EP1261346A1 - Modulation of cardiovascular injury - Google Patents

Abstract

A method of preventing ischemic and/or reperfusion injury after an ischemic or hemorrhagic episode by administering to a subject an effective amount of a heme oxygenase (HO) inhibitor, e.g., tin mesoporphyrin (SnMP), to ameliorate, reduce or prevent ischemic and post-ischemic reperfusion or hemorrhagic injury. In specific examples, SnMP protected heart tissue from injury due to free iron release from hemoglobin at the site of injury. A method of treating or preventing cardiac arrhythmia is also provided.

Description

MODULATION OF CARDIOVASCULAR INJURY

FIELD OF THE INVENTION

This invention relates to the heme oxygenase (HO) inhibitor tin- mesoporphyrin (SnMP) and its beneficial effects on the cardiovascular system. SnMP prevents the detrimental effects of free iron accumulation in tissue or organs in a mammal during ischemia and reperfusion or accompanying hemorrhage by inhibiting the release of free iron from heme in ischemic or hemorrhagic cardiac tissue. SnMP also acts as an antiarrhythmic agent by prolonging cardiac action potentials.

BACKGROUND OF THE INVENTION The Role Of Iron in Tissue Injury

Oxygen-derived free radicals play a significant role in myocardial reperfusion injury and in the expansion of myocardial infarct size; they damage cell membranes via lipid peroxidation. The presence of metal ions, most importantly iron (Fe), is required in the generation of the oxygen-derived free radicals. Free hydroxyl radicals (*OH) are produced from O₂ and H₂O₂ through the Haber- Weiss reaction catalyzed by iron.

Fe⁺³ + O₂ ^" → Fe⁺² + O₂ Fe⁺² + H₂O₂ → Fe⁺³ + *OH + OH Not only do free hydroxyl radicals damage cell membranes via lipid peroxidation, they also degrade DNA, proteins and matrix molecules such as hyaluronic acid.

Ischemic myocardium is tissue particularly enriched with iron as a result of the leakage of red cells from damaged small blood vessels, their subsequent hemolysis and the degradation of their constituent hemoglobin. The degradation of heme is controlled by the enzyme heme oxygenase (HO), which is present and also induced in ischemic as well as hemorrhagic tissue. Myoglobin and other heme proteins which are released from damaged muscle cells may also contribute heme as a substrate for HO and further add to the iron pool available for free radical production.

HO has been found to have protective properties such as inhibiting inflammation, protecting endothelium and smooth muscle from oxidant stress, protecting tissues from heme induced injury (Platt & Nath, Nature Medicine, 1998, 4: 1364-1365) and protecting grafted tissue from chronic rejection (Hancock et al., Nature Medicine, 1998, 4: 1392-1396). On the other hand, HO activity also has toxic effects. In addition to releasing iron, a powerful oxidant, heme degradation also produces bilirubin, implicated in tissue injury in jaundice and renal failure in hepatic disease. Another product of heme degradation is carbon monoxide (CO) which causes vasodilation.

Iron chelating agents, such as desferrioxamine, facilitate the binding and excretion of excess iron from tissue and thereby decrease free radical accumulation in tissue. Desferrioxamine has been used to limit infarct size and post-ischemic reperfusion injury (Bolli et al., Am J Physiol, 1987, 253:H1372-80). A major drawback of agents such as desferrioxamine as potential clinical pharmacologic agents in reducing reperfusion injury is that chelating agents are effective only after the iron has been released from heme and is already available for free radical production. In addition, it has been found that chelating agents like desferrioxamine must be present in very high concentrations before they can satisfactorily lower iron levels. Such effective concentrations can be difficult to achieve pharmacologically. For example, desferrioxamine typically must be administered continuously, i.e., via an infusion pump, to achieve results. Desferrioxamone is also known to produce allergic reactions in patients. The use of chelating agents for reducing reperfusion injury may be suitable in limited clinical environments, such as after surgery, however these agents are not practicable in situations of acute infarction or trauma.

Class HI antiarrhythmic drugs

Antiarrhythmic drugs have been grouped together according to the pattern of electrophysiological effects that they produce and/or their presumed mechanisms of action. Class III antiarrhythmic drugs such as dofetilide, azimilide and amiodarone exert their effect by prolongation of the cardiac action potential (AP) (Groh WJ, et al. J. Cardiovasc. Electrophysiol. 1997, 8:529-536). Reentrant rhythms in the heart are thought to be due to a spatial dispersion of repolarization and refractoriness (Restivo M, et al., Circ. Res. 1990, 66:1310-1327; Moe GK, Rev. Physiol. Biochem. Pharmacol. 1975, 72:55-81). The working hypothesis by which class III antiarrhythmic drugs operate is that prolongation of refractoriness increases the cycle length (CL) and thus the wavelength of potential reentrant wavelets.

There are two unwanted side effects of many, if not most, class III drugs. First, reverse use-dependence, whereby a class III drug's ability to prolong cardiac action potential duration (APD) is greater at slower heart rates, reduces the drug's potential effectiveness at the faster heart rates encountered during tachycardia (Groh WJ, et al., 1997). The second side effect is marked prolongation of cardiac APD which can ultimately lead to disturbances or oscillations in the repolarization phase of the AP, known as early afterdepolarizations (EADs). Early afterdepolarization-induced triggered activity, which conducts to hearts with prolonged and spatially dispersed APD, can cause potentially fatal polymorphic ventricular arrhythmia (El Sherif, et al., Circulation 1997, 96:4392-4399). The most common variant of drug induced polymorphic ventricular arrhythmias is torsades despointes. Although the class III drug amiodarone does not cause reverse use- dependence, it has many other side effects and is not well tolerated in all patients. Because of the potential aggravation of ventricular arrhythmias by the clinically available class III antiarrhythmic drugs such as sotalol, dofetilide and azimilide, their use has been limited generally to the treatment of supra ventricular arrhythmias.

Many of the developed class III compounds, including the two clinically available class III drugs sotalol and dofetilide, are methanesulfonimide compounds. It is believed that these act by blockade of the rapidly activating outward potassium current, IKr. The kinetics of blockade of this current is believed to be the cause of the reverse use- dependence. Regional differences in AP shape and duration are due to differences in ion channel densities and the ratio of ion channel subtypes.

