Method, composition, and formulation for preventing or reducing adverse effects in patient

Abstract

To provide means for preventing or reducing adverse effects in a patient.SOLUTION: The present invention provides a method, composition, formulation, and kit, which are related to acadesine or a prodrug, analog, or salt thereof, and / or a blood clotting inhibitor for preventing or reducing adverse side effects in a patient. The type of patient that may benefit includes a patient with decreased left ventricular function, a patient with a prior myocardial infarction, a patient undergoing non-vascular surgery, or a fetus during labor and delivery. In one aspect, the present invention provides a method for preventing or reducing adverse effects in a patient with decreased left ventricular function having an ejection fraction of less than 30% by administering an effective amount of acadesine, or a prodrug, analog or salt thereof. In the patient may be a female and / or or between the age of 65 and 95.SELECTED DRAWING: None

Description

Background Art

[0001] (Background of the Invention) Gruber (Patent Document 1) describes the prophylactic administration of AICA riboside compounds (including its analogs and prodrugs) for preventing tissue damage associated with undesirable blood flow reduction. The AICA riboside compounds are administered in an amount of 0.1 to 500 mg / kg / day. Prodrugs of AICA riboside (the name "AICA Riboside Prodrugs" in Patent Document 2 by the same applicant, the name "Methods and Compounds for AICA Riboside Delivery and for Lowering Blood Glucose" in U.S. Patent Application No. 07 / 408,107 (filed on September 15, 1989), and the name "Method and Compounds for AICA Riboside Delivery and for Lowering Blood Glucose" in U.S. Patent Application No. 07 / 466,979 (filed on January 18, 1990), the contents of which are incorporated herein by reference in their entirety) may be administered. Specific prodrugs of AICA riboside are defined in these specifications and are generally compounds that are metabolized to AICA riboside or an active metabolite (e.g., AICA riboside monophosphate) when taken into the body. Other prodrugs include mono-, di-, and tri-5'-phosphates of AICA riboside.

[0002] Adenosine, 9-β-D-ribofuranosyladenine (the nucleoside of purine adenine), belongs to a class of biochemical substances called purine nucleosides and is a key biochemical cell regulatory molecule as described in Fox and Kelly's Annual Reviews of Biochemistry, Vol. 47, p. 635, 1978. This molecule interacts with various cell types and is involved in diverse biological effects. For example, adenosine is a potent vasodilator, an inhibitor of immune cell function, activates mast cells to a certain level, is an inhibitor of oxygen radical production in granulocytes, is an antiarrhythmic drug, and is an inhibitory neurotransmitter. Considering this wide range of activities, considerable efforts have been made to establish practical therapeutic uses for adenosine and its analogs.

[0003] Since adenosine is thought to act at the level of the cell membrane by binding to receptors bound to the cell membrane, attempts have been made in the past to increase blood flow by administering adenosine to raise extracellular concentrations. Unfortunately, adenosine is toxic at the concentrations required to be administered to patients to maintain effective therapeutic levels extracellularly, so administration of adenosine alone can only be used for limited therapeutic applications. Furthermore, adenosine receptors undergo negative control feedback (including downregulation of the receptor) after contact with adenosine.

[0004] There are other ways to achieve locally high extracellular levels of adenosine and research has been conducted on them. Other ways include the following: (a) interfering with adenosine uptake using a reagent that specifically blocks adenosine transport, as described by Paterson et al. in Annals of the New York Academy of Sciences, Vol. 255, p. 402 (1975); (b) preventing the breakdown of adenosine, as described by Carson and Seegmiller in The Journal of Clinical Investigation Vol. 57, p. 274 (1976); and (c) using adenosine analogs constructed to bind to adenosine cell membrane receptors.

[0005] There are numerous chemicals that can inhibit the uptake of adenosine into cells. Some substances are those that specifically and essentially competitively inhibit adenosine uptake, and others are substances that inhibit non-specifically. P-nitrobenzylthionosine is considered a competitive inhibitor, and dipyridamole and various other chemicals (colchicine, phenethyl alcohol, and parabens) inhibit uptake non-specifically.

[0006] The extracellular concentration of adenosine can be increased by using chemicals that inhibit the enzymatic breakdown of adenosine. Past research has focused on identifying inhibitors of adenosine deaminase, which is involved in converting adenosine to inosine. The activity of adenosine deaminase is inhibited by coformycin, 2'-deoxycoformycin, and erythro 9-(2-hydroxy-3-nonyl)adenine hydrochloride.

[0007] Many adenosine receptor agonists and antagonists have been made using structural modifications of the purine ring, modification of substituents attached to the purine ring, and modification or alteration of the binding site of the carbohydrate moiety. Halogenated adenosine derivatives are considered to be the most promising agonists or antagonists as described in Wolff et al., the Journal of Biological Chemistry, Vol. 252, p. 681, 1977, and exhibit effects similar to those produced by adenosine in experimental systems.

[0008] The above three techniques have advantages when using adenosine alone, but also have some disadvantages. The most significant disadvantage is the reliance on chemical substances with side effects, which is mainly due to the fact that these substances have to be administered at dosages that are toxic and non-selectively affect most cell types. As described in Purine Metalolism in Man, (edited by De Bruyn, Simmonds and Muller), Plenum Press, New York, 1984, most cells in the body have adenosine receptors. As a result, using techniques to increase adenosine concentration will bring about undesirable drastic changes in the normal cell physiological functions of almost the entire body.

[0009] Regarding tissue and adenosine after ischemic myocardial infarction, Swain, J.L., J.J. Hines, R.L. Sabina and E.W. Holmes, Circulation Research 51:102-105 (1982), and U.S. Patent No. 4,575,498 (issued March 11, 1986) to Holmes et al. state that administration of the purine nucleoside 5-amino-4-imidazolecarboxamide riboside (AICA riboside) does not change adenosine concentration and blood flow in ischemic canine hearts. From these facts, it is considered that the exhaustion of accumulated purine nucleotides (especially adenosine triphosphate (ATP)) is one of the causes of such dysfunctions (e.g., ischemic events) occurring, and it is claimed that treatment of the myocardium after ischemic disease with the purine analog AICA riboside increases the amount of nucleotide synthesis and simultaneously increases the amount of ATP accumulation, which theoretically indicates that an increase in the amount of ATP accumulation can improve tissue damage.

[0010] However, other researchers have published results indicating that they were unable to show an increase in ATP accumulation in ischemic tissue by the methods of Swain et al. (supra); Mentzer, R.M., Ely, S.W., Lasley, R.D., Lee, B.K. and Berne, R.M., Fed. Proc. 43:903 (1984); Mitsos, S.E., S.R. Jolly and B.R. Lucchesi, Pharmacology 31:121-131 (1985); Hoffmeister, H.M., Nienaber, C., Mauser, M. and Schaper, W.E., Basic Research in Cardiology 80:445-458 (1985); Mauser, M., H.M. Hoffmeister, C. Nienaber, and W.E. Schaper, Circul. Res. 56:220-230 (1985). In fact, Hoffmeister et al. have shown that even if ATP accumulation is increased by another mechanism, cardiac dysfunction is not improved. Holmes and Swain have also shown that AICA riboside does not effectively reach ATP because the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) is inhibited. Sabina, R.L., Kernstine, K.H., Boyd, R.L., Holmes, E.W. and Swain, J.L., J. Biol. Chem. 257:10178 (1982); Amidon, T.M., Brazzamano, S., Swain, J.L., Circ. Suppl. 72:357 (1985); Swain, J.L., Hines, J.J., Sabina, R.L., Harburg, O.L. and Holmes, E.W., J. Clin. Invest. 74:1422-1427 (1984). Amidon et al. (supra) stated that, "These results suggest that adenylosuccinate synthetase and / or lyase activities are restricted in isolated hearts and that treatment designed to bypass IMP in AN (adenine nucleotide) synthesis would be beneficial for increasing AN accumulation.""Swain et al. (supra) (J. Biol. Chem.) showed that AICA riboside does not always increase ATP levels in non-ischemic myocardium.

