WO2005079803A1

WO2005079803A1 - Compounds for treatment of cardiovascular diseases

Info

Publication number: WO2005079803A1
Application number: PCT/US2005/004512
Authority: WO
Inventors: Ross W. Tracey
Original assignee: Pfizer Products, Inc.
Priority date: 2004-02-13
Filing date: 2005-02-11
Publication date: 2005-09-01

Abstract

Pharmaceutical compositions containing sodium-hydrogen exchanger type 1 (NHE-1) inhibitors in combination with Ca 2+ overload inhibitors, and the use of such combination inhibitors to treat, for example, cardiovascular disease, such as ischemia, particularly, perioperative myocardial ischemic injury in mammals, including humans, are disclosed.

Description

COMPOUNDS FOR TREATMENT OF CARDIOVASCULAR DISEASES FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions containing sodium-hydrogen exchanger type 1 (NHE-1) inhibitors in combination with Ca²⁺ overload inhibitors, and the use of such combination inhibitors to treat, for example, cardiovascular diseases, such as ischemia, particularly, perioperative myocardial ischemic injury in mammals, including humans.

BACKGROUND OF THE INVENTION The sodium-hydrogen exchangers (NHEs) are cell membrane transport proteins which exchange extracellular Na⁺ for intracellular H⁺ driven by concentration gradients. NHEs play a key role in intracellular pH regulation, ion-transport and control of cell volume. There are at least eight known isoforms that are designated NHE-1 through NHE-8. NHE-1 is expressed in virtually all cell types; NHE-2 is expressed in the intestine and kidney; NHE-3 is expressed in the kidney (proximal tubule, thick and thin loops of Henle) and intestine; NHE-4 is expressed in the stomach, kidney, intestine, and hippocampus; NHE-5 is expressed in the brain, testis, spleen, and skeletal muscle; NHE-6 is expressed by mitochondria; NHE-7 is expressed in the golgi network; and NHE-8 is expressed in the kidney. Each of the eight isoforms is a distinct gene product.

Cardiac cells rely mainly on NHE-1 , which is referred to as the myocardial Na⁺/H⁺ exchanger to prevent intracellular acidosis that inhibits contractility and mediates myocardial injury during periods of ischemia and reperfusion. See, e.g., Mentzer et al., Intracellular Sodium Hydrogen Exchange Inhibition and Clinical Myocardial Protection, Ann. Thorac. Surg., 2003; 75:S700-8; and Karmazyn ef al., The Myocardial Na⁺ - H⁺ Exchange, Structure, Regulation, and Its Role in Heart Disease, Circulation Research, AHA, Inc., pp. 777-86 (1999).

Consequently, inhibition of NHE-1, the sole isoform present in the myocardium, represents an attractive approach for reducing myocardial injury during ischemia/reperfusion. Selective NHE-1 inhibitors are desirable due to the important physiological functions played by the additional NHE isoforms in other tissues. In particular, NHE-2 and NHE-3 appear to have significant roles in the gastrointestinal and renal systems. The cardioprotective ability of a selective NHE-1 inhibitor could be extremely beneficial in hospital settings; for example, in patients that are at risk for myocardial ischemic injury during cardiac surgery. For surgical settings, an agent with high aqueous solubility that would allow for intravenous administration would be preferred. Selective inhibitors of NHE-1, such as zoniporide (which is known by the formula names [5-cyclopropyl-1-(quinolin-5-yl)-1 rY-pyrazole-4-carbonyl]guanidine and, alternatively, [1 -(quinolin-5-yl)-5-cyclopropyl-1 /-/-pyrazole-4-carbonyl]guanidine), cariporide, eniporide, and related guanidine based compounds, have been described in the literature (e.g., Guzman- Perez et al., Discovery of Zoniporide: A Potent and Selective Sodium - Hydrogen Exchanger Type 1 (NHE-1) Inhibitor with High Aqueous Solubility, Bioorg. Med. Chem. Lett. 11 : 803-807 (2001); and Tracey et al., Zoniporide: A Potent and Selective Inhibitor of Human Sodium Hydrogen Exchanger Isoform 1 (NHE-1), Cardiovascular Drug Reviews, 21 :1 , 17-32 2003), and have been the subject of various patents and published applications (e.g., Tracey et al., U.S. Patent Application Publication No. US 2002/0099075 A1; Tracey et al., U.S. Patent No. 6,423,705; Lambert et al., International Application Publication No. WO 2002/44133; Mylari, U.S. Patent Application Publication No. US 2001/0056095 A1 ; Tom, European Patent Application No. 1101763; Brostrom et al., International Application Publication No. WO 2001/30759; Masamune et al., International Application Publication No. WO 2001/23399; and Hamanaka et al., International Application Publication No. WO 99/43663). Other guanidine derivatives said to inhibit NHE and be useful for the treatment of, for example, arrhythmias are disclosed in Kleemann et al., US Patent No. 5,698,581 ; Kuno et al., US Patent No. 5,824,691 ; Kitano et al. US Patent No. 5,814,654; and Takahashi et al., European Patent Application Publication No. 953359.

A separate and distinct area of research into cardiovascular diseases has involved the study of the role that the sarcoplasmic reticulum, an organelle of each muscle cell, plays in excitation-contraction coupling and relaxation in cardiac muscle. See, e.g., Frank et al., Basic Res. Cardiol. 97; Suppl. 1 , 1/72 - 1/78 (2002). The sarcoplasmic reticulum reversibly sequesters Ca²⁺ ions at a very high concentration. Muscle contraction stimulates electrical potentials causing a rapid release of these sequestered Ca²⁺ ions into the sarcoplasmic fluid. Muscle relaxation is effectuated by ATP mediated calcium pump re-uptake into the reticulum. Numerous proteins, such as calsequestrin, troponin, and trypomyosin, are associated with maintaining appropriate calcium uptake and release.

Contraction and relaxation of cardiac muscle are dependent on cytosolic free Ca²⁺ concentration. The Ca²⁺ released from sarcoplasmic reticulum into cytoplasma during a systole is uptaken by the sarcoplasmic reticulum during a diastole. In diseased hearts, the Ca²⁺ uptake ability is reduced, resulting in higher level of intracellular Ca²⁺ during a diastole (referred to as Ca²⁺ overload). Ca²⁺ uptake into the sarcoplasmic reticulum is carried out by the Ca²⁺ pump, referred to as Ca²⁺ -ATPase, which is present on the membrane of endoplasmic reticulum. It has been reported that such reduction in Ca²⁺ uptake ability is accompanied by reduction in Ca²⁺ -ATPase activity in diseased hearts (See, e.g., Angel Zarain-Herzberg, Nasir Afzal, Vijayan Elimban and Naranjan S. Dhalla: Decreased expression of cardiac sarcoplasmic reticulum Ca²⁺ pump ATPase in congestive heart failure due to myocardial infarction. Molecular and Cellular Biochemistry 163/164: 285-290, (1996); and Ulrich Schmidt, Maria Carles, Roger H. Hajjar, Thomas G. DiSalvo, Marc J. Semigran, G. William Dec, Jagat Narula, Ban-An Khaw, Judith K. Gwathmey: Abnormal Sarcoplasmic Reticulum Ca²⁺ Activity and Uptake in Le Prigent and D. Feuvray: Altered Ca²⁺ handling in ventricular myocytes isolated from diabetic rates. Am. J. Physiol. 270: H1529-H1537, (1996)). Compounds that inhibit Ca²⁺ overload include sarcoplasmic reticulum calcium -

ATPase activators or "SERCA" activators, such as a 2-(1 -piperazinyl)-5-methylbenzene- sulfonic acid (i.e., MCC-135), and related aminobenzenesulfonic acid based compounds have been described in the literature. (See, e.g., Satoh et al., Lusitropic Effect of MCC-135 is Associated with Improvement of Sarcoplasmic Reticulum Function in Ventricular Muscles of Rats with Diabetic Cardiomyopathy, Journ. of Pharm. and Exp. Therap., vol. 298, No. 3, pp: 1161-1166 (2001)), and have been the subject of various patents and published applications (e.g., Inamori et al., International Application Publication No. WO 2003/11296; Satoh, International Application Publication No. WO 2003/09897; Kitada ef al., International Application Publication No. WO 2002/72097; Yuki et al., International Application Publication No. WO 2001/45739; Kawasumi et al., International Application Publication No. WO 99/40919; Kawazumi et al., Japanese Patent No. 10298077; Yamazaki et al., European Patent Application Publication No. 779283; Okujima et al., Japanese Patent No. 4139127; and Okushima ef al., European Patent Application Publication No. 390654). Other Ca²⁺ overload inhibitors are Ca²⁺ channel inhibitors (e.g., amlodipine), and Na⁺/Ca²⁺ exchanger (NCX) inhibitors (e.g., SEA0400, KB-R7943).

All of the documents discussed, referenced, or otherwise cited anywhere in this document are incorporated by reference herein in their entirety for all purposes.

SUMMARY OF THE INVENTION It has now surprisingly been found that a significant unexpected therapeutic benefit in the treatment of cardiovascular diseases, such as myocardial ischemia, can be obtained by combination therapy using at least one NHE-1 inhibitor and at least one Ca²⁺ overload inhibitor. For instance, it is possible using this combination therapy to considerably reduce the dosages of an NHE-1 inhibitor or a Ca²⁺ overload inhibitor that would, separately be required to obtain a given therapeutic effect, such as reducing the likelihood of myocardial ischemia. Through this combination of an NHE-1 inhibitor and a Ca²⁺ overload inhibitor, it is further possible to reduce infarct size following an ischemic event compared to the separate administration of an NHE-1 inhibitor or a Ca²⁺ overload inhibitor, and to accomplish that result with a combination dosage that is less than that which would be required were the same NHE-1 inhibitor or Ca²⁺ overload inhibitor administered separately and not in combination. Thus, in view of the decrease in amount of the combination of inhibitors that need be administered, it is also possible to minimize possible undesirable side effects. Even more surprising is that combining an NHE-1 inhibitor and a Ca²⁺ overload inhibitor markedly reduces the infarct size beyond that which either agent is capable of alone. Furthermore, the combination of these two inhibitors extends the duration of ischemia to which the heart can be exposed and yet still recover with minimal myocardial damage far beyond that observed for either inhibitor when administered not in combination.

This combination exhibits this effect when the two drugs are administered at the same time, e.g., in a composition containing both drugs, or when administered in combination separately or sequentially, so that medicaments of the invention facilitate the treatment of cardiovascular diseases. Medicaments of the invention can be used on demand in treatment (e.g., rescue treatment) of cardiovascular diseases and can facilitate treatment of such diseases with a single medicament.

As used herein, the terms "treat," "treats," "treated," "treating," and the like refer to reversing, alleviating (or reducing or ameliorating), inhibiting the progress of, and/or preventing the disease, condition, or syndrome being treated, including reversing, alleviating (or reducing or ameliorating), inhibiting the progress of, and/or preventing one or more symptoms of such disease, condition, or syndrome. The terms "reversing, "alleviating," "reducing," "ameliorating," "inhibiting," and "preventing" should be understood broadly and refer to partial as well as complete reversal, alleviation, reduction, amelioration, inhibition, and prevention. As used herein, the terms "treatment" and the like refer to the act of "treating." Thus, these terms are to be understood broadly and refer to, for example, prophylactic and palliative treatment.

The term "cardiovascular disease" should be understood broadly to mean a disease, condition, or syndrome in which the heart, pericardium, or blood vessels are directly or indirectly involved. Thus, the term "cardiovascular disease" includes, for example, endocarditis, myocarditis, cardiomyopathy, rheumatic heart disease, ischemic cardiac disease, non-cardiac ischemia, cerebrovascular disease (e.g., stroke), peripheral vascular disease, and hypertensive disease (e.g., high blood pressure). Examples of diseases, conditions, and syndromes in which the heart, pericardium, or blood vessels are directly involved include coronary artery disease, cardiomyopathy, valvular disease, congenital heart disease, arrhythmias, and conduction disturbances. Examples of diseases, conditions, and syndromes in which the heart, pericardium, or blood vessels are indirectly involved (including diseases, conditions, and/or syndromes with co-morbidity that could affect the heart) include systemic hypertension, pulmonary hypertension, and serum electrolyte abnormalities.

In one aspect, the present invention provides a pharmaceutical composition comprising a mixture of an NHE-1 inhibitor and a Ca²⁺ overload inhibitor, preferably in therapeutically effective amounts, optionally together with one or more pharmaceutically acceptable additional active ingredients (e.g., therapeutic agents) and/or inactive ingredients (e.g., carriers, vehicles, diluents).