While it has been long known that there is diversity in AP characteristics in the heart, ventricular muscle had been thought to be essentially electrically homogenous until Antzelevitch and co-workers demonstrated that there are subpopulations of ventricular cells with distinctly different electrophysiologic properties (Antzelevitch C et al., Circ.

Res. 1991, 69:1427-1449). There exists a molecular diversity of ion channel expression both across the ventricular wall and within the same layer. Voltage clamp studies have further validated that the constituent ion channels within the myocardium are different and account for the differences in AP shape and response. Salama et al. have shown heterogeneity in APD in the normal guinea pig epicardium (Salama G, et al., Am. J. Physiol. , 1987, 252:H384-H394). The APD gradient is uniform and oriented from apex to base and the total dispersion is less than 10 ms and not influenced by pacing site (Rosenbaum DS et al., Circulation, 1991, 84:1333-1345). Because of the minimal dispersion and the lack of nonuniform gradients, functional conduction block cannot be induced in the normal heart. However, other drugs which prolong APD can produce APD gradients due to regional differences in drug action. The neurotoxin anthopleurin-A can produce large APD gradients resulting in conduction block and reentry simulating characteristics of the long QT syndrome.

Heme oxygenase inhibitors

Metalloporphyrins, such as the tin (Sn) porphyrins, Sn-protoporphyrin (SnMP) and Sn-mesoporphyrin (SnMP), are known potent inhibitors of HO. They block binding of heme to HO, thus preventing the release of iron via heme catabolism. In contrast to iron chelating agents such as desferrioxamine, SnMP directly inhibits HO activity and prevents the degradation of heme and thus the release of its iron atom. SnMP has been used to inhibit HO activity in the intestine to reduce the absorption of iron from foodstuffs (U.S. Pat. No. 5,223,494); prevent hyperbilirubinemia by decreasing the rate of heme degradation (U.S. Pat. No. 4,657,902); and increase the rate of tryptophan metabolism in the liver of humans (U.S. Pat. No. 4,619,923). SnMP has also been shown to produce iron depletion in individuals taking an ordinary diet (Kappas et al., Pediatrics, 1993, 91: 537-539). It might be supposed on present knowledge that HO inhibitors such as SnMP would be contra-indicated for treating the types of injuries resulting from post-ischemic reperfusion and hemorrhage, because, as discussed above, HO levels increase after ischemia, and the enzyme has been thought to play a protective role in reducing damage to surrounding tissue. However, present knowledge may be misleading since it does not take into account the toxic impact of free iron released at the site of injury. A method to prevent or inhibit the release and accumulation of free iron in reperfused tissue, rather than removing already formed excess free iron, during or soon after the onset of ischemia, to prevent or inhibit deleterious tissue injury resulting from peroxidation in reperfused and hemorrhagic tissue would be highly desirable. In addition, the identification of a class of cardio-active compounds that do not produce the deleterious side effects of known anti-arrhythmic agents, such as the class III antiarrhythmic drugs, would also be desirable.

The present invention addresses these needs in a wholly unexpected fashion.

SUMMARY OF THE INVENTION The present invention provides a method for preventing or reducing post- ischemic reperfusion and hemorrhagic injury associated with free iron formation in tissue. The method comprises administering a heme oxygenase (HO) inhibitor, preferably tin mesoporphyrin (SnMP), in an amount effective to inhibit HO activity. Preferably the HO inhibitor is effective to prevent, reduce or ameliorate symptoms of ischemic, reperfusion or hemorrhagic injury. The SnMP may be administered before, during or after an ischemic or hemorrhagic episode. Preferably the SnMP is administered as soon as ischemia or hemorrhaging is detected such that HO activity may be reduced or inhibited and free iron release is minimized or prevented. The SnMP is administered parenterally, intravenously, intramuscularly or directly into the affected tissue. The SnMP is administered in an amount effective to inhibit HO activity to reduce or inhibit free iron release and accumulation associated with reperfusion or hemorrhagic injury. The SnMP can be administered at a dosage of from 1 to 50 mg/kg body weight, preferably from 2 to 25 mg/kg body weight.

The present invention further provides a method of inhibiting or reducing ventricular arrhythmia. The method comprises administering a heme oxygenase (HO) inhibitor, preferably tin mesopoφhyrin (SnMP), in an amount effective to prolong cardiac action potential duration (APD) and thereby preventing or reducing the occurrence of cardiac arrhythmia. In this embodiment, the SnMP is administered for effective regulation of cardiac rhythm. SnMP may be administered as soon as arrythmia is detected, or for example, in an acute coronary event such as heart failure or congestive heart failure, and/or may be administrated prophylactically over a course to time to regulate cardiac rhythm. SnMP can also be used for treatment of acute ischemia and may be administered during or after an ischemic episode. The SnMP is administered parenterally, intravenously, intramuscularly or directly into the affected tissue. The SnMP is administered in an amount effective to prolong APD. The SnMP can be administered at a dosage of from 1 to 50 mg/kg body weight, preferably from 2 to 25 mg/kg body weight.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows the ECG tracing of a rabbit heart. The heart was perfused with oxygenated Tyrode 's solution; the left coronary artery was clamped for 3 minutes; the ligature was loosened and the heart was reperfused. The ECG recording was made during and for 10 minutes after re-perfusion.

Figure 2 shows the ECG tracing of the rabbit heart treated as described in Figure 1, after which a 0.5g % hemoglobin in Tyrode 's solution was perfused into the heart. Figure 3 shows the ECG tracing of the rabbit heart treated as described in

Figure 2, then perfused with Tyrode 's containing 20μmol/L SnMP. The coronary artery was then closed for 3 minutes. The ligature was loosened and the heart was reperfused. The recording was made during and for 10 minutes after re-perfusion.

Figure 4 shows the ECG tracing of the rabbit heart treated as described in Figure 3 after perfusion for 15-20 minutes with hemoglobin/Tyrode's solution.

Figure 5 shows a schematic diagram of the perfusion chamber and optics system utilized for the experiments described in the Examples. A Langendorff perfused heart is stained with the voltage sensitive dye di-4-ANEPPS. The epicardium is illuminated with light from 2 halogen light sources (LS) which passes through 520+20 nm interference filters. Fluoresced light from the epicardial surface is gathered through a lens, filtered at 645 nm, and focused onto a 124-element photodiode array (lxl mm/photodiode).