[0011] Mitsos et al. (supra) claimed in their own research that when AICA riboside is inhaled into the coronary artery at a high dose, it can protect the ischemic heart as a whole from dysfunction related to ischemic injury. Hoffmeister et al., Basic Res. Cardiol. 80:445-458 (1985) showed that in reversible ischemia in dogs caused by coronary artery occlusion, application of AICA riboside did not improve after ischemic insufficiency and actually worsened. Swain et al. (supra) (J. Clin. Invest.) confirmed that high-dose AICA riboside has an adverse effect on myocardial contractility. Therefore, it is unclear whether the proposal that administration of AICA riboside increases ATP accumulation and is beneficial to patients after ischemic events is correct.

[0012] From the foregoing discussion, techniques for increasing the extracellular concentration of adenosine or adenosine analogs at a specific point in time during a pathological event, techniques for increasing the amount of these compounds without complex side effects, and techniques for increasing adenosine concentration so as to selectively target the most beneficial cells for the therapeutic use in question are understood. As an example, such techniques are particularly useful for the prevention of ischemic events (such as heart attacks or strokes) or the response during such events, or other events (such as atherosclerosis) involving undesirable restriction or reduction of blood flow, since adenosine is a vasodilator and prevents superoxide radical generation by granulocytes. Such techniques are also useful for the prophylactic or emergency treatment of pathological conditions involving an increase in intracellular excitatory states, such as (1) seizures or epilepsy, (2) arrhythmias, and (3) inflammation involved in the activation of granulocytes by contact of blood complement with artificial membranes during dialysis or when using a heart-lung machine, such as arthritis, autoimmune diseases, adult respiratory distress syndrome (ARDS). Furthermore, it is useful for the treatment of patients with chronically low adenosine levels (such as patients with autism, cerebral palsy, insomnia and other neuropsychiatric symptoms including schizophrenia). These objectives can be achieved using the compounds useful in the present invention (including AICA riboside).

[0013] Another medically important area is the treatment of allergic diseases, which can be achieved by suppressing the activation of mast cells or interfering with the mediators of allergic reactions secreted by mast cells. The activation of mast cells can be downregulated by immunotherapy (allergy shots) or mast cell stabilizers (such as cromolyn sodium, corticosteroids and aminophylline). There are also therapeutic agents (such as antihistamines and adrenergic agents) that interfere with the action of mast cell products. The mechanism of action of mast cell stabilization is not yet fully understood. In the case of aminophylline, it is thought to act as an adenosine receptor antagonist. However, the mechanism of action of drugs such as cromolyn sodium and corticosteroids is not well understood.

[0014] Therefore, it is of great significance and practicality to understand that effective allergy treatment using this compound does not exhibit the side effects of any of the above-mentioned compounds (for example, drowsiness in the case of antihistamines, excitatory states in the case of adrenergic agents, and Cushing's syndrome in the case of corticosteroids). Compounds useful in the present invention (such as AICA riboside and ribavirin) are not known or thought to be metabolized into purine nucleoside triphosphate or purine nucleoside monophosphate in cells among three known mast cell stabilizers in a controlled manner.

[0015] Gruber (U.S. Patent No. 5,817,640) describes specific therapeutic concentrations for preventing tissue damage associated with reduced human blood flow with respect to AICA riboside, and the determination of effective dosages while avoiding undesirable side effects. In one aspect, AICA riboside or its prodrug is administered to a human in an amount such that the concentration of AICA riboside in plasma is maintained at about 1 μg / ml to about 20 μg / ml over a sufficient period to reduce the risk of tissue damage in humans. In another aspect, AICA riboside is administered to a human at a dosage of about 0.01 mg / kg / min to about 2.0 mg / kg / min to reduce the risk of tissue damage. Another aspect is characterized in that the total dosage of AICA riboside is 10 mg / kg to 200 mg / kg to prevent tissue damage.

[0016] AICA riboside enters cells and is phosphorylated to AICA riboside monophosphate ("ZMP"), which is a naturally occurring intermediate in purine biosynthesis. AICA riboside increases extracellular adenosine concentration under net ATP hydrolysis conditions and may have potential therapeutic uses from the perspective that adenosine is cardioprotective and neuroprotective. However, AICA riboside is relatively low in efficacy and has a short half-life. Furthermore, the inventors of the present application have discovered that AICA riboside cannot sufficiently cross the blood-brain barrier and is not sufficiently absorbed from the gastrointestinal tract. These characteristics limit efficacy, limit oral bioavailability, limit permeability to the brain, and reduce the potential ability in therapeutic agent applications.

[0017] Treatment with AICA riboside has been reported to have beneficial effects in many experimental models of myocardial ischemia. In the dog model, pacing causes a significant decrease in wall thickness, a significant decrease in endocardial blood flow, and a large deviation in the ST segment of the intramyocardial EKG, but AICA riboside significantly reduces these changes and maintains systolic function. Young and Mullane, Am. J. Physiol. (in press, 1991). In another dog model, ischemia is induced by coronary artery occlusion, and AICA riboside has been reported to be beneficial by significantly reducing arrhythmias induced by ischemia and increasing blood flow to the ischemic regions of the myocardium (Gruber et al., Circulation 80(5):1400-1410 (1990)). Due to the effect of AICA riboside to increase local blood flow and maintain systolic function, it has also been reported to induce ischemia by directly administering microspheres to the coronary circulation in a dog model of coronary embolism (Takashima et al., Heart and Vessels 5(Supplement 4):41 (1990)). The potential result of this reported redistribution of blood flow by AICA riboside is said to reduce infarct size (McAllister et al., Clinical Research 35:303A (1987)). Treatment with AICA riboside has been reported to give favorable results in other experimental models of myocardial ischemia. For example, Mitsos et al. (Pharmacology It has been reported that in the hearts of cats perfused with isolated blood, the functional recovery after ischemia was improved by AICA riboside (Bullough et al., Jap. J. Pharmacol 52:85p (1990)), and Bullough et al. (Jap. J. Pharmacol 52:85p (1990)) reported that the degree of recovery was improved in the hearts of guinea pigs perfused with isolated buffer. Therefore, it has been reported that AICA riboside reduces the damage induced by ischemia in the hearts of various experimental models.

[0018] AICA riboside has also been reported to protect brain tissue from damage in two different experimental models of cerebral ischemia. In a gerbil model of widespread ischemia, AICA riboside has been reported to prevent the degeneration of hippocampal CA-1 cells that are substantially completely destroyed in control animals (Phillis and Clough-Helfman, Heart and Vessels 5 (Supplement 4):36 (1990)). In a rat model of focal ischemia, it has been reported that the infarct area is significantly reduced by AICA riboside treatment. The protective effect of AICA riboside has also been reported in other ischemia models (e.g., survival test of rat skin flaps) (Qadir et al., Fed Proc. A626 (1988); Salerno et al., Proceedings of 35th Annual Meeting of the Plastic Surgery Research Council, pp. 117-120 (1990)) and reperfusion injury of gastrointestinal ischemia (Kaminski & Proctor, Circulation Res. 66(6):1713-1729 (1990)).