In another aspect, the present invention provides a medicament which is a combination containing, separately or together, an NHE-1 inhibitor and Ca²⁺ overload inhibitor, for simultaneous, sequential, and/or separate administration in the treatment of a disease, condition, or syndrome, e.g., cardiovascular disease, such as ischemia.

The invention further provides a process for preparing a pharmaceutical composition or medicament that includes an NHE-1 inhibitor and a Ca²⁺ overload inhibitor.

The combination of this invention (i.e., the combination comprising an NHE-1 inhibitor and Ca²⁺ overload inhibitor, whether in a pharmaceutical composition or in a medicament) may be in the form of a kit that includes at least one NHE-1 inhibitor and at least one Ca²⁺ overload inhibitor in one or more dosage forms for simultaneous, sequential, or separate administration, and the kit may further include a container.

Another aspect of this invention is a method of treating a mammal (e.g., a human) having a disease, condition, or syndrome responsive to the combination of this invention of an

NHE-1 inhibitor and a Ca²⁺ overload inhibitor, such as a disease, condition, or syndrome identified herein, by simultaneously, sequentially, and/or separately administering to the mammal pharmaceutically acceptable amounts (preferably therapeutically effective amounts) of an NHE-1 inhibitor and of a Ca²⁺ overload inhibitor. In a preferred aspect, the present invention provides a method of treating a cardiovascular disease, such as ischemia, by combination therapy which comprises simultaneously, sequentially, and/or separately administering to a subject, such as a human, in need of such treatment a pharmaceutically acceptable amount (preferably therapeutically effective amount) of an NHE-1 inhibitor and a pharmaceutically acceptable amount (preferably therapeutically effective amount) of a Ca²⁺ overload inhibitor. The NHE-1 inhibitor and the Ca²⁺ overload inhibitor may be administered in an NHE-1 inhibitor/Ca²⁺ overload inhibitor ratio of from about 20:1 to about 1 :20, such as from about 10:1 to about 1 :10.

The NHE-1 inhibitor may be a compound of the following general formula wherein Z is a suitable substituent:

Formula I a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, and the Ca²⁺ overload inhibitor may be a compound of the following general formula wherein n is an integer from 1 to 4 and, R⁹ and R¹⁰ are suitable independent substituents:

Formula II or a pharmaceutically acceptable salt or solvate thereof.

Thus, more specifically, one aspect of this invention concerns a method of treating a mammal (e.g., a human) having a disease, condition, or syndrome responsive to the combination of an NHE-1 inhibitor and a Ca²⁺ overload inhibitor, such as a disease, condition, or syndrome identified herein, by simultaneously, sequentially, and/or separately administering to the mammal a pharmaceutically acceptable amount (preferably therapeutically effective amount) of an NHE-1 inhibitor, such as a compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt of said compound or of said prodrug, and a pharmaceutically acceptable amount (preferably therapeutically effective amount) of a Ca²⁺ overload inhibitor, such as a compound of Formula II, or a pharmaceutically acceptable salt or solvate of said compound. The method may further include the step of identifying a patient in need of such treatment. A more specific aspect of this invention concerns a method of treating to prevent or reduce tissue damage resulting from cardiovascular disease (e.g., ischemia) comprising simultaneously, sequentially, and/or separately administering to a mammal (e.g., a female or male human) in need of such treatment a pharmaceutically acceptable amount (preferably therapeutically effective amount) of an NHE-1 inhibitor in combination with a pharmaceutically acceptable amount (preferably therapeutically effective amount) of a Ca²⁺ overload inhibitor. The NHE-1 inhibitor may be a compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, and the Ca²⁺ overload inhibitor may be a compound of Formula II, or a pharmaceutically acceptable salt or solvate of said compound. Preferred ischemic tissues taken individually or as a group are ischemic cardiac, brain, liver, kidney, lung, gut, skeletal muscle, spleen, pancreas, nerve, spinal cord, retina tissue, the vasculature, and/or intestinal tissue. Especially preferred is ischemic cardiac tissue.

The combination of this invention may be administered to treat prophylactically, such as prior to an ischemic event, or to treat during an event, whether chronic or acute (e.g., an ischemic event). Alternatively, it may be administered upon reperfusion. The damage (e.g., ischemic damage) sought to be prevented or ameliorated (i.e., treated) may also occur during organ transplantation. Preferably, the combination of this invention is administered prior to, during, or shortly after cardiac surgery or non-cardiac surgery.

Myocardial tissue damage may be reduced by administration (e.g., chronic administration) of the combination of this invention to a patient. Thus, another specific aspect of this invention is a method of treating to reduce myocardial tissue damage, for example, (a) during surgery (e.g., coronary artery bypass grafting (CABG) surgeries, vascular surgeries, percutaneous transluminal coronary angioplasty (PTCA) or any percutaneous transluminal coronary intervention (PTCI), organ transplantation, or other non-cardiac surgeries), (b) in a patient presenting with ongoing cardiac (acute coronary syndromes, e.g., myocardial infarction or unstable angina) or cerebral ischemic events (e.g., stroke), and (c) in a patient who is at high risk for myocardial infarction (age > 65 and two or more risk factors for coronary heart disease).

It is especially preferred that the compounds be administered to prevent perioperative myocardial ischemic injury. The word "perioperative" refers to the time period a patient is in the operating suite and its adjoining facilities, in other words, the pre-operative, intra- operative, and post-operative time period.

Examples of diseases, conditions, and syndromes that may be treated using the combinations (compositions and medicaments) and methods of this invention include: arteriosclerosis, hypertension, arrhythmia, angina pectoris, renal diseases, diabetic complications, restenosis, diseases of cell proliferation, cancerous diseases, fibrotic diseases, glomerular nephrosclerosis, organ hypertrophies or hyperplasias, pulmonary fibrosis, cerebroischemic disorders, myocardial stunning, myocardial dysfunction, cerebrovascular diseases, and organ hypertrophies or hyperplasias.

In each case, the method of this invention for treating comprises simultaneously, sequentially, and/or separately administering to a mammal (e.g., a female or male human) a pharmaceutically acceptable (preferably therapeutically effective) amount of an NHE-1 inhibitor in combination with a pharmaceutically acceptable (preferably therapeutically effective) amount of a Ca²⁺ overload inhibitor. The NHE-1 inhibitor may be a compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, and the Ca²⁺ overload inhibitor may be a compound of Formula II, or a pharmaceutically acceptable salt or solvate of said compound.

The combinations of this invention may be combined with one or more additional therapeutic agents useful in the treatment of cardiovascular disease, e.g., myocardial ischemia-reperfusion injury. Furthermore, one skilled in the art will appreciate that when using the combinations of the invention in the treatment of diseases other than cardiovascular disease, the combinations of the invention may be combined with one or more additional therapeutic agents used for those other diseases. Thus, the combinations of this invention may further include one or more active ingredients such as one or more of any of those additional therapeutic agents. Examples of additional active ingredients are as follows.

1. PDE5 inhibitors, such as sildenafil, vardenafil or tadalafil;

2. standard non-steroidal anti-inflammatory agents (referred to as "NSAIDs"), such as piroxicam, diclofenac, propionic acids such as naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone, salicylates (e.g., aspirin), COX-2 inhibitors (e.g., celecoxib, valdecoxib, paracoxib, rofecoxib and etoricoxib), analgesics and intraarticular therapies (e.g., corticosteroids and hyaluronic acids, such as hyalgan and synvisc); 3. calcium channel blockers such as amlodipine diltiazem, verapamil, felodipine, isradipine, lacidipine, lercanidipine, nicardipine nifedipine, nimodipine and nisoldipine;

4. lipid lowering agents such as statins, e.g., atorvastatin, fluvastatin, pravastatin, simvastatin, lovastatin and rosuvastatin;

5. fibrates such as fenofibrate, clofibrate, gemfibrozil; 6. niacin; bile acids or cholestyramine resins (e.g., cholybat, Locholest, prevalite, and questran); colesevelam; colestipol; or IBAT inhibitors;

7. cholesterol absorption inhibitors (e.g., ezetimibe);

8. beta-blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, metoprolol, metipranolol, nadolol, nebivolol, oxprenolol, pindolol, propranolol, sotalolol and timolol);

9. ACE inhibitors (e.g., benazepril, captopril, cilazapril, enalapril, fosinopril, imidapril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril);

10. Angiotensin-2 receptor antagonists (e.g., telmisartan, olmesartan, candesartan, eprosartan, irbesartan, losartan and valsartan); 11. platelet aggregation inhibitors (e.g., clopidogrel, BM-531 , aspirins, NSAIDs, selective COX-2 inhibitors) and monocyte-endothelial cell adhesion inhibitors (e.g., K-7174);

12. aldosterone antagonists (e.g., eplerenone or spironolactone);

13. CETP inhibitors (e.g., JTT-705 or CP529414);

14. diuretics (e.g., hydrocholothiazide, amiloride, furosemide, bumetanide, torasemide, indapamide, metolazone, and triamterene);

15. antiarrhythmics (e.g., lidocaine);

16. alpha blockers (e.g., doxazosin, prazosin and terazosin); and

17. agents for the treatment of diabetes: insulin-like growth factor type I (IGF-1 ) mimetic; alpha-glucosidase inhibitors (e.g., acarbose or miglitol), biguanides (e.g., metformin), insulins, megltinides (e.g., nateglinide and repaglinide), sulfonylureas (e.g., acetohexamide, chlorpropamide, glimepride, glipizide, glyburide, tolazamide, or tolbutamide), or thiazolidinediones (e.g., pioglitazone or rosiglitazone). Still other therapeutic agents may also be used in the combination of this invention, provided they do not significantly adversely affect the action of the NHE-1 inhibitors and Ca²⁺ overload inhibitors. For example, it is believed that it may be possible to include TNF-α inhibitors, such as anti-TNF monoclonal antibodies (e.g., Remicade, CDP-870, and D2E7), TNF receptor immunoglobulin molecules (e.g., Enbrel®), and/or dual thromboxane synthase inhibitors/thromboxane receptor antagonists (e.g., BM-573) in the combination. To the extent not already mentioned above, the agents disclosed in Tracey et al., U.S. Patent No. 6,423,705 for use in combination with NHE-1 inhibitors may also be used in the combination of this invention (as previously indicated, that patent is incorporated herein in its entirety for all purposes). Use in the combination of many other therapeutic agents (e.g., CNS agents, anti-Parkinsonian drugs, osteoporosis agents, immunosuppressant agents, receptor antagonists for leukotrienes, anticholinergic agents, anticancer agents, and antiviral agents) may also be possible and desirable in some cases.

Preferred agents (most preferably cardiovascular agents) for use in the combination include, for example, β-blockers (e.g., acebutolol, atenolol, bopindolol, labetolol, mepindolol, nadolol, oxprenol, pindolol, propranolol, sotalol), calcium channel blockers (e.g., amlodipine, nifedipine, nisoldipine, nitrendipine, verapamil), potassium channel openers, adenosine, adenosine agonists, ACE inhibitors (e.g., captopril, enalapril), nitrates (e.g., isosorbide dinitrate, isosorbide 5-mononitrate, glyceryl trinitrate), diuretics (e.g., hydrochlorothiazide, indapamide, piretanide, xipamide), glycosides (e.g., digoxin, metildigoxin), thrombolytics (e.g., tPA), platelet inhibitors (e.g., reopro), aspirin, dipyridamol, potassium chloride, clonidine, prazosin, aldose reductase inhibitors, and adenosine A3 receptor agonists. A preferred aldose reductase inhibitor for use in the combination is zopolrestat: 1 -phthalazineacetic acid, 3,4-dihydro-4-oxo-3-[[5-trif luoromethyl)-2-benzothiazolyl]methyl]-. In view of this, the combination of this invention, which can be used in a method of treating to reduce tissue damage resulting from or which could result from ischemia, comprises: a. an NHE-1 inhibitor compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said Formula I compound or of said prodrug; b. a Ca²⁺ overload inhibitor compound of Formula II, or a pharmaceutically acceptable salt or solvate of said Formula II compound; and c. optionally a cardiovascular agent; wherein the amounts of the first, second, and optional cardiovascular agent result in a therapeutic effect. Furthermore, the combination in kit of this invention may comprise: a. a therapeutically effective amount of a Formula I compound, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug and a pharmaceutically acceptable carrier, vehicle, or diluent in a first unit dosage form; b. a therapeutically effective amount of a Formula II compound, or a pharmaceutically acceptable salt or solvate of said compound and a pharmaceutically acceptable carrier, vehicle, or diluent in a second unit dosage form; c. optionally a therapeutically effective amount of a cardiovascular agent and a pharmaceutically acceptable carrier, vehicle, or diluent in a third unit dosage form; and d. means for containing said first and second dosage forms, wherein the amounts of the first and second compounds and optional cardiovascular agent result in a therapeutic effect. In some cases, it may be possible to combine one or more of the first, second, and third dosage forms into a single dosage form.

