbon-carbon triple bond; R₃ is a three to ten atom linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms; and R₄ is a two to ten carbon linear or cyclic chain comprising substituted or unsubstituted carbon atoms and up to about three heteroatoms; or an optical or geometric isomer thereof, or a pharmaceutically acceptable salt thereof.

29. The method of claim 28, wherein R, is an alkyl or alkene moiety substituted with a hydroxyl, mercapto, lower alkyl, lower alkoxy, lower hydroxyalkyl, acyl, allyl, halogenated alkyl, -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, or a halogen.

30. The method of claim 28, wherein Rj is an unsubstituted alkene.

31. The method of claim 28, wherein a carbon in the two to ten carbon aryl or heteroaryl group or in the linear chain of R₂ is substituted with a hydroxyl, mercapto, lower alkyl, lower alkoxy, lower hydroxyalkyl, acyl, allyl, halogenated alkyl, -C(O)-, -C(S)-, -C(O)H, =O, -C(O)OH, or a halogen.

32. The method of claim 28, wherein R₂ is a linear chain of 5 unsubstituted carbon atoms and two non-conjugated carbon-carbon double bonds.

33. The method of claim 32, wherein the two double bonds are cis.

34. The method of claim 28, wherein a carbon in the linear or cyclic chain of R₃ is substituted with a hydroxy, hydroxyalkyl, alkyl, cycloalkyl, carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, halogenated alkyl, -C(O)-, - C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵, acyloxy, -OSO₂R⁶, or -NR⁷R⁸.

35. The method of claim 28, wherein the chain of R₃ is a substituted or unsubstituted hydrocarbon chain including one or more carbon-carbon double and/or triple bonds.

36. The method of claim 35, wherein R₃ is a three to seven carbon saturated or unsaturated alkyl or heteroalkyl moiety or a six to ten carbon aryl, heteroaryl, or substituted aryl moiety.

37. The method of claim 36, wherein R₃ is a n-pentyl moiety.

38. The method of claim 28, wherein a carbon in the linear or cyclic chain of R₄ is substituted with a hydroxy, hydroxyalkyl, alkyl, cycloalkyl, carboxyalkyl, acyl, aryl, arylalkyl, allyl, acyloxyalkyl, halogenated alkyl, -C(O)-, - C(S)-, -C(O)H, =O, -C(O)OH, -C(O)O-R⁵, acyloxy, -OSO₂R⁶, or -NR⁷R⁸.

39. The method of claim 28, wherein the chain of R₄ is a substituted or unsubstituted hydrocarbon chain including one or more carbon-carbon double and/or triple bonds.

40. The method of claim 39, wherein R₄ is a two to six carbon saturated or unsaturated alkyl or heteroalkyl moiety or a six to ten carbon aryl, heteroaryl, or substituted aryl moiety.

41. The method of claim 40, wherein R₄ has a terminal carboxyl group.

42. The method of claim 41 , wherein R₄ is a n-butyric acid moiety.

43. The method of claim 28, wherein the compound is 12- hydroperoxyeicosatetraenoic acid (12-HPETE), an optical isomer of 12-HPETE, or a geometric isomer of 12-HPETE.

44. The method of claim 43, wherein the compound is 12(R,S)- HPETE.

45. The method of claim 44, wherein the compound is 12(S)-

HPETE.

46. The method of claim 28, wherein the patient is susceptible to stroke, myocardial infarct, heart failure, an aneurysm, organ transplant, or organ donation.

47. The method of claim 28, wherein the compound is administered after occurrence of angina or a transient ischemic episode.

48. The method of claim 28, wherein the compound is administered before transplantation, angioplasty, or cardiopulmonary bypass surgery.

49. The method of claim 28, wherein the patient is to be a donor of a transplanted organ or tissue.

50. The method of claim 28, wherein the damage due to ischemia occurs in heart, brain, liver, lung, kidney, cornea, pancreas, stomach, or bowel.

51. The method of claim 50, wherein damage occurs in the heart or brain.

52. The method of claim 28, wherein the ischemia results from stroke, hemorrhage, heart attack, or aneurysm.

53. The method of claim 28, wherein the administration is parenteral.

54. The method of claim 53, wherein the administration is intravenous.