[0019] From many tests, the beneficial effect of AICA riboside at least partially increases the local adenosine concentration and has a similar cardioprotective effect (Olafsson et al., Circulation It is suggested that it is due to [[ID=]], neurotrophic (Dragunow & Faull, Trends in Pharmacol. Sci. 7:194 (1988); Marangos, Medical Hypothesis 32:45 (1990)) and neuroprotective effects. Evidence for the increase in adenosine concentration induced by AICA riboside comes from direct measurements of adenosine itself in animal and cell culture models (Gruber et al., Circulation 80(5):1400 - 1410 (1990); Barankiewicz et al., Arch. Biochem. Biophys., 283:377 - 385, (1990)) and indirect results, i.e., the reversal of the anti - ischemic effect of AICA riboside by removal of exogenous adenosine using adenosine deaminase (Young & Mullane, Am. J. Physiol. (in press, 1991)). In ischemic and reperfused hearts, cell injury is, in part, caused by neutrophil - mediated microvascular occlusion. Adenosine inhibits neutrophil adhesion to coronary endothelial cells and prevents neutrophil accumulation (Cronstein et al., J. Clin. Invest. 78:760 - 770 (1986)). Consequently, another feature of the adenosine - mediated cardioprotective effect of AICA riboside is that it can prevent neutrophil - dependent tissue injury in several ischemia and reperfusion models. This supports the fact that neutrophils accumulate in the ischemic areas of the heart in response to AICA riboside (Gruber et al., Circulation 80:1400 - 1410 (1990)).

[0020] By attempting to properly evaluate the cardioprotective and neuroprotective properties of adenosine, we sought to investigate the therapeutic use of administering adenosine itself exogenously. However, because the half-life of adenosine in the blood is short (less than 10 seconds), it is necessary to use continuous infusion at high doses to maintain levels suitable for most treatments. Adenosine itself causes hypotension, i.e., it lowers blood pressure, and adenosine is also a negative chronotropic and dromotropic agent, i.e., it decreases heart rate and cardiac electrical conduction. Therefore, adenosine has a significant impact on systemic blood flow at the concentrations required to exert cardioprotective or neuroprotective effects. These systemic cardiovascular effects are often contraindicated in most clinical conditions where adenosine would be useful. In contrast, administration of AICA riboside results in a local effect on adenosine concentration and does not produce such side effects even at doses much higher than the expected therapeutic concentration (Gruber et al., Circulation 80:1400-1410 (1990); Young & Mullane, Am. J. Physiol. (in press, 1991)).

[0021] Adenosine receptor agonists have been tested and effects similar to those of adenosine have been reported in many experimental models (Daly, J. Med. Chem. 25(3):197 (1982). Also, because most cell types have adenosine receptors, exogenously administered adenosine agonists exhibit significant effects in various tissues and organs other than the target organ, which limits their potential as therapeutic agents.

[0022] Other potential effects due to locally high extracellular concentrations of adenosine are being studied. These effects include: (a) interfering with the uptake of adenosine using reagents that specifically block adenosine transport, as described by Paterson et al. in the Annals of the New York Academy of Sciences, Vol. 255, p. 402 (1975); (b) preventing the modification of adenosine, as described by Carson and Seegmiller in The Journal of Clinical Investigation, Vol. 57, p. 274 (1976); and (c) using adenosine analogs constructed to bind to adenosine cell membrane receptors.

[0023] There is a repertoire of chemicals reported to be able to inhibit the cellular uptake of adenosine. Some chemicals are reported to inhibit specifically and are thought to essentially competitively inhibit adenosine uptake, while other chemicals are thought to inhibit non-specifically. p-Nitrobenzylthioinosine is thought to be a competitive inhibitor, and dipyridamole and various other chemicals (including colchicine, phenethyl alcohol, and papaverine) are thought to inhibit uptake non-specifically.

[0024] U.S. Patent No. 4,115,641 (Fischer et al.) relates to certain ribofuranosyl derivatives said to have dynamic properties with respect to the heart and circulatory system. In particular, the Fischer et al. patent relates to certain compounds said to have an essentially adenosine-like mode of action as determined by measuring a decrease in heart rate and a decrease in blood pressure.

[0025] In contrast, AICA riboside and AICA riboside-like compounds can increase adenosine concentration at specific times and specific locations of pathological events, and can occur selectively at locations where the increase in adenosine concentration is targeted without harmful side effects.

[0026] The present invention relates to AICA riboside analogs that exhibit good biological effects and, in many cases, enhanced effects compared to AICA riboside. The novel compounds typically exhibit one or more of the following improvements compared to AICA riboside: (1) functional superiority at low doses; (2) more potent adenosine modulating effects; (3) increased half-life; or (4) increased oral bioavailability and / or improved brain permeability.

[0027] Postoperative complications are a significant cause of morbidity and mortality and a major cause of healthcare expenditure.

[0028] In cardiac surgery, approximately 1 million patients undergo surgery each year, and approximately 1 in 6 develops serious major organ complications related to the heart, brain, kidney, GI tract, and lung (Mangano et al., 1997, J. Intensive Care Med. 12:148 - 160). Despite significant advances in monitoring and technology, no drugs have been shown to reduce or prevent these complications. The most notable is bleeding, and drugs are currently used to prevent bleeding. However, drugs that suppress bleeding generally cause thrombosis and, therefore, may induce ischemia and irreversible organ damage (Cosgrove et al., 1992, Ann. Thorac. Surg. 54:1031 - 36).

[0029] In non-cardiac surgery, approximately 2.5 million patients undergo surgery each year, and approximately 40% develop serious major organ complications related to the heart (Mangano et al., 1990, Anesthesiology 2:153-84; Mangano et al., 1990 NEJM 323:1781-88). Only one drug (atenolol) has been shown to reduce the damage (Mangano et al., 1996, NEJM 335:1713-20). Moreover, stopping bleeding is the top concern, and drugs that prevent thrombosis (platelet aggregation inhibitors, blood coagulation inhibitors) are virtually contraindicated (Eagle et al., 1999, JACC 34:1262-1347; Pearson et al., 1994, Circulation 90:3125-33; Baumgartner et al., 1994, Johns Hopkins Manual of Surgical Care, Mosby Yearbook, St. Louis).

[0030] However, in both cardiac and non-cardiac surgery, significant excitotoxicity and inflammatory responses occur not months but days after surgery (Silicano and Mangano, 1990, Mechanisms and Therapies. In: Estafanous, ed. Opioids in Anesthesia Butterworth Publishers, pp. 164-178). This significantly emphasized response is related to the activity of platelets and blood coagulation factors and may thereby cause thrombosis.

[0031] Although recognized as one possibility, this agent has an additional adverse, and in some cases (fibrinolytic agents) a completely opposite effect, due to the risk of excessive bleeding at the surgical site and other sites (Eagle et al., 1999, JACC 34:1262 - 1347; Pearson et al., 1994, Circulation 90:3125 - 3133; Baumgartner et al., 1994, Johns Hopkins Manual of Surgical Care, Mosby Yearbook, St. Louis). Furthermore, especially after cardiac surgery, some believe that the function of platelets and blood coagulation factors decreases postoperatively and thrombosis is not a problem (Kestin et al., 1993, Blood 82:107 - 117; Khuri et al., 1992, J. Thorac. Cardiovasc. Surg. 104:94 - 107). Therefore, no effort has been made to investigate in detail the use of blood coagulation inhibitors immediately after surgery.