Other features and advantages will be apparent from the specification and claims which describe the invention. BRIEF DESCRIPTION OF THE DRAWINGS

To aid in further describing the invention, the following drawings are provided. Figure 1 illustrates the maximum reduction of infarct size obtainable by a 30 minute/120 minute ischemia/reperfusion standard protocol by the administration of a Ca²⁺ overload inhibitor or an NHE-1 inhibitor. Figure 2 illustrates the maximum reduction of infarct size obtainable by a 30 minute/120 minute and 60 minute/120 minute ischemia/reperfusion protocol by the separate administration of a Ca²⁺ overload inhibitor or an NHE-1 inhibitor.

Figure 3 illustrates the maximum reduction of infarct size obtainable by a 60 minute/120 minute ischemia/reperfusion protocol by the administration of a Ca²⁺ overload inhibitor or an NHE-1 inhibitor separately and in combination.

These drawings are for illustrative purposes and should not be used to unduly limit the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a combination of at least one NHE-1 inhibitor, which preferably comprises at least one compound of Formula I, and at least one Ca²⁺ overload inhibitor, which preferably comprises at least one compound of Formula II, as defined below.

A. NHE - 1 Inhibitors

The NHE-1 inhibitor of the combination may be a compound of Formula I:

Formula I or a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug wherein

Z is a carbon of (a) a five-membered, diaza, diunsaturated ring having two contiguous nitrogens, said ring optionally mono-, di-, or tri-substituted with up to three substituents independently selected from R¹, R² and R³, or of (b) a five-membered, triaza, diunsaturated ring, said ring optionally mono- or di-substituted with up to two substituents independently selected from R⁴ and R⁵; wherein R¹, R², R³ , R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, hydroxy(CrC )alkyl, (Cι-C₄)alkyl, (C C₄)alkylthio, (C₃-C₄)cycloalkyl, (C₃-C₇)cycloalkyl(Cι-C₄)alkyl, (C C₄)alkoxy, (C C₄)alkoxy(C C₄)alkyl, mono-N-(C C₄)alkylcarbamoyl or di-^N^CrC-^alkylcarbamoyl, M or M(C C₄)alkyl, wherein any of said R¹, R², R³ , R⁴ and R⁵ (C-ι-C₄)alkyl moieties optionally have from one to nine fluorines; wherein said R¹, R², R³ , R⁴ and R⁵ (C C₄)alkyl moieties or said R¹, R², R³, R⁴ and R⁵ (C₃- C )cycloalkyl moieties are optionally independently substituted with one or two substituents selected from the group consisting of: hydroxy, (CrC )alkoxy, (Cι-C₄)alkylthio, (C C₄)alkylsulfinyl, (Cι-C₄)alkylsulfonyl, (Cι-C₄)alkyl, mono-N-(C₁-C₄)alkylcarbamoyl, di-N,N-(C C₄)alkylcarbamoyl, mono-N-(C₁-C₄)alkylaminosulfonyl, and di-N,N-(C₁-C₄)alkylaminosulfonyl, wherein said (C₃-C₄)cycloalkyl optionally contains from one to seven fluorines; wherein M is a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen, or, a bicyclic ring consisting of two fused partially saturated, fully saturated or fully unsaturated three to six membered rings, taken independently, optionally having one to four heteroatoms selected independently from nitrogen, sulfur and oxygen; said M is optionally substituted, on one ring if the moiety is monocyclic, or one or both rings if the moiety is bicyclic, on carbon or nitrogen with up to three substituents independently selected from R⁶, R⁷ and R⁸, wherein one of R⁶, R⁷ and R⁸ is optionally a partially saturated, fully saturated, or fully unsaturated three to seven membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen optionally substituted with (C C₄)alkyl and additionally R⁶, R⁷ and R⁸ are optionally hydroxy, nitro, halo, (C-|-C )alkoxy, (C C₄)alkoxycarbonyl, (C-ι-C )alkyl, formyl, (C C₄)alkanoyl, (C_rC₄)alkanoyloxy, (d-C^alkanoylamino, (CrC^alkoxycarbonylamino, sulfonamido, (C C₄)alkylsulfonamido, amino, mono-N-(C-|-C₄)alky!amino, di-N,N-(C C )alkylamino, carbamoyl, mono-N-(C C₄)alkylcarbamoyl, di-N,N-(C C )alkylcarbamoyl, cyano, thiol, (C_rC₄)alkylthio, (C_rC₄)alkylsulfinyl, (CrC₄)alkylsulfonyl, mono-N-(C C₄)alkylaminosulfonyl, di-N,N-(C₁-C₄)alkylaminosulfonyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl or (C₅- C₇)cycloalkenyl, wherein said (C C₄)alkoxy, (C C₄)alkyl, (C C₇)alkanoyl, (C C₄)alkylthio, mono-N- or di-N,N-(Cι-C₄)alkylamino or (C₃-C₇)cycloalkyl R⁶, R⁷ and R⁸ substituents are optionally mono- substituted independently with hydroxy, (Cι-C )alkoxycarbonyl, (C₃-C₇)cycloalkyl, (C C₄)alkanoyl, (CrC^alkanoylamino, (C C₄)alkanoyloxy, (CrC^alkoxycarbonylamino, sulfonamido, (C C )alkylsulfonamido, amino, mono-N- or di-N,N-(C₁-C )alkylamino, carbamoyl, mono-N- or di-N,N-(C₁-C₄)alkylcarbamoyl, cyano, thiol, nitro, (C-ι-C )alkylthio, (C C₄)alkylsulfinyl, (C C₄)alkylsulfonyl or mono-N- or di-N,N-(C C₄)alkylaminosulfonyl or optionally substituted with one to nine fluorines. Preferred Formula I compounds are: [1 -(2-chlorophenyl)-5-methyl-1 H-pyrazole-4-carbonyl]guanidine;

[5-methyl-1-(2-trifluoromethylphenyl)-1 H-pyrazole-4-carbonyl]guanidine; [5-ethyl-1 -phenyl-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1-(2-trifluoromethylphenyl)-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1 -phenyl-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1 -(2,6-dichlorophenyl)-1 H-pyrazole-4-carbonyl]guanidine;

[5-methyl-1 -(quinolin-6-yl)-1 H-pyrazole-4-carbonyl]guanidine; [5-methyl-1 -(naphthalen-1 -yl)-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4-carbonyl]guanidine; t5-cyclopropyl-1-(quinolin-8-yl)-1H-pyrazole-4-carbonyl]guanidine; [3-methyl-1 -phenyl-1 H-pyrazole-4-carbonyl]guanidine;

[3-methyl-1 -(naphthalen-1 -yl)-1 H-pyrazole-4-carbonyl]guanidine; [3-methyl-1-(isoquinolin-5-yl)-1 H-pyrazole-4-carbonyl]guanidine; [4-methyl-1 -phenyl-1 H-pyrazole-3-carbonyl]guanidine; [2-methyl-5-phenyl-2H-pyrazole-3-carbonyl]guanidine; [2-methyl-5-(naphthalen-1-yl)-2H-pyrazole-3-carbonyl]guanidine;

[5-methyl-2-phenyl-2H-1 ,2,3-triazole-4-carbonyl]guanidine; [5-methyl-2-(3-methoxyphenyl)-2H-1 ,2,3-triazole-4-carbonyl]guanidine; [2-(3-bromophenyl)-5-methyl-2H-1,2,3-triazole-4-carbonyl]guanidine; [2-(naphthalen-1-yl)-5-methyl-2H-1 ,2,3-triazole-4-carbonyl]guanidine; [2-(isoquinolin-5-yl)-5-methyl-2H-1 ,2,3-triazole-4-carbonyl]guanidine;

[5-methyl-2-(quinolin-5-yl)-2H-1,2,3-triazole-4-carbonyl]guanidine; [1 -(Naphthalen-1 -yl)-5-cyclopropyl-1 fV-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1-(2-trifluoromethylphenyl)-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1 -phenyl-1 H-pyrazole-4-carbonyl]guanidine; [5-cyclopropyl-1 -(2,6-dichlorophenyl)-1 H-pyrazole-4-carbonyl]guanidine;

[1-(2-Chloro-4-methylsulfonylphenyl)-5-cyclopropyl-1 -/-pyrazole-4- carbonyljguanidine; [1-(2-Chlorophenyl)-5-cyclopropyl-1 r/-pyrazole-4-carbonyl]guanidine;

[1-(2-Trifluoromethyl-4-fluorophenyl)-5-cyclopropyl-1 /--pyrazole-4-carbonyl]guanidine;

[1 -(2-Bromophenyl)-5-cyclopropyl-1 r/-pyrazole-4-carbonyl]guanidine;

[1 -(2-Fluorophenyl)-5-cyclopropyl-1 r-pyrazole-4-carbonyl]guanidine;

[1-(2-Chloro-5-methoxyphenyl)-5-cyclopropyl-1 AV-pyrazole-4-carbonyl]guanidine;

[1 -(2-Chloro-4-methylaminosulfonylphenyl)-5-cyclopropyl-1 /--pyrazole-4- carbonyljguanidine;

[1-(2,5-Dichlorophenyl)-5-cyclopropyl-1 /--pyrazole-4-carbonyl]guanidine;

[1-(2,3-Dichlorophenyl)-5-cyclopropyl-1 rV-pyrazole-4-carbonyl]guanidine;

[1 -(2-Chloro-5-aminocarbonylphenyl)-5-cyclopropyl-1 r/-pyrazole-4- carbonyljguanidine;

[1-(2-Chloro-5-aminosulfonylphenyl)-5-cyclopropyl-1 W-pyrazole-4-carbonyl]guanidine;

[1 -(2-Fluoro-6-trifluoromethylphenyl)-5-cyclopropyl-1 ry-pyrazole-4-carbonyl]guanidine;

[1 -(2-Chloro-5-methylsulfonylphenyl)-5-cyclopropyl-1 /-/-pyrazole-4- carbonyljguanidine;

[1 -(2-Chloro-5-dimethylaminosulfonylphenyl)-5-cyclopropyl-1 /-/-pyrazole-4- carbonyljguanidine;

[1-(2-Trifluoromethyl-4-chlorophenyl)-5-cyclopropyl-1 /--pyrazole-4- carbonyl]guanidine;

[5-cyclopropyl-1-(quinolin-8-yl)-1 H-pyrazole-4-carbonyl]guanidine;

[5-cyclopropyl-1-(quinolin-5-yl)-1 H-pyrazole-4-carbonyl]guanidine;

[1- 8-Bromoquinolin-5-yl)-5-cyclopropyl-1 /--pyrazole-4-carbonyl]guanidine;

[1- 6-Chloroquinolin-5-yl)-5-cyclopropyl-1 /-/-pyrazole-4-carbonyl]guanidine;

[1 - lndazol-7-yl)-5-cyclopropyl-1 H-pyrazole-4-carbonyl]guanidine; [1 - Benzimidazol-5-yl)-5-cyclopropyl-1 /- -pyrazole-4-carbonyl]guanidine;

[1- 1-lsoquinolyl)-5-cyclopropyl-1 /-/-pyrazole-4-carbonyl]guanidine;

[5-Cyclopropyl-1-(4-quinolinyl)-1 /-/-pyrazole-4-carbonyl]guanidine;

[1 -(lndazol-6-yl)-5-ethyl-1 /-/-pyrazole-4-carbonyl]guanidine;

[1 -(lndazol-5-yl)-5-ethyl-1 /-/-pyrazole-4-carbonyl]guanidine;

[1-(Benzimidazol-5-yl)-5-ethyl-1 Hpyrazole-4-carbonyl]guanidine;

[1 -(1 -Methylbenzimidazol-6-yl)-5-ethyl-1 --pyrazole-4-carbonyl]guanidine

[1 -(5-Quinolinyl)-5-n-propyl-1 H-pyrazole-4-carbonyl]guanidine;

[1-(5-Quinolinyl)-5-isopropyl-1 /- -pyrazole-4-carbonyl]guanidine;

[5-Ethyl-1 -(6-quinolinyl)-1 -/-pyrazole-4-carbonyl]guanidine;

[1 -(2-Methylbenzimidazol-5-yl)-5-ethyl-1 /--pyrazole-4-carbonyl]guanidine;

[1 -(1 ,4-Benzodioxan-6-yl)-5-ethyl-1 H-pyrazole-4-carbonyl]guanidine;

[1-(Benzotriazol-5-yl)-5-ethyl-1 r/-pyrazole-4-carbonyl]guanidine; [1 -(3-Chloroindazol-5-yl)-5-ethyl-1 H-pyrazole-4-carbonyl]guanidine;

[1 -(5-Quinolinyl)-5-butyl-1 H-pyrazole-4-carbonyl]guanidine;

[5-propyl-1 -(6-quinolinyl)-1 W-pyrazole-4-carbonyl]guanidine;

[5-lsopropyl-1 -(6-quinolinyl)-1 H-pyrazole-4-carbonyl]guanidine;

[1 -(lndazol-7-yl)-3-methyl-1 H-pyrazole-4-carbonyl]guanidine;

[1 -(2,1 ,3-Benzothiadiazol-4-yl)-3-methyl-1 -/-pyrazole-4-carbonyl]guanidine;

[3-Methyl-1 -(quinolin-5-yl)-1 H-pyrazole-4-carbonyl]guanidine; or the pharmaceutically acceptable salts of said compounds.