Figure 6 shows the AP prolonging effect of SnMP in the normal guinea pig heart. The left panel (A) shows optical APs before and after administration of 50μl SnMP at paced cycle lengths (CL) ranging from 250-450 ms. The right panel (B) shows a plot of APD as a function of CL before and after administration of SnMP. Optical APs are shown in control test (solid line) and in the presence of 50μl SnMP (dotted line).

Figure 7 shows the AP prolonging effect of SnMP in the normal rabbit heart. The left panel (A) shows optical APs before and after administration of lOOμl SnMP at paced cycle lengths (CL) ranging from 300-500 ms. The right panel (B) shows a plot of

APD as a function of CL before and after administration of SnMP. Optical APs are shown in control test (solid line) and in the presence of lOOμl SnMP (dotted line).

Figure 8 shows the long lasting effect of a single bolus of 50μl SnMP administered in a perfused rabbit heart. The upper trace is the control. The middle trace is the APD at 5 minutes after SnMP administration. The lower trace is the APD at 30 minutes after SnMP administration.

Figure 9 shows that SnMP does not affect the activation pattern of normal cardiac rhythm. Activation maps are shown from a rabbit heart during basic paced rhythm. Each shaded zone represents an isochronal region activated at successive 1 ms intervals. Sj activation maps are shown for control and after 200 μl SnMP was administered in a rabbit heart.

Figure 10 shows isochronal maps of APD distribution before and after administration of SnMP. Each shaded zone represents an isochronal region activated at successive 10 ms isochronal intervals during left ventricular (LV) pacing (400ms CL) of the rabbit heart. Panel A shows the control map, the LN AP and the ECG; Panel B shows these after administration of 100 μl SnMP.

Figure 11 shows that SnMP prevents ischemia-induced APD shortening in the ischemic zone. Base sites are proximal to ligation site; apex sites are distal to ligation site. The left panel shows the control, no ligation; the middle panel shows that coronary occlusion shortens APD in ischemic zone (apex) after 5 minutes of ischemia; the right panel shows that SnMP prolongs APD in normal zone (base) and 5 minutes of myocardial ischemia does not result in appreciable APD shortening.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the administration to animals of SnMP before, during, or after an ischemic cardiac episode inhibited or prevented the detrimental effects of ischemia and reperfusion. Experiments in a rabbit heart model showed that administration of SnMP prior to or following an ischemic event reduced, prevented and/or improved recovery from impaired myocardial function resulting from post-ischemic reperfusion. Administration of SnMP also inhibited deterioration of myocardial function following administration of hemoglobin directly to the heart during reperfusion. In one instance, the administration of SnMP to a heart which showed heart block with limited ventricular beats improved the heart function such that regular sinus rhythm (RSR) was achieved and full recovery from post ischemic reperfusion was obtained. Subsequent infusion of hemoglobin did not alter the RSR. Thus, SnMP ameliorated re-perfusion injury that occurred following ischemia. In addition, SnMP pre- treatment reduced or inhibited iron toxicity following direct infusion of hemoglobin.

Although not intending to be bound by any theory, these results seem to demonstrate that SnMP reduced or prevented the generation of free iron in animal heart tissue by inhibiting the activity of HO, interdicting subsequent iron toxicity at the site of injury. SnMP inhibited cellular HO (both preformed and induced) within minutes after parenteral administration and thereby prevented the release of free iron. Thus it has been found that SnMP can be administered as soon as the ischemic event has been detected and will effectively treat ischemic and post-ischemic reperfusion injury. Reperfusion injury occurring after ischemia in the presence of hemoglobin was minimized or prevented with SnMP pre-treatment, further suggesting a protective effect of SnMP.

In another embodiment, the present invention is based on the discovery that SnMP administered to isolated animal hearts during basic paced rhythm demonstrated an anti-arrhythmic effect similar to that of current class-Ill anti-arrhythmic agents, however without the deleterious side effects accompanying the use of these agents such as reverse- use dependence. Applicants found that the overall APD gradient was maintained throughout the epicardium with the administration of SnMP and the degree of APD increase was relatively uniform in the epicardial layer. When SnMP was administered to a perfused guinea pig heart, prolonged APD was observed within five minutes of administration and was maintained for the duration of the study. Experiments in a rabbit heart model showed that administration of SnMP during an ischemic event prolonged APD and the degree of APD shortening in the ischemic zone was blunted. Thus, it has been found that SnMP acts as an antiarrhythmic agent and provides a means for restoring rhythmic contraction, selectively prolongs the action potential duration and concomitantly increases the refractory period of heart cells without significant effect on cardiac conduction. SnMP is therefore suitable for the treatment of mammals suffering from arrhythmic disorders or disease.

Definitions

"Action potential" (AP) is a response, elicited by stimulus in excitable cells, which is measured from the beginning of membrane depolarization (upstroke) to return to baseline potential (repolarization). "Action potential duration" (APD) is the time interval between the upstroke of the action potential and its point of repolarization.

"Arrhythmia" is any variation from the normal rhythm of the heartbeat, including, without limitation, sinus arrhythmia, premature heartbeat, heartblock, fibrillation, flutter, tachycardia, and premature ventricular contractions. Arrhythmia occurs, for example, by deprivation of oxygen and other blood constituents in the myocardium. Production of oxygen derived free radicals during reperfusion also causes arrhythmia. Particularly dangerous is ventricular fibrillation whereby the heart loses its ability to contract in a coordinated fashion needed to pump blood properly.

"Reperfusion" is the return of the flow of blood through vessels, e.g., from an artery, through the vasculature to and over the bodily tissues after an episode of reduced blood flow, for example, ischemia. Reperfusion also occurs in transplanted organs; acute transplantation rejection, e.g., involving a complement mediated cascade, occurs from an immediate reaction after reperfusion. Reperfusion also occurs in cases of massive trauma or "crush injury" where limbs or other body areas may be crushed or mangled such as occurs in moving vehicle accidents and construction site accidents. Reperfusion causes tissue damage due in part to the generation of free hydroxyl radicals. Free hydroxyl radicals damage cell membranes via lipid peroxidation, and the degradation of DNA and proteins.