[0032] Finally, the applicants have clarified perioperative events over a period of 6 - 8 months or more (Mangano et al., 1992, JAMA 268:233 - 39); thus, the use of blood coagulation inhibitors was continued in the hospital, showing that the postoperative course after discharge was reasonable.

[0033] Currently, there are 40 million surgical patients in the United States alone each year, and they are aging at almost twice the rate of the overall population (see Mangano et al., 1997, J. Intensive Care Med. 12:148 - 160).

[0034] The current standard of care does not adequately address this critical problem, and there is an urgent need for new approaches to prevent postoperative complications in the elderly.

[0035] Electronic monitoring of fetal heart rate is an important part of the process from a woman's labor to childbirth. In some cases, decelerations of fetal heart rate (including persistent decelerations in which the variability of the heart rate interval is lost, which is associated with the loss of the variability of the heart rate interval; variable decelerations that are concerning and fluctuating; long-lasting severe bradycardia; sine wave patterns; fetal rest states; and confirmation of the loss of variability of the heart rate interval not related to drugs or severe prematurity) may require emergency in-utero fetal resuscitation and rapid delivery (Sweha et al., 1999. American Family Physician 59(9):2487-2507; Kripke 1999, American Family Physician 59(9):2416). There is a need for methods to prevent or reduce the side effects from these events on fetal health.

Prior Art Documents

Patent Documents

[0036]

Patent Document 1

Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0037] (Summary of the Invention) The present invention relates to a method for preventing or reducing side effects in a patient, which comprises administering to the patient acadesine or a prodrug, analog, or salt thereof, or administering to the patient acadesine or a prodrug, analog, or salt thereof and an anticoagulant. Further, the present invention includes pharmaceutical formulations, compositions, myocardial protective solutions, and kits related to preventing or reducing side effects in a patient. The present invention is beneficial for several types of patients, including patients with reduced left ventricular function, patients with a history of myocardial infarction, patients undergoing non-vascular surgery, or fetuses during labor and delivery.