Particularly preferred NHE-1 inhibitor compounds of the present invention are cariporide 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate, which has the following formula:

eniporide Λ/-(diaminomethylene)-5-(methylsulfonyl)-4-pyrrol-l-yl-otoluamide, which has the following formula:

zoniporide, which has the following formula:

crystalline zoniporide dihydrochloride, crystalline zoniporide hydrochloride monohydrate, and the monohydroxylated in vivo and in vitro metabolite of zoniporide, [5-cyclopropyl-1 -(2- quinolon-5-yl)-1 H-pyrazole-4-carbonyl] guanidine, and its hydrochloride, which has the formula:

sabiporide (Λ/-carbamimidoyl-4-[4-(1 /-/-pyrrol-2-ylcarbonyl)piperazin-1 -yl]-3-

(trifluoromethyl)benzamide) which has the following formula:

and BMS-284640, which has the following formula:

A most preferred NHE-1 inhibitor is zoniporide methanesulfonate (zoniporide mesylate), which has the formula:

Particularly preferred NHE-1 inhibitors are anhydrous crystalline zoniporide mesylate, the anhydrous crystalline Form A of zoniporide mesylate, the anhydrous crystalline Form D of zoniporide mesylate, and the hemihydrate crystalline Form C of zoniporide mesylate. Each of these crystalline forms can be prepared in accordance with the procedures set forth in Hamanaka et al., U.S. Patent No. 6,492,401 , and Brostrom et al., International Application Publication No. WO 2001/30759, both of which documents as previously noted are incorporated by reference herein in their entirety for all purposes.

B. Ca²⁺ Overload Inhibitors

Ca²⁺ overload inhibitor compounds of the combination of the invention may be aminobenzenesulfonic acid derivates of the formula:

Formula II wherein, in general, R⁹ represents hydrogen atom, a C C₆ alkyl group, a C₃-C₇ cycloalkyl group, a C C₄ halogenated alkyl group, a halogen atom or a C₆-C₁₂ aryl group, and R¹⁰ represents hydrogen atom, a C|-C₆ alkyl group or a C₇-Cι₂ aralkyl group which may also have at least one substituent selected from cyano group, nitro group, C C₆ alkoxy groups, halogen atoms, C Cβ alkyl groups and amino group, and n represents an integer of 1 to 4, or 2 to 3, or a pharmaceutically acceptable salt or solvate thereof. More particularly, as R⁹, there may be included hydrogen atom, a C C₆ straight or branched alkyl group such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl and n-hexyl groups, a C₃-C₇ cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups, a C C₄ halogenated alkyl group such as trifluoromethyl group, a halogen atom such as fluorine, chlorine and bromine atoms or C₆-Cι₂ aryl group such as phenyl, tolyl and naphthyl groups; and, as R¹⁰, there may be included hydrogen atom; a CrC-β straight or branched alkyl group such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl and n-hexyl groups; a C₇-C₁₂ aralkyl group such as benzyl, phen-ethyl and naphthylmethyl groups, which may also have at least one substituent selected from cyano group, nitro group, C_t-C₆ alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, pentoxy and hexyloxy groups, halogen atoms such as fluorine, chlorine and bromine atoms, C-ι-C₆ straight or branched alkyl group such as methyl, ethyl, n-propyi, iso-propyl, n-butyl, t-butyl, n-pentyl and n-hexyl groups, and amino group.

As specific Ca²⁺ overload inhibitor compounds of the present invention, for example, those set forth in the following Table 1 may be included. TABLEl

-CH₃

-(CHECKS

--^

(

— CH3 TABLE 1 - continued

61 3 -<CH2>4CH3 ϊ

-(CHifc

65 * -(CHώiCHj 2 OCHl

-(CH_J)-/Q OCHI

TABLE 1 - continued

71 3 -(CHjJaCHj 2

TABLE 1 - continued

Compound Substitution

No. position of R R n R₁₀

78 -(CHjhCHj

-^cB-(Q -_°

79 -(CHjJjCHj

80 -(CHjfcCHj

90 -(CHjhCHj ³

96 2 H

^■o

TABLE 1 - continued

Compound Substitution

Nα position of R« R9 n R,_α

103 5 .. 2 -CHj

104

-<g> ^'—©

103

^' -©

108

-O ^'-©

Pref erred Ca²⁺ overload inhibitor compounds of general Formula II include: aminobenzenesulfonic acid compounds wherein n is 1 to 4, 1 or 4, 2 or 3, or 2; aminobenzenesulfonic acid compounds wherein R¹⁰ is hydrogen atom, C₁-C₃ alkyl group or C₇-C₁₂ aralkyl group which may be substituted by C C₃ alkyl group, C C₃ alkoxy group or a halogen atom; aminobenzenesulfonic acid compounds wherein R¹⁰ is hydrogen atom or C₇-C₁₂ aralkyl group which may be substituted by CrC₃ alkyl group; aminobenzenesulfonic acid compounds wherein R⁹ is hydrogen atom, Cι-C₆ alkyl group, C₅-C₆ cycloalkyl group, trifluoromethyl group, a halogen atom or phenyl group; aminobezenesulfonic acid compounds wherein R⁹ is C₁-C₃ alkyl group, cyclohexyl group, trifluoromethyl group, chlorine atom, bromine atom or phenyl group; aminobenzenesulfonic acid compounds wherein said pharmaceutically acceptable salt is a nontoxic salt selected from alkali metal salts, alkaline earth metal salts and amine salts; aminobenzenesulfonic acid compounds wherein said pharmaceutically acceptable salt is a nontoxic salt selected from sodium salts, potassium salts, magnesium salts, calcium salts, aluminum salts, ammonium salts, lower alkylamine salts, hydroxy lower alkylamine salts, cycloalkylamine salts, benzylamine salts and dibenzylamine salts; and aminobenzenesulfonic acid compound wherein said pharmaceutically acceptable salt is a nontoxic salt selected from hydrochlorides, hydrobromides, sulfates, phosphates, fumarates, succinates, oxalates and lactates.

More preferably, the Ca²⁺ overload inhibitor compound is:

2-(1-piperazinyl)-5-methylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof; 2-(1-piperazinyl)-5-trifluoromethylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof;

2-(1 -piperazinyl)-5-n-propylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof;

2-(1-piperazinyl)-5-phenylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof;

2-(1 -piperazinyl)-5-chlorobenzenesulfonic acid or a pharmaceutically acceptable salt thereof;

2-(1-piperazinyl)-5-bromobenzenesulfonic acid or a pharmaceutically acceptable salt thereof; 2-(1-piperazinyl)-5-iso-propylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof; 2-(1-piperazinyl)-5-cyclohexylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof;

2-(1-homopiperazinyl)-5-n-propylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof; 2-[4-(2,3,4-trimethoxybenzyl)-1-piperazinyl]-5-n-propylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof; or

2-[4-(3,4-dimethoxybenzyl)-1-piperazinyl]-5-n-propylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof.

As is apparent from the discussion above, also within the medicaments and, more particularly, the compositions of the invention, are cardio-protective agents for ischemia- induced damage which include at least one of the aminobenzenesulfonic acid compounds of Formula II as an active ingredient and, more preferably, those subgenera and particular Ca²⁺ overload inhibitor compounds identified above.

As used herein, "medicament" means a combination of an NHE-1 inhibitor and a Ca²⁺ overload inhibitor, whether combined in a composition or separate as in individual unit dosage forms, such as in a kit. The medicament may include an NHE-1 inhibitor of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, and a Ca²⁺ overload inhibitor compound of Formula II, or a pharmaceutically acceptable salt or solvate of said compound. The term "damage resulting from ischemia" as employed herein refers to conditions directly associated with reduced blood flow to tissue, for example due to a clot or obstruction of blood vessels which supply blood to the subject tissue and which result, inter alia, in lowered oxygen transport to such tissue, impaired tissue performance, tissue dysfunction and/or necrosis. Alternatively, where blood flow or organ perfusion may be quantitatively adequate, the oxygen carrying capacity of the blood or organ perfusion medium may be reduced, e.g., in hypoxic environment, such that oxygen supply to the tissue is lowered, and impaired tissue performance, tissue dysfunction, and/or tissue necrosis ensues.

By "pharmaceutically acceptable" it is meant that the carrier, diluent, excipients, salt, etc. in question must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The expression "pharmaceutically acceptable salt" refers to nontoxic anionic salts containing anions such as (but not limited to) chloride, bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate and 4-toluene-sulfonate. Where more than one basic moiety exists the expression includes multiple salts (e.g., di-salt). The expression also refers to nontoxic cationic salts such as (but not limited to) sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,N'-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N- benzylphenethylamine), piperazine or tromethamine (2-amino-2-hydroxymethyl-1 ,3- propanediol).

The expression "prodrug" refers to compounds that are drug precursors which following administration, release the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form).

By "alkylene" is meant saturated hydrocarbon (straight chain or branched) wherein a hydrogen atom is removed from each of the terminal carbons. Exemplary of such groups (assuming the designated length encompasses the particular example) are methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene). By "halo" is meant chloro, bromo, iodo, or fluoro.

By "alkyl" is meant straight chain saturated hydrocarbon or branched saturated hydrocarbon. Exemplary of such alkyl groups (assuming the designated length encompasses the particular example) are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, neopentyl, tertiary pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, hexyl, isohexyl, heptyl and octyl.

By "alkoxy" is meant straight chain saturated alkyl or branched saturated alkyl bonded through an oxygen. Exemplary of such alkoxy groups (assuming the designated length encompasses the particular example) are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, neopentoxy, tertiary pentoxy, hexoxy, isohexoxy, heptoxy and octoxy.

As used herein the term "mono-N-(C C_x)alkyl" or "di-N,N-(C C_x)alkyl..." refers to the (CrC_x)alkyl moieties, taken independently when it is the di-N,N-(C C_x)alkyl... (x is an integer).

It is to be understood that if a carbocyclic or heterocyclic moiety may be bonded or otherwise attached to a designated substrate through differing ring atoms without denoting a specific point of attachment, then all possible points are intended, whether through a carbon atom or, for example, a trivalent nitrogen atom. For example, the term "pyridyl" means 2-, 3-, or 4-pyridyl, the term "thienyl" means 2-, or 3-thienyl, and so forth.

As used herein, the expressions "reaction-inert solvent" and "inert solvent" refer to a solvent or mixture of solvents which does not interact with starting materials, reagents, intermediates or products in a manner which adversely affects the yield of the desired product. The chemist of ordinary skill will recognize that certain compounds of this invention will contain one or more atoms which may be in a particular stereochemical or geometric configuration, giving rise to stereoisomers and configu rational isomers. All such isomers and mixtures thereof are included in this invention. Hydrates of the compounds of this invention are also included.

"DMF" means N,N-dimethylformamide. "DMSO" means dimethyl sulfoxide. "THF" means tetrahydrofuran. Some of the compounds of this invention have asymmetric carbon atoms and therefore are enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known βer se, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers and mixtures thereof are considered as part of this invention. Also, some of the compounds of this invention are atropisomers (e.g., substituted biaryls) and are considered as part of this invention.

Those skilled in the art will recognize that the compounds of Formula I can exist in several tautomeric forms. All such tautomeric forms are considered as part of this invention. For example, all of the tautomeric forms of the carbonylguanidine moiety of the compounds of Formula I are included in this invention. Also, for example, all enol-keto forms of the compounds of Formula I are included in this invention.

Some of the compounds of this invention are acidic and they form a salt with a pharmaceutically acceptable cation. All of the NHE-1 inhibitor compounds of this invention are basic and they form a salt with a pharmaceutically acceptable anion. All such salts, including di-salts, are within the scope of this invention and they can be prepared by conventional methods. For example, they can be prepared simply by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered either by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate. In addition, when the compounds of this invention form metabolites, hydrates or solvates, they are also within the scope of the invention.