"Ischemia" is a localized decrease in blood supply to an organ due to obstruction or constriction of a blood vessel due, for example, to a blood clot or other obstruction. The decrease in blood supply may result in stroke (if the organ is the brain), heart attack (if the organ is the heart) or mechanical organ failure. Ischemia can occur in an organ, tissue or parts thereof. As a result of deprivation of blood to these areas, necrosis (death) of cells or tissues can occur. Moreover, necrosis can also occur as a result of reperfusion by the action of, inter alia, free hydroxyl radicals. "Infarct" is an area of tissue that undergoes necrosis as a result of an obstruction of blood supply to the tissue due, for example, to a blood clot or other obstruction in the heart or blood vessel. A "myocardial infarction" is necrotic heart tissue. "Hemorrhage" is the release or escape of blood from the blood vessels into tissue and is typically manifested by an excessive loss of blood. Hemorrhages can occur as a result of traumatic injury, e.g., an automobile accident, or as the result of an infectious disease, e.g., as in hemorrhagic fever. Bruising, whether from trauma or surgery, is a localized hemorrhage (hematoma). Indeed, some of the discoloration (yellowness) associated with bruises is a direct result of bilirubin formation mediated by HO. Thus, the invention specifically addresses inhibition of extravasation that results from bruising. Certain synthetic metalloporphyrins are HO inhibitors. Such "compounds include metalloprotoporphyrins and metallomesoporphyrins wherein the metal group may be selected from tin, chromium, platinum, zinc, cobalt, nickel, copper, silver and manganese or other elements. The ring tetrapyrrole may be altered as well - viz proto- versus meso- porphyrins. Metallopoφhyrins may be obtained commercially or may be synthesized by described methods, see, e.g. U.S. Pat. No. 4,692,440 for synthesis of

SnMP. SnMP, which has received an IND from the FDA for treating hyperbilirubinemia in neonates, is preferred.

The present invention further provides other HO inhibitors useful for treatment of conditions arising from heme degradation by HO. Inhibitors that are contemplated include fragments, peptides, nucleic acids and oligonucleotides, carbohydrates, phospholipids and other lipid derivatives, steroids and steroid derivatives, antisense nucleic acids (including ribozymes) which may be used to inhibit expression of HO, anti-HO antibodies, small molecule inhibitors and vitamin B₁₂ and its derivatives. Conditions which may be treated with an HO inhibitor according to the present invention include hemorrhagic injury, which may result from surgery or other trauma to body tissue, reperfusion injury which may occur following resumption of blood flow in blood vessels following loosening or removal of blood clots either chemically (e.g., using tissue plasminogen activator) or mechanically (angioplasty), or resumption of blood flow after bypass surgery, or stroke, or other acute cardiac episodes; treatment during infarction, in order to minimize the damage to tissues surrounding the infarct area. In a specific embodiment, the HO inhibitor is provided prior to an elective surgery, e.g., plastic surgery of the face, to prevent extravasation associated with bruising.

In addition, it has been found that the HO inhibitors, particularly SnMP, can be used to treat or regulate arrhythmia in patients in need thereof. The present invention can be used as an effective first aid treatment for ischemic conditions or acute coronary events such as myocardial infarction or stroke, heart failure, particularly congestive heart failure, preferably when the HO inhibitor is a safety-tested compound like SnMP. Thus, when emergency personnel (emergency medical technicians, firefighters, police), or other responsible persons (flight attendants, conductors, event ushers, etc.) suspect an infarction, or observe symptoms such as chest pain, shortness of breath, fatigue, and anxiety, a bolus of HO inhibitor can be administered. Because, especially in the case of SnMP, the HO inhibitor chosen for such emergency use does not have any adverse side-effects, it will not be necessary for the emergency personnel to make or confirm a diagnosis.

This invention provides a valuable adjunct to infarct therapies, including but not limited to, administration of aspirin (acetylsalicylic acid) to prevent or inhibit clot formation, and administration of a "clot-busting" agent such as tissue plasminogen activator or streptokinase. It is also ideally used in combination with balloon angioplasty and other techniques that temporarily block blood flow.

In addition, HO inhibitors, like SnMP, are highly useful as components of perfusion solutions, e.g., for heart-lung machines during bypass surgery, or for infusing organs prior to transplantation. Furthermore, HO inhibitors can be administered to a subject prior to and during perfusion of the heart and lungs after completion of the surgery requiring use of a heart-lung machine, and prior to perfusion of a transplanted organ after grafting it into the host.

The HO inhibitors described herein can be administered to an individual in need of treatment as a therapeutically effective dose in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (18th Edition).

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. One example of a therapeutically effective amount of an HO inhibitor is an amount effective to inhibit HO activity in reperfused or hemorrhagic tissue. Another example of a therapeutically effective amount of an HO inhibitor such as SnMP is an amount effective to prolong cardiac action potential duration (APD) in a patient suffering from arrhythmia.

The actual dosage regimen (amount and frequency) of an HO inhibitor will be determined by the skilled physician, based on the condition, the age, sex, weight, and health of the patient, and other factors well within the ordinary level of skill in the art. In particular, the dosage range for SnMP employed for decreasing the rate of formation of free iron in reperfused blood or tissue is approximately from 1 to 50 mg/kg body weight, preferably 2 to 25 mg/kg body weight. The concentration of SnMP can be 2 to 25 g/liter to provide a dosage of from 2 to 25 mg/kg body weight. Accordingly the single dosage units will typically contain from 2 mg/ml to 25 mg/ml of SnMP solution. Compositions according to the present invention comprising HO inhibitors may be administered through various modes, for example, as described for metallopoφhyrins. See U.S. Pat. No. 4,692,440 and U.S. Pat. No. 4,657,902 (parenteral administration to increase the rate at which heme is excreted), U.S. Pat. No. 4,619,923 (parenteral administration to increase the rate of tryptophan metabolism in liver), U.S. Pat.