[0038] (Incorporation by reference) All publications and patent specifications mentioned in this specification are incorporated herein by reference as if each individual publication or patent specification were specifically incorporated by reference individually. The present invention provides the following items. (Item 1) A method for preventing or reducing side effects in a patient with reduced left ventricular function with an ejection fraction of less than 30%, comprising administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. (Item 2) A method for preventing or reducing side effects in a patient in a state selected from the group consisting of a history of at least one myocardial infarction, a history of at least two myocardial infarctions, a history of at least three myocardial infarctions, and a history of more than at least three myocardial infarctions, comprising administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. (Item 3) The method according to item 2, wherein the most recent myocardial infarction occurred at a time selected from the group consisting of within the most recent 24 months, within the most recent 36 months, and within the most recent 48 months. (Item 4) The method according to item 1 or 2, wherein the patient is female. (Item 5) The method according to item 1 or 2, wherein the age of the patient is between about 65 and about 95 years old. (Item 6) The method according to item 1 or 2, wherein the patient is female and the age of the patient is between about 65 and about 95 years old. (Item 7) A method for preventing or reducing side effects in a patient undergoing non-vascular surgery, comprising administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. (Item 8) The method according to item 7, wherein the non-vascular surgery is selected from the group consisting of abdominal non-vascular surgery, nerve non-vascular surgery, gynecological non-vascular surgery, orthopedic non-vascular surgery, urological non-vascular surgery, and otolaryngological non-vascular surgery. (Item 9) The method according to item 7, wherein the non-vascular surgery is selected from the group consisting of resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringectomy, placement of tympanostomy tubes, polyp removal from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc. (Item 10) A method for preventing stroke in a patient undergoing CABG surgery, comprising administering to the patient an effective amount of acadine or a prodrug, analog or salt thereof. (Item 11) A pharmaceutical formulation comprising acadine or a prodrug, analog or salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient, which is administered to a patient in need thereof and is adapted to be lipophilic, wherein the plasma concentration of acadine is from 1 μg / ml to 20 μg / ml over about 7 hours. (Item 12) The pharmaceutical formulation according to item 11, wherein the formulation is in micellar form. (Item 13) A method for reducing tissue damage associated with decreased blood flow in a patient, comprising administering to the patient an effective amount of acadine or a prodrug, analog or salt thereof, wherein the patient is a fetus during labor and delivery. (Item 14) The method according to item 13, wherein the acadine is administered to the mother of the fetus during labor and delivery. (Item 15) A method for preventing or reducing side effects in a patient undergoing surgery, comprising a first step of administering to the patient an effective amount of acadine or a prodrug, analog or salt thereof, and a second step of administering to the patient an effective amount of a blood coagulation inhibitor. (Item 16) The method according to item 15, wherein the blood coagulation inhibitor is administered during the administration of acadine. (Item 17) The method according to item 15, wherein the acadine is administered at a total dosage of 10 mg / kg to 200 mg / kg. (Item 18) A method for preventing or reducing side effects in a patient undergoing non-vascular surgery, comprising a first step of administering to the patient an effective amount of acadine or a prodrug, analog or salt thereof, and a second step of administering to the patient an effective amount of a blood coagulation inhibitor. (Item 19) The method according to item 18, wherein the non-vascular surgery is selected from the group consisting of non-vascular surgery of the abdomen, non-vascular surgery of the nerves, non-vascular surgery of the gynecology, non-vascular surgery of the orthopedics, non-vascular surgery of the urology, and non-vascular surgery of the otorhinolaryngology. (Item 20) The method according to item 18, wherein the non-vascular surgery is selected from the group consisting of resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringotomy, placement of tympanostomy tubes, polyp removal from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc. (Item 21) The method according to item 15 or 18, wherein the blood coagulation inhibitor is aspirin. (Item 22) The method according to item 15 or 18, wherein the acadine is administered at 0.1 mg / kg / min. (Item 23) The method according to item 15 or 18, wherein the acadine is administered for 7 hours during the operation. (Item 24) The method according to item 18, wherein the aspirin is administered at a dosage of 400 mg to 5 g. (Item 25) The method according to item 18, wherein the aspirin is administered at least once within 48 hours after the operation. (Item 26) The method according to item 15 or 18, wherein the blood coagulation inhibitor is selected from the group consisting of antiplatelet agents, thrombolytic enzymes, aggregation inhibitors, glycoprotein IIb / IIIa inhibitors, glycosaminoglycans, thrombin inhibitors, anticoagulants, heparin, low molecular weight heparin, coumarin, indandione derivatives, and tissue plasminogen activator. (Item 27) The method according to item 15 or 18, wherein the patient has a medical history of a condition selected from the group consisting of at least one history of myocardial infarction, at least two histories of myocardial infarction, at least three histories of myocardial infarction, and more than three histories of myocardial infarction. (Item 28) The method according to item 27, wherein the most recent myocardial infarction occurred within a period selected from the group consisting of within the most recent 24 months, within the most recent 36 months, and within the most recent 48 months. (Item 29) A method for preventing or reducing side effects in a patient undergoing CABG surgery, comprising a first step of administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof, and a second step of administering to the patient an effective amount of a blood coagulation inhibitor. (Item 30) The method according to item 29, wherein the blood coagulation inhibitor is administered during the administration of acadine. (Item 31) The method according to item 29, wherein the acadine is administered at a total dosage of 10 mg / kg to 200 mg / kg. (Item 32) A pharmaceutical formulation comprising acadine or a prodrug, analog, or salt thereof at a concentration such that the concentration of acadine in the patient's plasma is 1 μg / ml to 20 μg / ml over about 7 hours, aspirin at a dosage of 40 mg to 5 g, and at least one pharmaceutically acceptable carrier, diluent, or excipient. (Item 33) A method for preventing or reducing side effects in a patient undergoing surgery, comprising administering to the patient the pharmaceutical formulation according to item 32 within 48 hours after the surgery. (Item 34) A method for preventing or reducing side effects in a patient who has previously undergone CABG surgery, comprising administering to the patient a pharmaceutical formulation as described in item 32 in an effective amount. (Item 35) A cardioplegic solution containing 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt at a concentration of about 5 μM to about 100 μM. (Item 36) (a) Lyophilized 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt for use in preparing a solution of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt for injection into a patient undergoing cardiac surgery, and (b) A kit for administering 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt to a patient undergoing cardiac surgery, comprising a solution of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt for use in preparing a cardioplegic perfusion solution for perfusion of the heart of a patient undergoing cardiac surgery. (Item 37) (a) A kit for administering 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt to a patient, comprising a sterile container of non-pyrogenic lyophilized 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt. (Item 38) A method for preventing or reducing side effects in patients with reduced left ventricular function with an ejection fraction of less than 30%, comprising administering to the patient an effective amount of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt. (Item 39) A method for preventing or reducing side effects in patients in a state selected from the group consisting of a history of at least 1 myocardial infarction, a history of at least 2 myocardial infarctions, a history of at least 3 myocardial infarctions, and a history of more than at least 3 myocardial infarctions, comprising administering to the patient an effective amount of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt. (Item 40) The method according to item 38 or 39, wherein the most recent myocardial infarction occurred at a time selected from the group consisting of within the most recent 24 months, within the most recent 36 months, and within the most recent 48 months. (Item 41) The method according to item 38 or 39, wherein the patient is female. (Item 42) The method according to item 38 or 39, wherein the patient is female and the patient's age is between about 65 years and about 95 years. (Item 43) The method according to item 38 or 39, wherein 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt is administered at a concentration such that the concentration in the patient's plasma is 1 μg / ml to 20 μg / ml over about 7 hours. (Item 44) The method according to item 38 or 39, wherein 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt is administered at 0.1 mg / kg / min. (Item 45) The method according to item 38 or 39, wherein 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt is administered over about 7 hours. (Item 46) A method for preventing or reducing side effects in a patient undergoing non-vascular surgery, comprising administering to the patient an effective amount of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt. (Item 47) The method according to item 46, wherein the non-vascular surgery is selected from the group consisting of non-vascular surgery of the abdomen, non-vascular surgery of the nerves, non-vascular surgery of the gynecology, non-vascular surgery of the orthopedics, non-vascular surgery of the urology, and non-vascular surgery of the otorhinolaryngology. (Item 48) The method according to item 46, wherein the non-vascular surgery is selected from the group consisting of resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringectomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc. (Item 49) A pharmaceutical formulation comprising 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide, its prodrug, analog or salt and at least one pharmaceutically acceptable carrier, diluent or excipient, and is administered to a patient in whom it is necessary for the plasma concentration of 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide to be 1 μg / ml to 20 μg / ml over about 7 hours, and is adapted to be lipophilic. (Item 50) The pharmaceutical formulation according to item 49, wherein the formulation is in micellar form. (Item 51) A cardioplegic solution containing 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt at a concentration of about 5 μM to about 100 μM. (Item 52) (a) Lyophilized 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt for use in preparing a solution of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt for injection into a patient undergoing cardiac surgery, and (b) A kit for administering 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt to a patient undergoing cardiac surgery, comprising a solution of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt for use in preparing a cardioplegic perfusion fluid for perfusion of the heart of a patient undergoing cardiac surgery. (Item 53) (a) A kit for administering 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt to a patient, comprising a sterile container containing a non-pyrogenic lyophilized 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt. (Item 54) A method for preventing or reducing side effects in patients with reduced left ventricular function with an ejection fraction of less than 30%, comprising administering to the patient an effective amount of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt. (Item 55) A method for preventing or reducing side effects in a patient in a state selected from the group consisting of a history of at least 1 myocardial infarction, a history of at least 2 myocardial infarctions, a history of at least 3 myocardial infarctions and a history of more than at least 3 myocardial infarctions, comprising administering to the patient an effective amount of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt. (Item 56) The method according to item 55, wherein the most recent myocardial infarction occurred at a time selected from the group consisting of within the most recent 24 months, within the most recent 36 months and within the most recent 48 months. (Item 57) The method according to item 54 or 55, wherein the patient is female. (Item 58) The method according to item 54 or 55, wherein the patient is female and the patient's age is between about 65 years and about 95 years. (Item 59) The method according to item 54 or 55, wherein 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt is administered at a concentration such that the concentration in the patient's plasma is 1 μg / ml to 20 μg / ml over about 7 hours. (Item 60) The method according to item 54 or 55, wherein 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt is administered at 0.1 mg / kg / min. (Item 61) The method according to item 54 or 55, wherein 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt is administered over about 7 hours. (Item 62) A method for preventing or reducing side effects in a patient undergoing non-vascular surgery, comprising administering to the patient an effective amount of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt. (Item 63) The method according to item 62, wherein the non-vascular surgery is selected from the group consisting of non-vascular surgery of the abdomen, non-vascular surgery of the nerves, non-vascular surgery of the gynecology, non-vascular surgery of the orthopedics, non-vascular surgery of the urinary organs, and non-vascular surgery of the otorhinolaryngology. (Item 64) The method according to item 62, wherein the non-vascular surgery is selected from the group consisting of resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringotomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc. (Item 65) A pharmaceutical formulation comprising 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt and at least one pharmaceutically acceptable carrier, diluent or excipient, and is provided to a patient in whom the plasma concentration of 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboximidamide, its prodrug, analog or salt needs to be 1 μg / ml to 20 μg / ml, and is adapted to be lipophilic. (Item 66) The pharmaceutical formulation according to item 65, wherein the formulation is in micellar form. (Item 67) Formula (Ia): [Chemical formula] A composition comprising a compound represented thereby, or any prodrug or salt thereof. (Item 68) (a) The composition according to item 67 in lyophilized form for use in preparing a solution containing the composition according to item 67 for injection into a patient undergoing cardiac surgery, and (b) A kit for administering the composition according to item 67 to a patient undergoing cardiac surgery, comprising the composition according to item 67 for use in preparing a myocardial protective perfusion fluid for perfusing the heart of a patient undergoing cardiac surgery. (Item 69) (a) A kit for administering the composition according to item 67 to a patient, comprising a sterile container of the non - pyrogenic lyophilized composition according to item 67. (Item 70) Formula (IIa): [Chemical formula] A myocardial protective fluid comprising a compound represented thereby, and a pharmaceutically acceptable salt thereof, and comprising a composition in which the compound, its prodrug, analog or salt has a concentration of 5 μM to 100 μM, wherein In the formula,[[]] R 2 is hydrogen, --CN and the following groups: [Chemical formula] selected from the group consisting of, T is oxygen, sulfur, NOH, NH and NO(CH 2 ) n CH 3 selected from, n is 0 to 2, and U is lower alkoxy, amino, a 3 - to 6 - membered heterocyclic ring optionally condensed with a 3 - to 6 - membered aryl ring and the following groups: [Chemical formula] selected from wherein A is either NH or S, n is from 0 to 3, i is from 0 to 2, Q is either hydrogen or hydroxy, E represents a nitro group or a hydroxy group, provided that when U is amino, T is not sulfur, NOH, NH or NOCH 3 ; when T is amino, U is not lower alkoxy; when A is amino and n is 1, Q is not hydroxy; R 3 is hydrogen, halogen and S--W, where W is phenyl or substituted phenyl, or when T is not oxygen and U is not amino, is hydrogen; R 4 and R 5 are each independently selected from hydrogen, --COCH 3 and lower alkyl, or together form a cyclic carbonate; R 6 is hydroxy, phosphate ester, --OSO 2 NH 2 sulfhydryl, halogen, --OCOCH 3 --SCH 3 --SOCH 3 NH 2 and N 3 selected from provided that when R 2 is CONH 2 CONH - para - iodophenyl, hydrogen, CN or CONHCH 2 --φ, R 3 is hydrogen or halogen, and when R 4 and R 5 are hydrogen, acyl, or together form a cyclic carbonate, R 6 is not halogen, phosphate ester, OH or --O - acyl, Myocardial protective solution. (Item 71) A myocardial protective solution comprising a composition containing the compound according to Item 67. (Item 72) The kit according to item 68, wherein the myocardial protective perfusion fluid is the solution described in item 70. (Item 73) A method for preventing or reducing side effects in a patient with reduced left ventricular function with an ejection fraction of less than 30%, comprising administering to the patient an effective amount of the composition according to item 67 or a prodrug, analog or salt thereof. (Item 74) A method for preventing or reducing side effects in a patient undergoing CABG surgery, comprising administering to the patient an effective amount of the composition according to item 67 or a prodrug, analog or salt thereof during the operation. (Item 75) A method for preventing or reducing side effects in a patient in a state selected from the group consisting of at least one history of myocardial infarction, at least two histories of myocardial infarction, at least three histories of myocardial infarction, and more than at least three histories of myocardial infarction, comprising administering to the patient an effective amount of the composition according to item 67 or a prodrug, analog or salt thereof. (Item 76) The method according to item 90, wherein the most recent myocardial infarction occurred at a time selected from the group consisting of within the most recent 24 months, within the most recent 36 months, and within the most recent 48 months. (Item 77) The method according to item 73, 74 or 75, wherein the patient is female. (Item 78) The method according to item 73, 74 or 75, wherein the patient is female and the patient's age is between about 65 years and about 95 years. (Item 79) The method according to item 73, 74 or 75, wherein the composition according to item 67, its prodrug or salt is administered at a concentration such that the concentration in the patient's plasma is 1 μg / ml to 20 μg / ml over about 7 hours. (Item 80) The method according to item 73, 74 or 75, wherein the composition according to item 67, its prodrug or salt is administered at 0.1 mg / kg / min. (Item 81) The method according to item 73, 74 or 75, wherein the composition according to item 67, its prodrug or salt is administered over about 7 hours. (Item 82) A method for preventing or reducing side effects in a patient undergoing non-vascular surgery, comprising administering to the patient an effective amount of the composition according to item 67, its prodrug, analog or salt. (Item 83) The method according to item 82, wherein the non-vascular surgery is selected from the group consisting of non-vascular surgery of the abdomen, non-vascular surgery of the nerves, non-vascular surgery of the gynecology, non-vascular surgery of the orthopedics, non-vascular surgery of the urinary organs, and non-vascular surgery of the otorhinolaryngology. (Item 84) The method according to item 83, wherein the non-vascular surgery is selected from the group consisting of resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringectomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc. (Item 85) A pharmaceutical formulation comprising the composition according to item 67 and at least one pharmaceutically acceptable carrier, diluent or excipient, which is administered to a patient in need of a plasma concentration of the composition according to item 67 of 1 μg / ml to 20 μg / ml over about 7 hours and is adapted for oral administration in solid dosage form.