A preferred dosage of an NHE-1 inhibitor, such as an NHE-1 inhibitor of the Formula I compound, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, for treating a patient (e.g., for ischemic protection) is about 0.001 to 100 mg/kg/day. An especially preferred dosage of an NHE-1 inhibitor, such as compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, is about 0.01 to 50 mg/kg/day. A preferred dosage of an Ca²⁺ overload inhibitor, such as a Ca²⁺ inhibitor of Formula II a prodrug thereof, or a pharmaceutically acceptable salt or solvate of said compound or of said prodrug, for treating a patient (e.g., for ischemic protection) is about 0.001 to 100 mg/kg/day. A preferred dosage of an Ca²⁺ inhibitor, such as a compound of Formula II, or a pharmaceutically acceptable salt or solvate of said compound, when the compound is used orally, is within the range from 0.01 mg to 1000 mg (preferably from 0.1 mg to 100 mg) for human adult per day, but more preferably be increased or decreased suitably depending on the age, sex, condition, symptom, and presence of simultaneous treatment. When the Ca²⁺ overload inhibitor of the present invention is used as an injection agent, whether or not the NHE-1 inhibitor is so used, it should be preferably administered continuously or intermittently to a human adult in an amount of from about 0.01 mg to about 100 mg per dose.

The number of administrations may be once per day of either or both essential ingredients of the combination, in several divided doses per day with appropriate intervals of either or both essential ingredients of the combination, or using any other appropriate regimen.

Administration of the compounds of this invention can be via any method which delivers a compound of this invention preferentially to the desired tissue (e.g., liver and/or cardiac tissues). These methods include oral routes, parenteral (e.g., intravenous, intramuscular, subcutaneous or intramedullary), intraduodenal routes, etc. Topical administration may also be indicated, for example, where the patient is suffering from gastrointestinal disorders or whenever the medication is best applied to the surface of a tissue or organ as determined by the attending physician. Administration may be localized or systemic. The compounds of the present invention are usually administered in single (e.g., once daily) or multiple doses or via constant infusion. The amount and timing of compounds administered will, of course, be dependent on the subject being treated, on the severity of the affliction, on the manner of administration and on the judgment of the prescribing physician. Thus, because of patient to patient variability, the dosages set forth herein are a guideline and the physician may titrate doses of the drug to achieve the treatment that the physician considers appropriate for the patient. In considering the degree of treatment desired, the physician must balance a variety of factors such as age of the patient, presence of preexisting disease, as well as presence of other diseases (e.g., different cardiovascular diseases).

Thus, for example, in one mode of administration the compounds of this invention may be administered just prior to surgery (e.g., within twenty-four hours before surgery for example cardiac surgery) during or subsequent to surgery (e.g., within twenty-four hours after surgery) where there is risk of myocardial ischemia. The compounds of this invention may also be administered in a chronic daily mode. The compounds of this invention (i.e., the NHE-1 inhibitor/Ca²⁺ overload inhibitor combination) can be co-administered simultaneously or sequentially in any order or as a single pharmaceutical composition. The compounds of this invention are generally administered in the form of a pharmaceutical composition comprising at least one NHE-1 inhibitor and at least one Ca²⁺ overload inhibitor together with a pharmaceutically acceptable carrier, vehicle, or diluent. Thus, the compounds of this invention can be administered individually or together in any conventional oral, parenteral, rectal or transdermal dosage form.

For oral administration a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

For purposes of parenteral administration, solutions, for example, in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.

The combination of this invention may be administered with other drug therapies to mammals (e.g., humans, male or female) using, for example, conventional methods.

The NHE-1 inhibitor compounds of the present invention inhibit the sodium/proton (Na+/H+) exchange transport system and hence are useful as a therapeutic or prophylactic agent for diseases caused or aggravated by the acceleration of the sodium/proton (Na+/H+) exchange transport system. Similarly, as explained above, the Ca²⁺ overload inhibitor compounds of the present invention inhibit Ca²⁺ overload and hence are useful as a therapeutic or prophylactic agent for diseases caused or aggravated by the increase in Ca²⁺. Thus, the medicaments of the present invention that contain these compounds are useful in treating a variety of diseases, conditions, and syndromes, for example, arteriosclerosis, hypertension, arrhythmia (e.g., ischemic arrhythmia, arrhythmia due to myocardial infarction, myocardial stunning, myocardial dysfunction, arrhythmia after PTCA or after thrombolysis, etc.), angina pectoris, cardiac hypertrophy, myocardial infarction, heart failure (e.g., congestive heart failure, acute heart failure, cardiac hypertrophy, etc.), restenosis after PTCA, PTCI, shock (e.g., hemorrhagic shock, endotoxin shock, etc.), renal diseases (e.g., diabetes mellitus, diabetic nephropathy, ischemic acute renal failure, etc.), organ disorders associated with ischemia or ischemic reperfusion [(e.g., heart muscle ischemic reperfusion associated disorders, acute renal failure, or disorders induced by surgical treatment such as coronary artery bypass grafting (CABG) surgeries, vascular surgeries, organ transplantation, non-cardiac surgeries or percutaneous transluminal coronary angioplasty (PTCA)], cerebrovascular diseases (e.g., ischemic stroke, hemorrhagic stroke, etc.), and cerebroischemic disorders (e.g., disorders associated with cerebral infarction, disorders caused after cerebral apoplexy as sequelae, or cerebral edema), many of which diseases, conditions, and syndromes are within the definition of "cardiovascular disease" set forth above. The compounds of this invention can also be used as an agent for myocardial protection during coronary artery bypass grafting (CABG) surgeries, vascular surgeries, percutaneous transluminal coronary angioplasty (PTCA), PTCI, organ transplantation, or non- cardiac surgeries. In addition, the compounds of this invention are notable for their strong inhibitory effect on the proliferation of cells, for example the proliferation of fibroblast cells and the proliferation of the smooth muscle cells of the blood vessels. For this reason, the compounds of this invention are valuable treating or treatment (therapeutic) agents for use in diseases in which cell proliferation represents a primary or secondary cause and may, therefore, be used as antiatherosclerotic agents, and as agents against diabetic late complications, cancerous diseases, fibrotic diseases such as pulmonary fibrosis, hepatic fibrosis or renal fibrosis, glomerular nephrosclerosis, organ hypertrophies or hyperplasias, in particular hyperplasia or hypertrophy of the prostate, pulmonary fibrosis, diabetic complications or recurrent stricture after PTCA, or diseases caused by endothelial cell injury. Methods of preparing various pharmaceutical compositions with a certain amount of active ingredients are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples of methods of preparing pharmaceutical compositions, see Reminαton's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).

Pharmaceutical compositions according to the invention may contain for example 0.0001 %-95% of the compound(s) of this invention. In any event, the composition or medicament to be administered will contain a quantity of one or more compounds according to the invention in an amount effective to treat the disease, condition, or syndrome of the subject being treated.

Since the present invention has an aspect that relates to the treatment of diseases, conditions, and syndromes with a combination of active ingredients which may be administered separately, the invention also relates to combining separate pharmaceutical compositions in kit form. The kit may comprise two separate pharmaceutical compositions: a compound of Formula I, a prodrug thereof, or a pharmaceutically acceptable salt or solvate of such compound or prodrug, and a second compound of Formula II or a pharmaceutically acceptable salt or solvate thereof. The kit may comprise means for containing the separate compositions such as a container, a divided bottle or a divided foil packet. Typically the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested. Another example of such a memory aid is a calendar printed on the card, e.g., as follows "First Week, Monday, Tuesday, ..., etc. Second Week, Monday, Tuesday, ... etc." Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several pills or capsules to be taken on a given day. Also, a daily dose of a combination of a Formula I compound and a Formula II compound can consist of one or more tablets, capsules, or other dosage forms, a daily dose of the first compound can consist of one or more tablets, capsules, or other dosage forms, and a daily dose of the second compound can consist of one or more tablets, capsules, or other dosage forms. The memory aid should reflect this.

In another specific embodiment of the invention, a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided. Preferably, the dispenser is equipped with a memory aid, so as to further facilitate compliance with the regimen. An example of such a memory aid is a mechanical counter which indicates the number of daily doses that has been dispensed. Another example of such a memory aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

The compounds of this invention generally will be administered in a convenient formulation. The following formulation examples are illustrative only and are not intended to limit the scope of the present invention.

In the formulations which follow, "active ingredients" means a combination of the compounds of this invention.

Hard gelatin capsules are prepared using the following: Formulation 1 : Gelatin Capsules Ingredients Quantity (mg/capsule)

Active ingredients 0.25-100

Starch, NF 0-650

Starch flowable powder 0-50

Silicone fluid 350 centistokes 0-15

A tablet formulation is prepared using the ingredients below: Formulation 2: Tablets

Ingredients Quantity (mg/tablet)

Active ingredients 0.25-100

Cellulose, microcrystalline 200-650

Silicon dioxide, fumed 10-650

Stearate acid 5-15

The components are blended and compressed to form tablets. Alternatively, tablets each containing 0.25-100 mg of active ingredients are made up as follows:

Formulation 3: Tablets Ingredients Quantity (mg/tablet)

Active ingredients 0.25-100

Starch 45

Cellulose, microcrystalline 35

Polyvinylpyrrolidone (as 10% solution in water) 4

Sodium carboxymethyl cellulose 4.5

Magnesium stearate 0.5

Talc 1

The active ingredients, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50° - 60°C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.25-100 mg of active ingredients per 5 ml dose are made as follows:

Formulation 4: Suspensions Ingredients Quantity (mg/5 ml)

Active ingredients 0.25-100 mg .

Sodium carboxymethyl cellulose 50 mg

Syrup 1.25 mg

Benzoic acid solution 0.10 mL

Flavor q.v.

Color q.v.

Purified Water to 5 mL

The active ingredients are passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.

An aerosol solution is prepared containing the following ingredients: Formulation 5: Aerosol

Ingredients Quantity (% by weight)

Active ingredients 0.25

Ethanol 25.75

Propellant 22 (Chlorodifluoromethane) 74.00

The active ingredients are mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to 30°C, and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remaining propellant. The valve units are then fitted to the container.

Suppositories are prepared as follows:

Formulation 6: Suppositories Ingredients Quantity (mg/suppository)

Active ingredients 250

Saturated fatty acid glycerides 2,000

The active ingredients are passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimal necessary heat. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool. An intravenous formulation is prepared as follows:

Formulation 7: Intravenous Solution Ingredients Quantity

Active ingredients 25 mg-10,000 mg

Isotonic saline 1 ,000 mL

The solution of the above ingredients is intravenously administered to a patient. The active ingredients above may also be a combination of agents. DETAILED DESCRIPTION OF THE

DRAWING FIGURES AND EFFICACY ANALYSES

The following examples demonstrate and can be used to demonstrate the superior and unexpected cardioprotective efficacy of a combination of a Ca²⁺ overload inhibitor (i.e.,

MCC-135) and an NHE-1 inhibitor compared to the separate efficacies of the NHE-1 inhibitor and the Ca²⁺ overload inhibitor. The NHE-1 inhibitor utilized in the comparative example efficacy analysis immediately below is CP-566,827, and it is represented by the formula:

The following examples demonstrates that NHE-1/Ca²⁺ overload inhibitor combination therapy is a clinically viable approach for providing greater cardioprotection compared to that afforded by either agent individually and not in combination. 1. Comparative Example

An in vitro model of ischemia/reperfusion injury was conducted by exposing isolated rabbit hearts to 30 minutes of regional ischemia, followed by 120 minute of reperfusion, at which point infarct size was determined (control). MCC-135 and CP-566,827 were individually evaluated under these conditions and found to elicit similar reductions in infarct size of approximately 80%.

Male New Zealand White rabbits (3-4 kg) were anesthetized by i.v. administration of sodium pentobarbital (30 mg/kg; marginal ear vein), followed by intubation and ventilation with 100% O₂ using a positive pressure ventilator. A left thoracotomy was performed, the heart exposed, and a snare (2-0 silk) placed loosely around a branch of the left coronary artery. The heart was rapidly removed from the chest, mounted on a Langendorff apparatus, and maintained by retrograde perfusion (non-recirculating) with a modified Krebs solution (NaCI 118.5 mM, KCI 4.7 mM, Mg S0₄ 1.2 mM, KH₂P0₄ 1.2 mM, NaHCO₃ 24.8 mM, CaCI₂ 2.5 mM, and glucose 10 mM) at a constant pressure of 80 mmHg and a temperature of 37°C. Perfusate pH was maintained at 7.4 -7.5 by bubbling with 95% O₂/5% CO₂. The temperature of the hearts was maintained by suspending them in heated, water jacketed organ baths. A fluid-filled latex balloon was inserted in the left ventricle and connected by stainless steel tubing to a pressure transducer; the balloon was inflated to provide a systolic pressure of 80-120 mmHg, and a diastolic pressure ≤ 10 mmHg. Heart rate and left ventricular diastolic and systolic pressures were recorded using a PO-NE-MAH Data Acquisition and Archive System; left ventricular developed pressures (LVDP) were calculated by subtracting the left ventricular diastolic pressure from the left ventricular systolic pressure. Total coronary flow rates (CF) were determined using an in-line flow probe; all coronary flows were normalized for heart weight. These parameters were continuously monitored for the duration of the experiment. Hearts were allowed to equilibrate for 30 min.; if stable left ventricular pressures within the parameters outlined above were not observed, the heart was discarded. Hearts were not paced, unless the heart rate fell below 180 bpm prior to the 30 min. period of regional ischemia; in this case, the heart was paced at 200 bpm, which was the average spontaneous rate observed.