No. 4,782,049 (parenteral administration for treatment of psoriasis), WO94/28906 and U.S. Pat. No. 4,684,637 (parenteral administration, esp. intramuscular and intravenous, to prevent hyperbilirubinemia), and U.S. Pat. No. 5,063,223 (parenteral administration, esp. intravenous, subcutaneous, intramuscular, for controlling steroidal hormone levels).

Kits

It is contemplated that compositions according to the present invention may be included as part of a kit, such as a medical emergency kit. Such a kit may itself be a component of an emergency first aid kit accompanying a portable defribillator, such as are presently used to equip police cars, aiφlanes, etc. In this embodiment, a therapeutically effective amount of a HO inhibitor, e.g., SnMP, is provided in a solution, or in lyophilized powder for reconstitution as a solution (in which case the kit also preferably includes a reconstitution buffer). The composition may be packaged in a container, preferably a syringe or other injection device, preferably in a pre-measured unit dosage form. The compound SnMP is known to be stable in solution at room temperature for several years.

EXAMPLES

The present invention is demonstrated by the following non-limiting examples.

The procedures in this study conform to guidelines set forth in the Declaration of Helsinki. All experimental protocols were approved by the Animal Studies Subcommittee of the Research and Development Department of the Department of Veterans

Affairs, New York Harbor Healthcare System, and all procedures related to animal use comply with the "Guiding Principles for Research Involving Animals and Human Beings" published by the U.S. National Institutes of Health. EXAMPLE 1

Modulation of Reperfusion and Ischemic Injury

Materials and Methods

The model system used for the experiments in Examples 1-3 was the Langendorff-perfused rabbit heart preparation. Surgical procedures were performed as previously described (Salama G et al., Am. J. Physiol 1987, 252:H384-H394).

Briefly, rabbits (approximate weight 2-4 kg, of either sex) were anesthetized by intraperitoneal injection of sodium phenobarbital and heparinized. A mid-thoracotomy was performed and the hearts were rapidly excised and placed in cold oxygenated Tyrode 's containing 100 U/ml heparin. The excised hearts were rapidly annulated at the aorta and retrogradely perfused in a modified Langendorff apparatus. The Tyrode 's solution comprised (in mM): NaCl 130, KC1 4.75, CaCl₂ 1.0, MgSO₄ 1.2, NaHCO₃ 12.5, and glucose 15.0. The solution was continuously bubbled with 95%-O₂/ 5%CO₂ through a fitted glass tube. Temperature was maintained at 36 +0.3C by monitoring the temperature of the efusate within the closed chamber. A small incision was made in the pulmonary artery for drainage. A variable speed roller. pump was used to maintain reperfusion pressure of 80 to 90 cm H₂O using a perfusion flow rate of 2.0ml/min/g heart weight.

A custom designed perfusion chamber was used to study the isolated rabbit hearts (Salama G et al., 1987). The heart in the chamber was immersed in perfusate. Bipolar surface electrocardiograms were recorded using Teflon-coated silver wires. Recording and stimulating electrodes were positioned on the epicardial surfaces or perfusion chamber side pads.

Hearts were paced at a fixed cycle length SI ranging from 200 to 1200 ms with bipolar Ag-AgCl electrodes placed at the left ventricular free wall and the right ventricle near the anterior septum. Ventricular stimulation was applied using constant current pulses of 2.5ms duration at 1.5 times diastolic threshold. Steady state APD measurements were determined after a minimum of 30 beats.

Toxicity of hemoglobin on isolated perfusal rabbit heart. During perfusion of the heart with Tyrode 's solution, the left coronary artery was clamped for three minutes, after which the clamp was removed (reperfusion). There was no significant ECG change noticed upon reperfusion.

After 15-20 minutes, the preparation was perfused with a 0.5 gm% hemoglobin solution in Tyrode's. Within 1-2 minutes the ECG showed only small amplitude QRS cycles. The heart was non-functional within 5 minutes after hemoglobin perfusion.

This experiment demonstrated the potent toxicity of hemoglobin (at a lower than physiological concentration) for an isolated reperfused heart. The toxic effect was attributable to the pro-oxidant effects of iron released from heme in situ. Protective effect of SnMP against reperfusion injury and associated hemoglobin toxicity. The rabbit heart was initially perfused with Tyrode's solution. The left coronary artery was clamped for three minutes after which the ligature was loosened (reperfusion). The ECG was recorded during and for ten minutes after reperfusion. No change in ECG was recorded. The ECG tracing is shown in Figure 1. Starting at 20 minutes after reperfusion a 0.5g % hemoglobin/ Tyrode's solution was perfused into the heart. The ECG almost immediately (within 2 minutes) showed increasing A-V block followed by ventricular asystole. The ECG tracing is shown in Figure 2. Subsequently the heart was reperfused with Tyrode's solution alone. After 2- 5 minutes, ventricular rhythm returned to normal sinus rhythm. The heart was then perfused with Tyrode's solution containing 20μmol/L SnMP for 5-10 minutes to allow full exposure of the cardiac tissue to the SnMP. The coronary artery was then ligated for three minutes after which the ligature was loosened (reperfusion). The ECG was recorded during and for ten minutes after reperfusion. No rhythm changes were observed. The ECG tracing is shown in Figure 3. After another 15-20 minutes the heart was again perfused with 0.5% hemoglobin Tyrode's solution. No deleterious effect on the ECG was recorded; the heart demonstrated normal sinus rhythm (NSR). The ECG tracing is shown in Figure 4.

In this experiment, the protective effect of SnMP against the deleterious consequences of coronary artery ligation and later release (reperfusion) as well as the toxic effects of hemoglobin in this reperfusion circumstance were demonstrated. Protective effects of SnMP injected directly into the heart against reperfusion injury and hemoglobin toxicity. During initial perfusion of the heart with Tyrode's solution, the left anterior descending coronary artery (LAD) was clamped just distal to the origin of the diagonal branch. The ischemia period lasted three minutes, after which the clamp was removed (reperfusion). Two minutes following reperfusion, progressively increasing heart block developed resulting in only occasional ventricular beats occurring at 5-6 minutes.

At 7-8 minutes post-reperfusion, 1.5 ml (30mg) of an SnMP solution was injected directly into the chamber leading to heart intake (aorta). Within 1 to 2 minutes after injection, an increasing ventricular response reappeared followed at 4 to 5 minutes by restoration of regular sinus rhythm (RSR).