Brief Description of the Drawings

[0039]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20A

Figure 20B

Figure 20C

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Mode for Carrying Out the Invention

[0040] (Detailed Description of the Invention) The "ejection fraction" is a measurement of the function of the left ventricle and is also called the left ventricular ejection fraction (LVEF). The ejection fraction is the proportion of blood ejected from the left ventricle with each heartbeat. An LVEF of 50% indicates that half of the volume of the left ventricle is ejected from the left ventricle with each contraction. A normal ejection fraction is 50% or more. A decrease in the ejection fraction is a sign indicating the presence of cardiomyopathy.

[0041] In one aspect, the present invention provides a method for preventing or reducing side effects in a patient with reduced left ventricular function having an ejection fraction of less than 30% by administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. Another embodiment provides a method wherein the patient is female and / or is between 65 and 95 years of age.

[0042] Another aspect of the present invention provides a method for reducing tissue damage associated with reduced blood flow in a patient who is a fetus during labor and delivery, the method comprising administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. In one embodiment, the effective amount of acadine or a prodrug, analog, or salt thereof is administered to a woman delivering the fetus.

[0043] In one aspect, the present invention provides a pharmaceutical embodiment, and the present invention provides a pharmaceutical formulation comprising acadine or a prodrug, analog or salt thereof for use in administering to a fetus during labor and delivery to prevent or reduce tissue damage associated with reduced blood flow in the patient.

[0044] Particularly, a novel method for enhancing adenosine release during net ATP catabolism (i.e., the time or period during which the ratio of ATP synthesis to ATP breakdown in a cell or cell compartment decreases or has decreased) is described.

[0045] A novel method for stabilizing mast cells is also described.

[0046] Also within the scope of the present invention is a method for screening a purine nucleoside compound or analog for its ability to enhance cell synthesis and adenosine release, comprising administering a first composition comprising the purine nucleoside compound or analog to be screened to cultured cells, administering a second composition comprising a compound that promotes net catabolism of adenosine triphosphate to the cultured cells, and determining the concentration or amount of adenosine released by the cultured cells.

[0047] The last-written method may further include a first control set of cultured cells to which neither the first composition nor the second composition is added, a second control set to which the first composition is added, and a third control set to which the second composition is added. The cultured cells may be derived from a human malignant cell line (e.g., B lymphocytes transformed with Epstein - Barr virus as used in Example II herein, or the WI - L2 human spleen lymphoblast cell line). Compounds used to create a composition for promoting net catabolism of adenosine triphosphate include calcium ionophore and 2 - deoxyglucose.

[0048] A method for enhancing adenosine release is utilized by administering a compound that is thought to alter one or more of the biochemical pathways of adenosine metabolism, resulting in an increase in the extracellular concentration of adenosine (arising from one or more processes including intracellular production and / or release of adenosine) as a net result. Examples of compounds useful in the present invention include compounds broadly classified as purine nucleosides and related analogs, such as AICA riboside, AICA ribotide, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide (ribavirin), ribavirin monophosphate, and various pro-forms of the above-mentioned compounds. This compound is taken up by cells and, if necessary, converted to monophosphate, and a small amount is further converted to the triphosphate form. Also included are (1) agents that enhance the endogenous synthesis of AICA ribotide or metabolites, such as purine intermediate metabolites or compounds capable of forming this metabolite, such as succinylaminoimidazole carboxamide (SAICA) riboside, (2) agents that accumulate AICA-ribotide or its metabolites (including methotrexate), and (3) agents that produce a bacterial flora that increases AICA riboside production (e.g., sulfonamides). These compounds can be administered prophylactically to patients and, in some cases, respond directly to other physical conditions. Purine nucleosides that enhance the cellular secretion of adenosine and / or adenosine analogs may be administered to a biological system in a concentration range of 0.5 μM to 0.5 M, typically administered at a concentration up to 0.5 M.