Hearts were subjected to either thirty or sixty minutes of regional ischemia (produced by tightening the snare around the coronary artery) followed by 120 min. of reperfusion. Regional ischemia was confirmed in all groups by a significant drop in CF and LVDP (see Table II) during occlusion of the coronary artery. CP-566,827, or MCC-135 (dissolved in DMSO, final concentration 0.1%) either alone, or in combination, were constantly perfused through each heart beginning 30 min. prior to the regional ischemia, and continuing until the end of the experiment. At the end of the 120 min. reperfusion period, the coronary artery snare was tightened and a 0.5% suspension of fluorescent zinc cadmium sulfate particles (1 -10 μm) was perfused through the heart to delineate the left ventricular area-at-risk (no crystals present) for infarct development. The heart was removed from the Langendorff apparatus, blotted dry, weighed, wrapped in aluminum foil and stored overnight at -20°C. Frozen hearts were sliced into 2 mm transverse sections and incubated with 1% triphenyl tetrazolium chloride in phosphate-buffered saline for 20 min. at 37°C to delineate non-infarcted (stained) from infarcted (non-stained) left ventricular tissue. The infarct area and the area-at-risk were calculated for each slice of left ventricle using a precalibrated image analyzer, followed by adding the values for each tissue slice to obtain the total infarct area and total area-at-risk for each heart. To normalize the infarct area for differences in the area-at-risk between hearts, the infarct size was expressed as the ratio of infarct area vs. area-at-risk (% IA/AAR). The area-at-risk as a percent of left ventricular area (% AAR/LV) was not significantly different (P > 0.05) between groups.

As illustrated in Figure 1 , employing the 30 minute/120 minute ischemia/reperfusion protocol, the maximum reduction in infarct size with the administration of MCC-135 was obtained at an amount of between about 1x10^"7M and 1x10^"6M, where infarct size plateaued at about 10%.

As illustrated in Figure 2, the maximum reduction in infarct size that could be obtained in the 30 minute/120 minute ischemia/reperfusion protocol by administration of either CP- 566,827 or MCC-135 was a reduction to about 10%. Thus, based upon these data, the maximum reduction in infarct size achievable in this model is a % IA/AAR of about 10%, and it would not be expected that infarct size could be reduced any further by the separate administration of the NHE-1 inhibitor or the Ca²⁺ overload inhibitor regardless of the amount(s) administered. Following that understanding, the ischemic insult to the hearts was increased by employing a novel 60 minute/120 minute ischemia/reperfusion protocol as further illustrated in Figure 2, it was observed that the maximum reduction in infarct size produced by the separate administration of the NHE-1 inhibitor or the Ca²⁺ overload inhibitor could not exceed the 45% - 52% size range. Given this observation, and the knowledge that both of these compounds ultimately act via a similar mechanism, i.e., lowering intracellular Ca²⁺ concentrations in the cardiomyocyte, it was not to be expected that infarct size could be decreased any further than the 45% - 52% size range by the combined administration of the NHE-1 inhibitor and/or the Ca²⁺ overload inhibitor regardless of the amount(s) administered.

As illustrated in Figure 3, however, the combined effect of administration of the NHE- 1 inhibitor with the Ca²⁺ overload inhibitor dramatically reduced infarct size far beyond that which was obtained by the separate administration of those inhibitors. TABLE II. Hemodynamic data from isolated rabbit hearts.

Pre-φφ sipn Post-60 min occlusion

HR CF LVDP HR CF LVDP

Group n

(beats-min^*1) (mlmin^'V') (mmHg) (beats-mitf¹) (mhnitf'-g"¹) (mmHg)

Control 5 215 ± 15 6.9 ± 0.3 105 ± 6 193 ± 13 3.9 ± 02* 56 ±4*

CP-566.827.50 nM 5 225 ± 11 7.3 ± 0.7 99 ±3 205 ± 10 4.9 ± 0.7* 53 ± 5*

MCC-135, 1 μM 6 222 ±7 5.8 ± 0.3 101 ± 4 191 ± 8 3.5 ± 0.4* 54 ± 6*

MCC-135 + 9 206 ± 6 5.8 ± 0.3 97 ± 3 197 ± 6 3.5 ± 0.2* 51 ± 4*

CP-566.827

HR: heart rate; CF: total coronary flow; LVDP: left ventricular developed pressure; P < 0.05 vs. pre- occlusion values.

2. Efficacy Example A

Male New Zealand White rabbits (3-4 kg) are anesthetized with sodium pentobarbital (30 mg/kg, i.v.), the heart removed, and isolated cardiomyocytes prepared in accordance with the procedure disclosed in Ladilov et al., Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na⁺/H⁺ exchange, Am. J. Physiol. (Heart Circ. Physiol.) 268: H1531-39 (1995). Glass coverslips are preincubated overnight with 4% fetal calf serum. Ventricular muscle cells are isolated and plated on the glass cover slips in medium 199 with 4% fetal calf serum. Four hours after the plating, the coverslips are washed twice with medium 199. As a result of the medium change, broken cells are removed, leaving a homogeneous population of rod-shaped cells attached to the coverslip.

Determination of fura 2, sodium-binding benzofuran isophthalate, and 2',7'-bis(2- carboxyethyl)-5,6-carboxyfluorescein fluorescence and cell shortening. For measurement of cytosolic Ca²⁺, Na⁺, or H⁺ concentrations, the cardiomyocytes are loaded at 37°C with fura 2, sodium-binding benzofuran isophthalate (SBFI), or 2',7'-bis(2-carboxyethyl)-5,6- carboxyfluorescein (BCECF), respectively. For loading, the cells, attached to glass coverslips, are incubated in medium 199 for 15 min with the acetoxymethylesters of fura 2 (5 μM fura 2-AM), for 30 min with 10 μM SBFI, or for 30 min with 3 μM BCECF. After the loading, the cells are washed twice with medium 199 containing 1% (wt/vol) bovine serum albumin. This washing is followed by a 30-min postincubation period, to allow hydrolysis of the acetoxymethylesters within the cells. These loading protocols are selected from a large number of variations because they provided the highest yield in fluorescence and minimal dye compartmentation. To assess the extent of intracellular dye compartmentation, the cells are chemically "skinned" by use of digitonin. The fluorescence retained in skinned cells is <15% of previous fluorescence for fura 2, <5% for SBFI, and <12% for BCECF (after subtraction of background fluorescence of the cell before dye exposure). These percentages indicate only a small extent of dye compartmentation.

The coverslip with the loaded cells is then introduced into an airtight, temperature- controlled (37°C), transparent perfusion chamber positioned in the light beam of an inverted microscope (Diaphot TMD, Nikon, Dϋsseldorf, Germany). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura 2 and SBFI and 450 to 490 nm for BCECF are performed with the use of the AR-CM Cation Measurement System (Spex Industries, Grasbrunn, Germany) adapted to the microscope. The emitted light (490-520 nm for fura 2 and SBFI and 520-560 nm for BCECF) from a 10 x 10 μm area within a single fluorescent cell is collected by a photomultiplier of the Spex system. The signal is recorded and analyzed with the use of an IBM PC/AT-based data analyzing system (model DM3000CM, Spex Industries). Autofluorescence intensity is measured before loading a cell with the fluorescent dyes and subtracted from the fluorescence signals.

In the analysis of the fura 2 signal in reoxygenated cardiomyocytes, the "diastolic" fura 2 signal, defined as the fura 2 signal when not oscillating, the curve of minima when the signal is oscillating, and the frequency of oscillations of the fura 2 signal at given times are determined.

Because of the inherent problems with all calibration protocols, data are generally expressed in arbitrary units of fluorescence ratios. To facilitate understanding, calibration protocols are performed to obtain numerical relations between selected ratio values and ion concentrations. Fura 2 signals are calibrated by exposing the cells to 5 μM ionomycin in modified Tyrode solution containing either 3 mM Ca²⁺ or 5 mM ethylene glycol-bis(β- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to obtain the maximum (R_max) and the minimum (Rn,i_n) ratio of fluorescence, respectively. The free cytosolic Ca²⁺ concentration ([Ca ⁺]₁) is calculated according to the equation [Ca^2"1"]_! = K_d x β x (R - R_min)/(Rm_ax - R)_> with the use of K_d, the dissociation constant of fura 2, and β, the ratio of the 380-nm excitation signals of ionomycin-treated cells at 5 mM EGTA and at 3 mM Ca²⁺. SBFI-ratio signals are calibrated in loaded cells with 6 μM gramicidin D, a Na⁺ ionophore, and incubation media with various Na⁺ concentrations. Calibration of the BCECF ratio signals is performed, with 10 μg/ml nigericin, a K⁺/H⁺ ionophore, and incubation media with various pH values.

The cell's microscopic picture is recorded at the time of measurement of the fluorescence signal with the aid of a video camera and is stored on tape. The changes in the cell's length are determined from this recording. In the case of hypercontracted cells, the cell diameter is determined along with its previous longitudinal axis.

Media. The perfusion chamber on the microscopic stage (1 ml filling volume) is perfused at a rate of 0.5 ml/min with modified, glucose-free Tyrode solution containing (in mM) 140.0 NaCI, 2.6 KCI, 1.2 KH₂PO₄, 1.2 MgS0₄, 1.0 CaCI₂, and 25.0 N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH is 7.4 at 37°C. Media are made anoxic by autoclaving and by equilibrating before and during use with 100% N₂. Oxygenated media are equilibrated with air.

Incubation protocols. In the standard experiment for Ca²⁺ measurements, the anoxic cardiomyocyte is allowed to accumulate Ca²⁺ until the saturation of the fura 2 ratio is reached and then is reoxygenated. During the anoxic incubation, the cells undergo rigor shortening. The time to rigor shortening varies among individual cells, but the changes after rigor shortening follow a reproducible time course. The time between rigor shortening and saturation of the fura 2 signal differs between anoxic media of pH 7.4 and 6.4. Different times for reoxygenation are therefore chosen for these different anoxic protocols. 1 ) In anoxic medium with pH 7.4, saturation of the fura 2 signal is obtained < 30 min after the onset of rigor shortening of the cells. Under these conditions, cells are therefore reoxygenated 30 min after rigor shortening. 2) In media with pH 6.4, it takes about 50 min to reach saturation of the fura 2 signal (n = 20 cells). Consequently, reoxygenation is started about 50 min after rigor. Incubation protocols are the same for cells loaded with fura 2, SBFI, or BCECF. Additions of zoniporide, MCC-135, or both compounds in combination are done simultaneously with reoxygenation. 3. Efficacy Example B

In accordance with the procedure disclosed in Armstrong et al., Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A₃ receptor involvement, Cardiovascular Research (1994:28:1049-1056), isolated, calcium tolerant, adult rabbit cardiomyocytes are prepared by collagenase perfusion. Each isolate provides about 12 million calcium tolerant myocytes. For the experimental studies, cells are resuspended in a Kreb-Henseleit (KH) buffer, containing (in mmol-litre^"1); CaCI₂ 1.25, NaCI 115, KCL 5, NaHC0₃, 25, KH₂P0₄ 1.2, MgCI₂ 1.2, glucose 11 , creatine 20, taurine 60, and 1 mg-ml^"1 bovine serum albumin (BSA, Pentex Fraction V). There are four experimental groups which use the majority of the isolate to provide sufficient cells for pelleting. The four control groups use fewer cells, as these groups are not to be pelleted. Incubations are at 30°C. A separate isolate is used for each experiment, and each series consists of five experiments. Ischemia model