Twenty minutes post-reperfusion, a filtered 0.5 gm % hemoglobin solution in Tyrode's was perfused into the heart for fifteen minutes. No change in ECG was recorded, and RSR was maintained. After the fifteen minute hemoglobin solution perfusion, the LAD was clamped again for three minutes. After the three minute ischemia period, the clamp was removed (reperfusion). Immediately following reperfusion, ventricular tachycardia developed. Within 2-3 minutes following reperfusion idioventricular rhythm occurred with approximately 40-50 beats per minute, and at 5-6 minutes RSR was restored. The results showed that SnMP injected directly into the main perfusion vessel (aorta and coronary arteries) ameliorated the reperfusion injury that occurred following the initial clamping of the LAD. In addition, the toxicity that was seen following the infusion of hemoglobin was abolished by pre-treatment with SnMP. Also, the re-perfusion injury that occurred after clamping of the LAD in the presence of hemoglobin was minimal consistent with the continual protective effect of SnMP.

EXAMPLE 2 SnMP as an Antiarrythmic Agent Materials and Methods Instrumentation Details of the optical and recording apparatus have been described elsewhere (Salama G, et al. 1987; Kanai A and Salama G 1995; Efimov IR et al. 1994; Choi BR and Salama G, 2000; Efimov IR et al. , J.Cardiovasc. Electrophysiol. 1996, 7:512-530). Figure 5 illustrates the testing system used in the experiments described below. Briefly, the perfusion chamber containing a Langendorff perfused heart was mounted on a micromanipulator and positioned along the optical axis of a photodiode array scanning apparatus. The epicardial surface of the heart was illuminated with light from two 45 W tungsten halogen lamps (LS). The light was collimated and passed through 520+20 nm interference filters. A 45° mirror in the optical apparatus was used to focus the grid pattern on the region of interest using a 35 mm camera lens (50 mm, Fl : 1.4, Nikon). Epi-fluorescent light from the stained heart was gathered through a lens (L), projected through a 645 nm cutoff filter, and focused to form an image of the heart on a 12x12 element photodiode array. The photodiode array consisted of 144 square diode elements, with each diode having dimensions of 1.0 x 1.0 mm separated by 0.1 mm. 124 diodes were current to voltage converted and sampled. The depth of field of the optics was approximately 150 μm.

Langendorff Preparation and Perfusion Chamber Pacing Protocol Hearts were paced at a fixed cycle length (SI) ranging from 200 to 800 ms with bipolar Ag-AgCI electrodes placed at the left ventricular free wall and the right ventricle near the anterior septum. Ventricular stimulation was applied using constant current pulses of 2.5 ms duration at 1.5 times diastolic threshold. Steady state APD measurements were determined after a minimum of 30 beats. Fluorescent Dye Staining A voltage sensitive styryl dye, di-4-ANEPPS

(Molecular Probes; Eugene, OR), was used as the potentiometric fluorescent probe. Dye fluorescence was measured at wavelengths above the 645-nm cutoff filter when excited with a 520+20 nm interference filter. Because the dye exhibits a fractional decrease in fluorescence (6% to 9% per 100 mV) in response to depolarization, the signals were inverted to display optical APs. This dye does not produce detectable pharmacological effects and remains optically stable as evidenced by a high signal-to-noise ratio, lasting for 2 to 4 hours (Choi BR and Salama G, J. Physiol., 2000, 529 Pt 1 :171-188).

Hearts were stained by gradual injection of 40 to 60 μl from a 2.5 mM stock solution of dye into a 5ml bubble trap situated directly above the aortic cannula. The final dye concentration was approximately 1.8 μM; 10 to 15 minutes was allowed for the staining to be completed. The procedure resulted in homogeneous staining throughout the heart because the dye was efficiently delivered via coronary vessels. For longer protocols for which photobleaching (prolonged exposure of the dye to light degrades its usefulness) and/or dye washout may reduce the optical signal amplitudes, hearts were restained with smaller amounts of dye (5-10 ml) to restore the original signal-to-noise ratio. Signal Acquisition and Data Analysis Each data acquisition epoch comprised a scan of 128 simultaneously recorded traces (124 photodiodes plus 4 instrumentation channels). The multiplexed instrumentation channels monitored the stimulus pulses and surface electrogram signals from the ventricle. The duration of each stored acquisition epoch ranged from 1-15 seconds. The photodiode currents from each of the 124 sites were fed to a current-to- voltage converter, amplified, and high pass filtered to remove background fluorescence. The filtered signals were further amplified using a selectable gain amplifier stage then digitized with 12-bit resolution with a sampling rate of 0.64 ms/channel (1.6 kHz) using a Microstar A/D converter board in an IBM compatible PC computer.

A custom designed analysis system (IDL 4.0, Research Systems, Inc; Boulder, CO) was used for detection of activation and repolarization times at each site in the array.

Activation time was defined as the peak temporal derivative of the AP upstroke and recovery was defined as the point of maximum second derivative during repolarization. A good concordance has been previously shown between the latter value and the refractory period of normal guinea pig myocardium (Efimov et al 1994). Animals The studies were performed in 9 guinea pig hearts and 13 rabbit hearts. For study of the electrophysiologic effect of SnMP in normal myocardium, each animal served as its own control. Steady-state rate analysis was performed in those experiments in which high fidelity signals were obtained in control and after SnMP and with a 1:1 activation pattern at all cycle lengths. Guinea Pig Model: Surgical Preparation The surgical procedure has been previously described (Salama G et al., 1987; Kanai A, and Salama G, Circ. Res. 1995, 77:784- 802). Briefly, Dunkin-Hartley guinea pigs of either sex, weighing between 300 and 400 g, were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg) and heparinized (1000 U/kg). A mid-thoracotomy was performed on each animal, and the heart was rapidly excised and placed in cold oxygenated Tyrode's solution (NaCl 130 mM, KC1

4.75 mM, CaCl₂ 1.0 mM, MgSO₄ 1.2 mM, NaHCO₃ 12.5 mM, and glucose 15.0 mM) containing 1,000 U/l heparin. The excised heart was rapidly cannulated at the aorta and retrogradely perfused in a modified Langendorff apparatus. The solution was continuously bubbled with 95% O₂/ 5% CO₂ through a fritted glass tube. The temperature of the efusate within the closed chamber was monitored and maintained at 36±0.3°C. In a previous study, it was shown that the epicardial temperature varies less than 1 °C even during illumination. A small incision was made in the pulmonary artery for drainage.