[0049] Adenosine or inosine is made from adenosine triphosphate in a series of rapid cellular energy utilization, such as during the activation of seizures, arrhythmias or conditions that reduce blood flow (ischemia), such as stroke, heart attack or angina. Usually, during such events, more inosine is produced than adenosine. In areas with low blood flow during coronary artery occlusion, for example, the venous inosine to adenosine ratio is 100 to 1. A certain percentage of inosine and adenosine exits the cell and is immediately present in the extracellular environment. Compounds useful in the methods described herein and claimed are shown to increase the extracellular concentration of adenosine and decrease the production of inosine. The change in adenosine production occurs only in the net ATP utilization area and time, and since adenosine is rapidly degraded, the adenosine concentration does not change significantly throughout the patient's body. Thus, the methods described herein and claimed do not increase adenosine systemically or globally, but rather locally increase the extracellular concentration of adenosine.

[0050] The oxidation of low-density lipoprotein (LDL) is one of the first (not necessarily the first) steps in the process of atherosclerosis, and this process is thought to be involved in inflammation and is caused by the activation of monocytes and / or granulocytes. Oxidized lipids are taken up by macrophages to form atherosclerotic plaques. Since adenosine prevents the production of superoxide radicals by granulocytes, the compounds of the present invention that increase adenosine release slow down, prevent, or reverse the progression of atherosclerosis.

[0051] The patient is a patient with (1) autoimmune disease, (2) arthritis, (3) psoriasis, (4) organ transplant rejection, (5) granulocyte activation mediated by complement after contact with a heart-lung or dialysis membrane, (6) ARDS or other inflammatory conditions. Since ATP catabolism is thought to occur during the inflammatory response, whether caused by granulocyte activation (such as in (1)-(6) above) or by mononuclear cell activation, symptoms should be alleviated by treatment with the compounds useful in the present invention.

[0052] Patients with diseases that are thought to be associated with chronic adenosine depletion (such as insomnia, autism, schizophrenia, and cerebral palsy) may also benefit from using the present invention to increase adenosine concentration.

[0053] Furthermore, treatment with the compounds of the present invention is also beneficial to patients with various diseases associated with mast cell degranulation. Examples of patients include those with allergies (especially asthma, hay fever, chronic urticaria, pigmented urticaria, and eczema). AICA riboside and ribavirin, for example, suppress the activation of mast cells (including prevention of mast cell degranulation). Since agents released from mast cells can spread damage during ischemia following processes such as arrhythmia or vasospasm, a decrease in mast cell activity is also beneficial to patients with reduced blood flow.

[0054] The compounds useful in the present invention are expected to be effectively administered in an amount in the range of about 0.1 mg / kg / day to about 500 mg / kg / day, preferably about 15 mg / kg / day to about 200 mg / kg / day. This dosage range should be particularly suitable for the compounds useful in the present invention as prophylactic agents for preventing tissue damage associated with undesirably restricted blood flow or reduced blood flow. It is further expected to use at least 0.1 mg / kg / day of AICA riboside or AICA ribotide, preferably about 1.0 mg / kg / day to about 500 mg / kg / day for prophylaxis, and more preferably about 20 mg / kg / day to about 100 mg / kg / day. For the above prophylaxis, at least about 0.1 mg / kg / day of ribavirin or ribavirin monophosphate is preferably administered at about 1.0 mg / kg / day to about 20 mg / kg / day. In the case of treating brain diseases (such as stroke, seizure, epilepsy, transient ischemic attack, autism, schizophrenia, cerebral palsy, and insomnia), due to the blood-brain barrier, a dosage exceeding 200 - 500 mg / kg / day may be required. However, when using a prodrug directly in the brain, a lower dosage may be sufficient.

[0055] Figure 1 shows the pathway by which adenosine is produced and degraded within cells. Adenosine either moves into cells or is released from cells. Adenosine metabolism may utilize some of the following pathways: 1 S-adenosylmethionine methyltransferase; 2 S-adenosylhomocysteine hydrolase; 3 adenosine deaminase; 4 purine nucleoside phosphorylase; 5 and 6 xanthine oxidase; 7 transport mechanisms; 8 adenosine phosphorylase (not established in humans); 9 adenosine kinase: 10 5’-nucleotidase and non-specific phosphatases; 11 adenylate kinase; 12 nucleoside diphosphate kinase; 13 adenylate cyclase; 14 AMP deaminase; and 15 adenylosuccinate synthetase and adenylosuccinate lyase.

[0056] As described in further detail below, the effects on extracellular adenosine concentration when using the described compounds (including purine nucleosides ribavirin and AICA riboside) are shown in vitro and in vivo. Since the compounds of the present invention are not readily degraded by extracellular enzymes in the body or when exposed to the low pH of the stomach, it is expected that these molecules are often administered orally to deliver them to patients. These drugs can also be administered by direct intramuscular injection for intravenous administration, subcutaneous administration, topical administration to the skin or mucosa, rectal administration, or inhalation. Compositions acceptable for pharmaceutical use are well known. Prodrugs (i.e., compounds that, when introduced into the body, are metabolized into the active form of the compounds of the present invention) may be used.

[0057] Since purine nucleoside AICA riboside is metabolized to uric acid, this agent may be used together with allopurinol or other drugs that prevent uric acid synthesis, or may be used together with a uric acid excretant (e.g., probenicid). Certain agents (e.g., methotrexate and ribavirin) inhibit ACIA ribotide transformylase, but this agent may increase the amount of endogenously synthesized AICA ribotide and produce the same effect as administering purine nucleoside. Co - administration of AICA riboside or AICA ribotide and an AICA ribotide transformylase inhibitor should have at least an additive effect. Furthermore, any one of the de novo purine nucleotide synthesis intermediates (after the first involved step of purine synthesis) or its nucleoside or base is presumed to be rapidly converted to AICA ribotide. An example is SAICA ribotide or its nucleoside or base.

[0058] The above - mentioned compounds can be used to increase the extracellular concentration of adenosine and thus can be used to treat diseases that result from or are exacerbated by insufficient blood flow to a particular organ or a part thereof. For example, heart attack or stroke, microvascular diseases due to diabetes (which may affect the brain, kidney, heart, skin, retina, and peripheral nerves and the microvascular system associated therewith), or events resulting from chronically reduced blood flow, such as angina pectoris, transient ischemic attack, intestinal ischemia, renal ischemia, intermittent claudication of skeletal muscle, migraine, and Raynaud's syndrome can be treated by administering the compounds of the present invention. Adenosine is a potent vasodilator that acts by suppressing the contraction of vascular smooth muscle and is also known to be an inhibitor of granulocyte - free radical generation, a process involved in ischemic disorders. As already described, this should be useful for the treatment of atherosclerosis.

[0059] Upon contact with cells, the compounds useful in the present invention enter the cells and are phosphorylated by adenosine kinase intracellularly, or when a base is administered, are converted to nucleotides by phosphoribosyltransferase enzymes, becoming purine nucleotide monophosphates and ultimately nucleotide triphosphates. This triphosphate form may accumulate as it is broken down to the monophosphate form.