At the end of each experimental protocol, cells are resuspended into fresh KH buffer with glucose, placed in a 1.8 ml microcentrifuge tube, and are centrifuged into a pellet. Each cell pellet occupies a volume of about 0.25 ml, and measures 0.8-1 cm in thickness. Excess supernatant is removed to leave a fluid layer above the pelleted cells of about one third the volume of the pellet. After layering with mineral oil to exclude gaseous exchange, the cell pellets are incubated without agitation at 37°C for up to 4 hours. A 25 μl sample of the cell pellet is removed through the oil layer at the appropriate time points and is resuspended for 3- 5 min at 30°C, in 200 μl of hypotonic (85 mOsm) buffer, consisting of KH buffer diluted to the appropriate osmolarity. A 25 μl sample is mixed on a microscope slide with an equal volume of 85 mOsm counting media (0.5% glutaraldehye in modified Tyrode solution, with reduced NaCI, containing 1% trypan blue and 3mM amytal) for 3-5 min at room temperature. Microscopic examination at 100X magnification determines the morphology (rod, round, or square) and the permeability of the cells to trypan blue. With ischemia the rods convert almost entirely to squares with little rounding. Experimental design Each isolation provides sufficient cells for four experimental groups and four oxygenated control groups. Each experimental protocol usually contains two internal control groups. A negative (untreated) control group is preincubated for 5 min at 30°C with 11 mM glucose, with oxygenation in 1.5 ml of 1.25 mM calcium containing KH buffer. The positive control (preconditioned) is similarly incubated for 5 min but in the absence of glucose. The two experimental groups are suspended in glucose containing or glucose-free media, according to the particular protocol to be followed. Following the preincubation periods, all groups are subjected to a post incubation period in glucose containing medium lasting in most experiments for 30 min prior to ischemic pelleting. Experimental groups are treated during the 30 min post incubation period with either zoniporide, MCC-135, or both compounds in combination; the compound(s) remain present during the 4 hours of ischemic pelleting. Four parallel oxygenated control groups are subjected to the same pre- and postincubation protocols as the experimental groups, but are then resuspended into oxygenated, glucose containing media for 240 min at 37°C, in place of ischemic pelleting. All groups, including oxygenated controls, are swollen prior to determination of viability.

PREPARATIVE EXAMPLES Reference to the hydrochloride salt in the Example names below includes mono-or di- salts as appropriate in the particular Example.

A. Preparation Of NHE-1 Inhibitors The NHE-1 inhibitor compounds of the present invention can be prepared in accordance with the teachings of Hamanaka et al., U.S. Patent No. 6,492,401 , and Brostrom et al., International Application Publication No. WO 2001/30759, both of which documents as previously noted are incorporated by reference herein in their entirety for all purposes. The following examples illustrate the preparation of preferred NHE-1 inhibitors.

EXAMPLE 1 r5-Cvclopropyl-1 -(quinolin-5-yl)-1 /- -pyrazole-4-carbonvnquanidine dihydrochloride

A mixture of 5-cyclopropyl-1-(quinolin-5-yl)-1 r/-pyrazole-4-carboxylic acid (4.08 g,

14.6 mmol) and 25 ml SOCI₂ was heated to reflux for 1 hour. The excess SOCI₂ was removed in vacuo via codistillation with toluene. The solid residue was added portionwise over 45 minutes to a vigorously stirred 40 °C solution of guanidine hydrochloride (5.02 g, 52.6 mmol) in 59 ml of 2 N NaOH and 29 ml THF. The resulting mixture was heated at reflux for 1 hour and then cooled to 23 °C. The organic solvent and 40 ml of the H₂0 were removed in vacuo. The tan solid that precipitated was filtered and washed with 2X5 ml portions of cold H₂0. This solid was air-dried for 1 hour and then dried for 24 h under high vacuum at 40 °C to afford 3.5 g of the free base of the title compound. This solid was dissolved in 25 ml of hot methanol and treated with 1.85 ml of cone. HCI. This pale yellow solution was stirred for 15 min at room temperature and concentrated in vacuo to a light amber gum. The residual H₂0 was removed in vacuo via codistillation with 3x25 ml portions of anhydrous ethanol. The resulting pale yellow solid was recrystallized from hot ethanol to afford 3.58 g of the title compound (62% yield).

APCIMS 319 [M-1]^"

¹H NMR (DMSO-cfe) 5 9.16 (m, 1 H), 8.86 (s, 1 H), 8.85 (bs, 2H), 8.50 (bs, 2H), 8.37 (d, J=8.4, 1 H), 8.08-7.97 (m, 3H), 7.78 (dd, J=4.4, 8.4, 1 H), 1.99-1.93 (m, 1 H), 0.64-0.62 (m, 2H), 0.42 (m, 2H).

EXAMPLE 2 ri-(Quinolin-5-yl)-5-cvclopropyl-1 H-pyrazole-4-carbonyllquanidine hydrochloride monohydrate

A solution of guanidine hydrochloride (3.11 g, 32.6 mmol) in warm anhydrous ethanol (8 mL) under a nitrogen atmosphere was treated in one portion with sodium methoxide (1.76 g, 32.6 mmol). The resulting slurry was concentrated in vacuo. The residue was treated with anhydrous toluene (10 mL) and concentrated to dryness in vacuo (twice). Each time the vacuum was released to a nitrogen atmosphere. The residue was treated in one portion with ethyl 1 -(quinolin-5-yl)-5-cyclopropyl-1 /- -pyrazole-4-carboxylate (1.00 g, 3.26 mmol) in anhydrous ethanol (8 mL). The resulting mixture was concentrated in vacuo (rotary evaporator, 80 °C water bath). The residue was treated with anhydrous toluene (10 mL) and the resulting mixture was concentrated in vacuo (three times). The resulting solid was triturated with water (85 mL) and filtered. The solid was air-dried to provide 0.880 g (76% yield) of [1-(quinolin-5-yl)-5-cyclopropyl-1 H-pyrazole-4-carbonyl]guanidine dihydrate. APCIMS 321 [M+1]⁺

¹H NMR (400 MHz, DMSO-cfe) δ 0.51 -0.53 (m, 4H), 1.88-1.95 (m, 1 H), 7.52-7.60 (m, 2H), 7.73 (d, J=8, 1 H), 7.86 (t, J=9, 1 H), 7.94 (s, 1 H), 8.16 (d, J=9, 1 H), 8.95 (t, J=1.8, 1 H).

A suspension of [1-(quinolin-5-yl)-5-cyclopropyl-1 H-pyrazole-4-carbonyl]guanidine dihydrate (1.28 g, 3.59 mmol) in tetrahydrofuran (38.4 mL) with vigorous stirring was treated with concentrated hydrochloric acid (0.30 mL, 3.6 mmol). The mixture became homogeneous within one minute and then a solid began to precipitate. The resulting mixture was stirred vigorously for 1 h and filtered. The solid was air-dried to provide 1.11 g (82% yield) of the title compound.

APCIMS 321 [M+1]⁺

¹H NMR (400 MHz, DMSO-d₆) δ 0.42 (m, 2H), 0.59-0.61 (m, 2H), 1.88-1.95 (m, 1 H), 7.57 (dd, J=9, 4, 1 H), 7.67 (d, J=4, 1 H), 7.82 (d, J=7, 1 H), 7.90 (t, J=8, 1 H), 8.22 (d, J=8, 1 H), 8.38 (bs, 2H), 8.69 (bs, 2H), 8.72 (s, 1 H), 8.98 (dd, J=4, 1.4, 1 H).

EXAMPLE 3 4-lsopropyl-3-methylsulfonylbenzoylquanidine methanesulfonate Synthesis route for 4-lsopropyl-3-methylsulfonylbenzoylguanidine methanesulfonate: a) 4-lsopropyl-3-chlorosulfonylbenzoic acid (m.p. 203-204 C^°) by heating 4- isopropylbenzoic acid with chlorosulfonic acid at 95 C° for 3 hours; b) 2-lsopropyl-5-carboxybenzenesulfinic acid (m.p. 205-207C^") from a) by reduction with sodium sulfite at 60° C. in aqueous NaOH, at pH 9-10; c) 4-lsopropyl-3-methylsulfonylbenzoic acid (m.p.: 209 -211 C°) from b) by alkylation with methyl bromide in the presence of NaOH in DMF at 60 C^° for 3 hours; d) 4-lsopropyl-3-methylsulfonylbenzoylguanidine methanesulfonate from c) by reaction with thionyl chloride in toluene (reflux) for 1 hour.

After evaporating the solvent, the residue is dissolved in THF and the solution is added to a mixture of guanidine hydrochloride, 2N NaOH and THF. After 4 hours at 30-40 ^°C, the THF is removed by distillation and the crystalline 4-isopropyl-3- methylsulfonylbenzoylguanidine (m.p.: 226°-228 ^°C) is filtered off. Reaction with methanesulfonic acid follows to form the salt.

B. Preparation Of Ca²⁺Overload Inhibitors EXAMPLE 4 Preparation of 2-(1 -Piperazinyl)-5-Methylbenzenesulfonic Acid Derivative Monohydrate

According to the method described in Example 1 of Japanese Patent Application Laid-Open No. 3-7263, 2-fluoro-5-methylbenzene sulfonic acid (0.76 g) was reacted with piperazine (3.44 g) in the co-presence of cuprous iodide (0.76 g) and copper powder (0.26 g) in a sealed tube at 160 ^°C. for 8 hours. Then, the reaction product was purified on silica gel column chromatography (eluent=chloroform: methanol: acetic acid=100: 100: 3) to give anhydrous crystal of 2-(1-piperazinyl)-5-methylbenzenesulfonic acid derivative (0.67 g, yield=65.0%).

The anhydrous crystal of 2-(1-piperazinyl)-5-methylbenzenesulfonic acid derivative (0.4506 g) and distilled water (1.35 ml) were added to a 5 ml flask, and stirred at 5 °C. for 2 hours. Crystals were recovered from the suspension by suction filtration and then the residual crystals remaining in the flask were recovered by washing with filtrate. The crystals were combined and dried at 50 ^°C, 90 mmHg for 3 hours to give white product, 2-(1 - piperazinyl)-5-methylbenzenesulfonic acid derivative monohydrate (0.4485 g, yield 93.0%). This compound was verified as the monohydrate from the following results of elemental analysis.

Anal.: Theoretical value (monohydrate crystal): C: 48.16, H: 6.62, N: 10.21 , S:11.69; Found: C: 48.16, H: 6.55, N: 10.09, S:11.87; (Reference): Theoretical value (anhydrate crystal): C: 51.54, H: 6.29, N: 10.93. S:12.51.

EXAMPLE 5

A solution of 31.4 g of p-n-propylaniline dissolved in 116 ml of acetic acid and 232 ml of water was added dropwise into 200 ml of a mixed solution comprising 130 g of sodium isocyanate and 900 ml of water, and then stirred in an ice-bath for 30 minutes. The crystals precipitated were filtered, washed with water and dried to give 38.88 g of 4-n- propylphenylurea. After the 4-n-propylphenylurea was added in portions into 107.6 ml of 20% fuming sulfuric acid, the reaction was carried out at 60 °C. for 2 hours. Under cooling in an ice-bath, about 400 ml of ice was added into the reaction mixture, followed by heating under reflux for 4 hours. The crystals precipitated by cooling were filtered, washed with water and then dried to give 30.09 g of the above title product (yield: 64.1%). Melting point: 261.7°-

262.3 ^°C.

To 500 ml of water are added 50.0 g of 2-amino-5-n-propylbenzenesulfonic acid and

6.28 g of sodium carbonate and the mixture was dissolved by heating. To the solution was added 88.13 g of bis(2-chloroethyl)amine hydrochloride and the mixture was refluxed by heating for 3 hours, and then, a suspension containing 26.25 g of sodium carbonate suspended in 63 ml of water was added thereto, and the mixture was refluxed by heating for 12 hours. The reaction mixture was condensed under reduced pressure, extracted with methyl alcohol and then methyl alcohol was removed under reduced pressure. To the residue was added 200 ml of water, and after a pH of the mixture was adjusted to about 8.0 with sodium carbonate, 500 ml of chloroform was added thereto and the mixture was stirred. Precipitated crystals were collected by filtration, washed with chloroform and dried to give 44.83 g (yield: 67.9%) of the above title product.

The invention has been described in an illustrative manner and the terminology that has been used is intended to be in the nature of description. Modifications and variations that can be made should be apparent in light of the teachings herein. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described and that the claims are intended to cover all modifications and variations falling within the true spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A medicament comprising an NHE-1 inhibitor and a Ca²⁺ overload inhibitor.