Rabbit Model: Surgical Preparation The rabbit model has been extensively described (Gillis AM, et al., Am.J.Physiol 1996, 271:H784-H789). The electrophysiologic properties of the ionic currents that constitute the rabbit AP have been well characterized and may better represent the human heart rather than smaller animals. The dimensions of the rabbit heart are well suited for the spatial resolution of the optical system and permit imaging of large planar surfaces of the heart and specific cardiac structures. The optical resolution of the system can be adjusted from 400-1800 mM/pixel, depending on the dimension of the region of interest. Electrophysiologic observations can be correlated at high resolution with the anatomic features of the heart.

For the studies described herein, New Zealand young rabbits of either sex, weighing between 1.5-2 Kg, were anesthetized by intravenous injection of fentanyl citrate (100 mg/kg + 15 mg/kg/hr) and heparinized (1000 U/kg). A tracheotomy was performed on each animal and the animal was intubated with an endotracheal tube. The rabbit was ventilated with room air via a positive pressure ventilator (MD Industries, Mobile, AL). A midline thoracotomy was performed and the heart was exposed in a pericardial cradle. The heart was rapidly excised and placed in cold oxygenated Tyrode's solution, containing 1 ,000 U/l heparin. The heart was then cannulated and retrogradely perfused in a modified Langendorff setup and optical perfusion chamber. The hearts were perfused with oxygenated (95% O₂, 5% CO₂) modified

Tyrode's solution containing NaCl 130 mM, NaHCO₃ 25 mM, MgSO₄ 1.20 mM, KC1 4.75 mM, dextrose 10 mM, glucose 15 mM, and CaCl₂ 1 mM (pH 7.4, 37°C). Data were acquired after a 15 minute stabilization period.

For the acute ischemia experiments a ligature was placed around the left anterior descending artery using a 5-0 suture with a non-cutting needle. A snare comprising a small piece of polyethylene tubing and a small hemostat was used to reversibly ligate the coronary artery.

Results

The findings of these studies demonstrate that SnMP prolongs APD in both the rabbit and guinea pig heart. Although only a 2-D image of activation and repolarization could be obtained, the technique allows a quantitative description of the spatial changes in AP characteristics in response to heart rate within what has been considered the same population of cells.

Normal guinea pie and rabbit hearts Because of the optical density of SnMP, continuous infusion of the drug seriously degraded the optical signals. Because SnMP has a long tissue half-life and usually requires only a bolus dose in newborns to control severe jaundice, the drug was administered as single bolus dose in the perfused heart. A single bolus dose of 50 μl SnMP solution (20 mg/ml) was added to the bubble trap, proximal to the aortic cannula. The AP prolonging effect of SnMP in the normal guinea pig heart is shown in Figure 6. The mean increase was 32.4 + 4.1 ms. As seen in panel (A) of Figure 6, SnMP prolonged

APD at all cycle lengths (CL). As seen in panel (B) of Figure 6, the increase in APD was equivalent at all CLs indicating little or no reverse use dependence in the guinea pig.

SnMP had a similar effect in the rabbit heart. The shape of the rabbit AP is different from that of the guinea pig and may be related to differences in the density and ratios of the delayed rectifier repolarizing currents, IK,, and IK_r ( the slow and rapid activating components, respectively). In this experiment, a dose of 100 μl SnMP was administered. The results of this study are shown in Figure 7. As in the guinea pig, SnMP prolonged APD at all CLs and the amount of prolongation was slightly greater than in the guinea pig. The mean increase was 41.4 + 1.5 ms. Within the CL range used in this protocol, there appeared to be no reverse use dependence associated with SnMP.

The effect of SnMP did not degrade during the course of the experiments. Figure 8 shows that the effect of a single bolus of SnMP is long lasting. SnMP delivered as a single bolus dose of 50 μl prolonged APD in a guinea pig heart within 5 minutes (Figure 8, middle trace). The effect was maintained for the duration of the experiments. The lower trace in Figure 8 shows that the APD at 30 minutes was similar to that observed 5 minutes after the administration of SnMP.

These results show clearly that there was little if any reverse use-dependence associated with SnMP action in both the guinea pig and rabbit heart. In the cycle ranges used in the present study, the APD increase was similar at all cycle lengths. The lack of this side- effect is in contrast with most of the currently available class III drugs used clinically, with the exception of amiodarone. However, amiodarone has many other side effects and is not well tolerated in all patients. Because of the potential aggravation of ventricular arrhythmias by the clinically available class III antiarrhythmic drugs such as sotalol, dofetilide and azimilide , their use has been limited generally to the treatment of supraventricular arrhythmias .

Activation Mapping Isochronal maps of APD were constructed to assess the spatial organization of APD prolongation by SnMP in the heart (Salama G et al. , Am. J. Physiol 1987, 252.H384-H394). Activation maps during basic paced rhythm were obtained. In the maps shown in Figure 9, each shaded zone represents an isochronal region activated at successive 1 ms intervals. Activation maps for the basic paced rhythm S_j are shown for control and after 200 μl SnMP was administered in a rabbit heart. Hearts were paced from the right ventricle. The shape of the isochrones and total activation time were similar before and after SnMP and indicated a lack of effect on conduction in the heart.

The APD maps shown in Figure 10 were drawn at 10 ms isochronal intervals. Representative optical APs from the 124 recordings made are shown below each map along with an ECG. In the normal heart (A) there is an apical to basal gradient of APD with the shorter APD at the apex of the heart (lower portion of grid). In the control, APD ranged from 100-150 ms. After administration of SnMP, overall APD in the imaged area (B) increased significantly (130-180 ms) but the dispersion of APD remained similar to the control. Thus, the orientation of the overall APD gradient was preserved and the degree of APD increase was relatively uniform in the epicardial layer such that the magnitude of APD gradient was similar to control. The drug increased APD but did not cause gradients of APD dispersion to occur, which are potentially arrhythmogenic.