2. A medicament according to claim 1 wherein the NHE-1 inhibitor is a compound of Formula I

Formula I a prodrug thereof or a pharmaceutically acceptable salt of said compound or of said prodrug, wherein Z is a carbon of (a) a five-membered, diaza, diunsaturated ring having two contiguous nitrogens, said ring optionally mono-, di-, or tri-substituted with up to three substituents independently selected from R¹, R² and R³, or of (b) a five-membered, triaza, diunsaturated ring, said ring optionally mono- or di-substituted with up to two substituents independently selected from R⁴ and R⁵; wherein R¹, R², R³ , R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, hydroxy(CrC₄)alkyl, (C C₄)alkyl, (C C₄)alkylthio, (C₃-C₄)cycloalkyl, (C₃-C₇)cycloalkyl(C₁-C )alkyl, (C C₄)alkoxy, (C₁-C₄)alkoxy(C₁-C₄)alkyl, mono-N-(C C₄)alkylcarbamoyl or di-N,N-(C₁-C₄)alkylcarbamoyl, M or M(C C )alkyl, wherein any of said R¹, R², R³ , R⁴ and R⁵ (C₁-C₄)alkyl moieties optionally have from one to nine fluorines; wherein said R¹, R², R³ , R⁴ and R⁵ (d-C₄)alkyl moieties or said R¹, R², R³, R⁴ and R⁵ (C₃- C₄)cycloalkyl moieties are optionally independently substituted with one or two substituents selected from the group consisting of: hydroxy, (C C )alkoxy, (C_rC₄)alkylthio, (C C₄)alkylsulfinyl, (C C₄)alkylsulfonyl, (C C₄)alkyl, mono-N-(C₁-C )alkylcarbamoyl, di-N,N-(Cr C₄)alkylcarbamoyl, mono-N-(C₁-C₄)alkylaminosulfonyl, and di-N,N-(C₁-C₄)alkylaminosulfonyl, wherein said (C₃-C₄)cycloalkyl optionally contains from one to seven fluorines; wherein M is a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen, or, a bicyclic ring consisting of two fused partially saturated, fully saturated or fully unsaturated three to six membered rings, taken independently, optionally having one to four heteroatoms selected independently from nitrogen, sulfur and oxygen; said M is optionally substituted, on one ring if the moiety is monocyclic, or one or both rings if the moiety is bicyclic, on carbon or nitrogen with up to three substituents independently selected from R⁶, R⁷ and R⁸, wherein one of R⁶, R⁷ and R⁸ is optionally a partially saturated, fully saturated, or fully unsaturated three to seven membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen optionally substituted with (C_rC₄)alkyl and additionally R⁶, R⁷ and R⁸ are optionally hydroxy, nitro, halo, (C C₄)alkoxy, (CrC₄)alkoxycarbonyl, (C C₄)alkyl, formyl, (C C₄)alkanoyl, (Cι-C₄)alkanoyloxy, (d-C^alkanoylamino, (CrC₄)alkoxycarbonylamino, sulfonamido, (C C^alkylsulfonamido, amino, mono-N-(C C₄)alkylamino, di-N,N-(C C₄)alkylamino, carbamoyl, mono-N-(CrC₄)alkylcarbamoyl, di-N,N-(C₁-C₄)alkylcarbamoyl, cyano, thiol, (C C₄)alkylthio, (CrC₄)alkylsulfinyl, (CrC₄)alkylsulfonyl, mono-N-(C C₄)alkylaminosulfonyl, di-N,N-(CrC₄)alkylaminosulfonyl, (C₂-C )alkenyl, (C₂-C )alkynyl or (C₅- C₇)cycloalkenyl, wherein said (Cι-C )alkoxy, (CrC )alkyl, (C C₇)alkanoyl, (C C₄)alkylthio, mono-N- or di-N,N-(CrC₄)alkylamino or (C₃-C₇)cycloalkyl R⁶, R⁷ and R⁸ substituents are optionally mono- substituted independently with hydroxy, (CrC₄)alkoxycarbonyl, (C₃-C₇)cycloalkyl, (C C₄)alkanoyl, (Cι-C₄)alkanoylamino, (C C₄)alkanoyloxy, (Cι-C₄)alkoxycarbonylamino, sulfonamido, (Cι-C₄)alkylsulfonamido, amino, mono-N- or di-N,N-(C₁-C₄)alkylamino, carbamoyl, mono-N- or di-N,N-(CrC₄)alkylcarbamoyl, cyano, thiol, nitro, (C-|-C )alkylthio, (C C₄)alkylsulfinyl, (C C₄)alkylsulfonyl or mono-N- or di-N,N-(C₁-C₄)alkylaminosulfonyl or optionally substituted with one to nine fluorines.

3. A medicament according to claim 1 wherein the Ca²⁺ overload inhibitor is a compound of Formula II

Formula II wherein R⁹ is selected from the group consisting of a hydrogen atom, a C C₆ alkyl group, a C₃-C₇ cycloalkyl group, a C C₄ halogenated alkyl group, a halogen atom, and a C₆-Cι₂ aryl group, and wherein R¹⁰ is selected from the group consisting of a hydrogen atom, a C C₆ alkyl group, and a C₇-C₁₂ aralkyl group optionally substituted with at least one substituent selected from the group consisting of a cyano group, a nitro group, a C|-C₆ alkoxy group, a halogen atom, a C C₆ alkyl group and an amino group, and wherein n represents an integer of 1 to 4, or a pharmaceutically acceptable salt or solvate thereof.

4. A medicament according to claim 1 wherein the NHE-1 inhibitor is a compound of Formula I

Formula I a prodrug thereof or a pharmaceutically acceptable salt of said compound or of said prodrug, wherein

Z is a carbon of (a) a five-membered, diaza, diunsaturated ring having two contiguous nitrogens, said ring optionally mono-, di-, or tri-substituted with up to three substituents independently selected from R¹, R² and R³, or of (b) a five-membered, triaza, diunsaturated ring, said ring optionally mono- or di-substituted with up to two substituents independently selected from R⁴ and R⁵; wherein R¹, R², R³ , R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, hydroxy(C C )alkyl, (Cι-C₄)alkyl, (C C₄)alkylthio, (C₃-C₄)cycloalkyl, (C₃-C₇)cycloalkyl(C C₄)alkyl, (C C₄)alkoxy, (C C₄)alkoxy(C₁-C₄)alkyl, mono-N-(C C₄)alkylcarbamoyl or di-N,N-(Cι-C₄)alkylcarbamoyl, M or M(CrC₄)alkyl, wherein any of said R¹, R², R³ , R⁴ and R⁵ (CrC₄)alkyl moieties optionally have from one to nine fluorines; wherein said R¹, R², R³ , R⁴ and R⁵ (C C₄)alkyl moieties or said R¹, R², R³, R⁴ and R⁵ (C₃- C₄)cycloalkyl moieties are optionally independently substituted with one or two substituents selected from the group consisting of: hydroxy, (C C )alkoxy, (Cι-C₄)alkylthio, (d- C₄)alkylsulfinyl, (C C₄)alkylsulfonyl, (Cι-C₄)alkyl, mono-N-(CrC₄)alkylcarbamoyl, di-N,N-(C C )alkylcarbamoyl, mono-N-(C₁-C₄)alkylaminosulfonyl, and di-N,N-(C₁-C₄)alkylaminosulfonyl, wherein said (C₃-C₄)cycloalkyl optionally contains from one to seven fluorines; wherein M is a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen, or, a bicyclic ring consisting of two fused partially saturated, fully saturated or fully unsaturated three to six membered rings, taken independently, optionally having one to four heteroatoms selected independently from nitrogen, sulfur and oxygen; said M is optionally substituted, on one ring if the moiety is monocyclic, or one or both rings if the moiety is bicyclic, on carbon or nitrogen with up to three substituents independently selected from R⁶, R⁷ and R⁸, wherein one of R⁶, R⁷ and R⁸ is optionally a partially saturated, fully saturated, or fully unsaturated three to seven membered ring optionally having one to three heteroatoms selected independently from oxygen, sulfur and nitrogen optionally substituted with (Cι-C )alkyl and additionally R⁶, R⁷ and R⁸ are optionally hydroxy, nitro, halo, (C-|-C₄)alkoxy, (C C₄)alkoxycarbonyl, (C C₄)alkyl, formyl, (C C )alkanoyl, (CrC₄)alkanoyloxy, (CrC₄)alkanoylamino, (C C )alkoxycarbonylamino, sulfonamido, (Cι-C₄)alkylsulfonamido, amino, mono-N-(C₁ -C₄)alkylamino, di-N,N-(C|- C₄)alkylamino, carbamoyl, mono-N-(Cι-C₄)alkylcarbamoyl, di-N,N-(C C )alkylcarbamoyl, cyano, thiol, (Cι-C₄)alkylthio, (Cι-C )alkylsulfinyl, (C C )alkylsulfonyl, mono-N-(C₁- C₄)alkylaminosulfonyl, di-N,N-(CrC₄)alkylaminosulfonyl, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl or (C₅- C₇)cycloalkenyl, wherein said (C C₄)alkoxy, (C C₄)alkyl, (C C₇)alkanoyl, (C C₄)alkylthio, mono-N- or di-N,N-(C₁-C₄)alkylamino or (C₃-C₇)cycloalkyl R⁶, R⁷ and R⁸ substituents are optionally mono- substituted independently with hydroxy, (d-C₄)alkoxycarbonyl, (C₃-C₇)cycloalkyl, (C C )alkanoyl, (C C₄)alkanoylamino, (C C₄)alkanoyloxy, (CrC₄)alkoxycarbonylamino, sulfonamido, (C C₄)alkylsulfonamido, amino, mono-N- or di-N,N-(C₁-C₄)alkylamino, carbamoyl, mono-N- or di-N,N-(C₁-C₄)alkylcarbamoyl, cyano, thiol, nitro, (CrC₄)alkylthio, (C C₄)alkylsulfinyl, (Cι-C₄)alkylsulfonyl or mono-N- or di-N,N-(C₁-C₄)alkylaminosulfonyl or optionally substituted with one to nine fluorines, and the Ca²⁺ overload inhibitor is a compound of Formula II

Formula II wherein R⁹ is selected from the group consisting of a hydrogen atom, a C C₆ alkyl group, a C₃-C₇ cycloalkyl group, a C C₄ halogenated alkyl group, a halogen atom, and a C₆-C ₂ aryl group, and wherein R¹⁰ is selected from the group consisting of a hydrogen atom, a Cι-C₆ alkyl group, and a C₇-C ₂ aralkyl group optionally substituted with at least one substituent selected from the group consisting of a cyano group, a nitro group, a C Cβ alkoxy group, a halogen atom, a Cι-C₆ alkyl group and an amino group, and wherein n represents an integer of 1 to 4, or a pharmaceutically acceptable salt or solvate thereof.

5. A composition comprising [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4- carbonyljguanidine or a pharmaceutically acceptable salt thereof and 2-(1 -piperazinyl)-5- methylbenzenesulfonic acid or a pharmaceutically acceptable salt thereof.

6. A composition according to claim 5 wherein the pharmaceutically acceptable salt is a nontoxic salt selected from sodium salts, potassium salts, magnesium salts, calcium salts, aluminum salts, ammonium salts, lower alkylamine salts, hydroxyl lower alkylamine salts, cycloalkylamine salts, benzylamine salts and dibenzylamine salts.

7. A composition according to claim 5 wherein the pharmaceutically acceptable salt is a nontoxic salt selected from hydrochlorides, mesylates, hydrobromides, sulfates, phosphates, fumarates, succinates, oxalates and lactates.

8. A composition comprising crystalline [5-cyclopropyl-1-(quinolin-5-yl)-1H- pyrazole-4-carbonyl]guanidine hydrochloride monohydrate and crystalline 2-(1-piperazinyl)-5- methylbenzenesulfonic acid monohydrate.

9 A composition comprising [5-cyclopropyl-1-(quinolin-5-yl)-1H-pyrazole-4- carbonyljguanidine mesylate and 2-(1-piperazinyl)-5-methylbenzenesulfonic acid.

10. A composition according to claim 5 wherein the 2-(1-piperazinyl)-5- methylbenzenesulfonic acid is anhydrous 2-(1 -piperazinyl)-5-methylbenzenesulfonic acid.

11. A medicament comprising 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate and 2-(1 -piperazinyl)-5-methylbenzenesulfonic acid.

12. A medicament comprising Λ/-(diaminomethylene)-5-(methylsulfonyl)-4-pyrrol- l-yl-otoluamide and 2-(1 -piperazinyl)-5-methylbenzenesulfonic acid.

13. A medicament comprising (Λ/-carbamimidoyl-4-[4-(1 H-pyrrol-2- ylcarbonyl)piperazin-1-yl]-3-(trifluoromethyl)benzamide) and 2-(1-piperazinyl)-5- methylbenzenesulfonic acid.

14. A medicament comprising BMS-284640 and 2-(1-piperazinyl)-5- methylbenzenesulfonic acid.