Anti-Ischemic Effect The focus of this study was the acute ischemia phase of coronary occlusion. AP shortening in the ischemic zone is the electrophysiologic hallmark of acute ischemia and is believed to be the substrate for arrhythmia formation during this phase. In hearts pretreated with SnMP, not only was APD prolonged, but the degree of APD shortening in the ischemic zone was considerably blunted compared to untreated hearts as is demonstrated in the results shown in Figure 7. These results define a significant cardioprotective effect of SnMP in the acute phase of coronary occlusion. The left map in Figure 11 shows the typical shortening of APD induced by coronary artery occlusion. The center map in Figure 11 shows that APD was uniformly increased throughout the epicardial layer. When the heart was subjected to occlusion of the left anterior descending coronary artery, APD were shortened but to a much lesser degree than the control (right map).

Examples 3-10 provide a number of clinical circumstances in which the immediate administration of a dose of HO inhibitor such as SnMP may prove useful in ameliorating the damaging sequelae of heme-iron tissue injury. In all these circumstances, a single dose, or multiple doses (at approximately 24 hours apart) of the inhibitor may be administered at the slightest indication of need since the inhibitor has been shown to be innocuous in human newborns subjects for other puφoses.

EXAMPLE 3

Treating a patient suspected of a cerebrovascular hemorrhage or infarction by administering immediately, either intramuscularly, subcutaneously or intravenously, a suitable dose of SnMP (1-50 mg/ kg body weight).

EXAMPLE 4

Treating a subject suspected of undergoing a myocardial infarction or impending coronary occlusion, as above

EXAMPLE 5

Treating a subject in shock, in which volume repletion will inevitably lead to reperfusion injury; or a patient with "crush injury" or massive bodily trauma, in which reperfusion as well as extravasation of blood occurs, in which cases the SnMP would be administered as above by emergency personnel as on-site treatment or in the hospital emergency department.

EXAMPLE 6

Patients undergoing angioplasty in which case the restitution of coronary blood flow will lead to some reperfusion injury; to be treated immediately prior to undertaking the procedure, as above.

EXAMPLE 7

Patients being treated for coronary thromboses with fhrombolytic therapy, i.e. , TPA, streptokinase etc., in which case reperfusion also occurs, to receive SnMP prior to undertaking the procedure, as above.

EXAMPLE 8

Patients undergoing emergency or scheduled coronary bypass surgery who, when removed from the heart-lung machine with normal circulation restored, may experience reperfusion injury. Treated prior to surgery, as above.

EXAMPLE 9

Organs to be used for transplantation (heart, kidney, etc.) undergo reperfusion injury when implanted into recipients. In this case, the solutions in which the organs are maintained should contain SnMP in a concentration range of 1-20 μmol/liter.

EXAMPLE 10 Patients undergoing cosmetic or other forms of plastic and reconstructive surgery will have tissue extravasation of blood with extensive bruising; pre-emptive treatment, as above (pre-operatively). The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

All values provided herein are for illustration and comparison only, and are not intended to be limiting.

Various patents, patent applications, and publications are cited herein, the disclosures of which are incoφorated by reference in their entireties.

Claims

What is claimed is:

1. A method for inhibiting heme oxygenase activity in ischemic, reperfused or hemorrhagic tissue, which method comprises administering tin mesopoφhyrin (SnMP) to a subject who may be suffering from an ischemic condition or a hemorrhage in an amount effective to reduce the release of free iron by the action of heme oxygenase.

2. The method of claim 1, wherein the SnMP is administered intravenously.

3. The method of claim 1, wherein the SnMP is administered subcutaneously.

4. The method of claim 1, wherein SnMP is administered intramuscularly.

5. The method of claim 1, wherein the SnMP is administered during ischemia.

6. The method of claim 1, wherein the SnMP is administered in response to an apparent stroke or myocardial infarction.

7. The method of claim 1, wherein the tissue is myocardial tissue.

8. The method of claim 1 , wherein the SnMP is administered for massive trauma.

9. A method of treating cardiac arrythmia which method comprises administering an effective amount of SnMP to a subject in need of such treatment.

10. The method of claim 9, wherein the SnMP is administered intravenously.

11. The method of claim 9, wherein the SnMP is administered subcutaneously.

12. The method of claim 9, wherein SnMP is administered intramuscularly.

13. A method of regulating or preventing cardiac arrhythmia which method comprises administering an effective amount of SnMP to a subject in need of such treatment.

14. A method of treating heart failure which method comprises administering an effective amount of SnMP to a subject in need of such treatment.

15. The method of claim 14, wherein the SnMP is administered intravenously.

16. The method of claim 14, wherein the SnMP is administered subcutaneously.

17. The method of claim 14, wherein SnMP is administered intramuscularly.

18. The method of claim 14, wherein the subject is suffering from congestive heart failure.

19. A method for reducing the extent of reperfusion or hemorrhagic injury associated with release of free iron in tissue, which method comprises administering SnMP to a subject in need thereof in an amount effective to ameliorate, reduce or prevent post-ischemic reperfusion or hemorrhagic injury.

20. The method of claim 19, wherein the SnMP is administered intravenously.

21. The method of claim 19, wherein the SnMP is administered subcutaneously.

22. The method of claim 19, wherein the SnMP is administered intramuscularly.

23. The method of claim 19, wherein the SnMP is administered during ischemia.

24. The method of claim 19, wherein the SnMP is administered in response to an apparent stroke or myocardial infarction.

25. The method of claim 19, wherein the tissue is myocardial tissue.

26. The method of claim 19 , wherein the SnMP is administered for massive trauma .

27. A kit for treating arrhythmia, an infarct or a hemorrhage, comprising a heme oxygenase inhibitor in a container.

28. The kit of claim 27 , which is labeled for use in treating an apparent heart attack or stroke.

29. The kit of claim 28, further comprising a clot-disrupting agent.

30. The kit of claim 29, wherein the clot-disrupting agent is acetylsalicylic acid (aspirin).

31. The kit of claim 27 , wherein the heme oxygenase inhibitor is provided in a unit dosage form.

32. The kit of claim 31, wherein the container is a syringe.

33. The kit of claim 31, wherein the heme oxygenase inhibitor is SnMP.