[0060] Although not wishing to be bound by the following proposed modes of action, the compounds of the present invention or their metabolites are thought to inhibit one or more enzymes (including AMP deaminase) in the adenosine biosynthetic pathway, whereby ATP is not generated intracellularly, not released, adenosine is not reabsorbed, and at the same time inosine is not generated intracellularly or released.

[0061] It is important that ribavirin cannot be normally metabolized to purines (i.e., cannot be converted to AMP, ADP, ATP, IMP or guanosine phosphate GMP, GDP or GTP). In other words, the compounds useful in the present invention can increase the amount of adenosine released without being directly metabolized to adenosine. AICA riboside has biochemical properties similar to ribavirin and is thought to increase the amount of adenosine released by a mechanism similar to ribavirin, rather than being converted to adenosine in a roundabout way. This compound has been shown not to act by increasing the amount of ATP accumulated.

[0062] Figure 1 shows that adenosine is mainly metabolized in one of two modes. First, as shown by pathway 3, adenosine may be catabolized by adenosine deaminase enzyme to produce inosine. This inosine is then mostly further broken down to any substance by the enzymes represented by pathways 4, 5 and 6, or is excreted extracellularly through the plasma membrane. A transport mechanism 7 is shown by which adenosine can be transported in both directions through the cell membrane.

[0063] Adenosine may be assimilated by the adenosine kinase enzyme represented by 9 to become adenosine monophosphate (AMP), or may become S-adenosylhomocysteine by the S-adenosylhomocysteine hydrolase represented by 2 (depending on the availability of homocysteine). The former reaction is an energy-requiring reaction. AMP may act on the AMP deaminase enzyme (14) to produce inosine monophosphate (IMP), or may be further assimilated by various enzymatic reactions to produce adenosine triphosphate (ATP) or cyclic AMP. Inhibiting adenosine kinase or S-adenosylhomocysteine hydrolase can indirectly reduce the absorption of adenosine.

[0064] Referring to Examples I to III regarding the adenosine release amount, from the results of FIGS. 2, 3, 7 to 9 and Table 1, it is shown that when AICA riboside is present during net ATP catabolism, the cellular release amount of adenosine increases, and at the same time, the cellular release amount of inosine decreases (see Example IV and FIG. 5), which suggests that the conversion of AMP to IMP is inhibited or the conversion of adenosine to inosine is inhibited.

[0065] From the cell culture experiments of Examples I and II, it is shown that AICA riboside increases the cellular release amount of adenosine even in the presence of 2-deoxycoformycin, a potent inhibitor of adenosine deaminase. Therefore, the compounds of the present invention are considered to have the above effects at some point in the adenosine pathway other than or in addition to the reaction catalyzed by adenosine deaminase. The compounds of the present invention are considered to inhibit the conversion from AMP to IMP by interfering with the action of the AMP deaminase enzyme. The ability of the metabolites of the compounds of the present invention to inhibit the AMP deaminase enzyme was evaluated in Example VII, and the results are shown in FIG. 6. AICA riboside and ribavirin monophosphate, a structurally similar compound, were shown to have a similar inhibitory effect on AMP deaminase.

[0066] However, it is also possible that the compounds of the present invention inhibit the conversion of IMP to inosine by 5'-nucleotidase enzyme, reduce the amount of IMP degradation, and as a result increase the cellular release amount of adenosine. Furthermore, the compounds of the present invention may act to directly or indirectly inhibit the reabsorption, phosphorylation, or deamination of adenosine into cells.

[0067] In summary, it is considered that the compounds useful in the present invention can provide beneficial effects through a pathway in which the compounds enter cells, are ribosylated (when no sugar ring is present) and phosphorylated (when not yet phosphorylated), and become in the monophosphate form. The monophosphate form of this compound inhibits AMP deaminase. During ATP catabolism, since AMP is not easily converted to IMP, the amount of AMP accumulation in treated cells is increased compared to untreated cells. When purine monophosphate cleaves, the cellular release amount of adenosine increases, and at the same time the cellular release amount of inosine decreases. Since adenosine is considered a natural beneficial mediator during certain pathological events, the method of increasing the adenosine release amount by being converted to adenosine rather than being converted from ATP to inosine is a novel and particularly important treatment method.

[0068] During a heart attack, adenosine is released as usual, maintains the patency of ischemic blood vessels as described below, inhibits granulocyte free radical production, and at the same time helps prevent microvascular blockage. The compounds useful in the present invention increase the adenosine release amount, and the normal protective effect of adenosine is enhanced during such ischemic events.

[0069] Adenosine release can sometimes be a beneficial event, but high adenosine concentrations in areas where it is not needed can have adverse effects. One advantage of the invention described herein and in the claims is that, without treating the patient with adenosine itself, the amount of adenosine released from cells that undergo net ATP breakdown by the compounds useful in the present invention can be selectively increased. Thus, only the surrounding cells are treated. When a patient is treated with a compound useful in the present invention, the amount of adenosine released can be specifically increased in tissues that undergo net ATP catabolism (i.e., tissues where adenosine release is required). Systemic effects due to adenosine administration can be avoided. Furthermore, adenosine is released only at the specific times when it is needed. Any disease and condition described or disclosed herein is or is thought to be associated with local net ATP catabolism.

[0070] Furthermore, cells that respond beneficially to adenosine are more responsive than when continuously exposed to high concentrations of adenosine. Since adenosine is available only at the moment it is needed, the receptors on the cell surface (e.g., granulocytes and smooth muscle cells) are not continuously in contact with adenosine, so the adenosine receptors are not downregulated by continuous contact with adenosine and are more responsive.

[0071] In addition to acting to cause vasodilation by adenosine release, the compounds of the present invention can increase the blood flow in collateral blood vessels by a second mechanism. Tests have shown that in regions where blood flow is restricted, granulocytes are activated, oxygen free radicals are released, and the microvessels are clogged and destroyed. The drugs useful in the present invention increase the amount of adenosine released and prevent the generation of free radicals from granulocytes, thus suppressing the clogging of microvessels (see Example VIII), and blood flows from collateral blood vessels to the occluded region. As shown in Example IX, indium-labeled granulocytes flow out from the hearts of dogs treated with AICA riboside at 1 hour after ischemia. This outflow is significantly higher compared to dogs treated with saline, and the blood flow in collateral blood vessels also increases. Therefore, by absorbing the compounds of the present invention into muscle cells and / or endothelial cells and releasing adenosine during ischemia, vasodilation is achieved and / or the activation of granulocytes is suppressed, the clogging and damage of microvessels are suppressed, and the damage to the myocardium is reduced.

[0072] As shown in Examples I-VI, IX, XIII, and XIV, an important aspect of the compounds of the present invention is that they can be administered as prophylactic agents. When this drug is present in a beneficial state for an ischemic event, seizure activity, or other physical conditions that are treatment targets, the amount of adenosine is measured to be higher than the amount of inosine due to the net breakdown of ATP.

[0073] When this drug is administered to a patient so that it reaches the ischemic region after or during an ischemic event, the target amount of ATP accumulation is used up relatively quickly, so there is little or no ability for ATP to react with adenosine at this site. Also, since many of the damaging events during ischemia occur rapidly, this drug should ideally be present as early as possible. If this drug is present as a prophylactic agent, the process to be blocked can be delayed early enough to prevent permanent damage. For example, when vasodilation is induced to increase the blood flow in microvessels and reduce the clogging of white blood cells, the patency of the microvessels is maintained, blood clots are washed away from adjacent atherosclerotic regions, and substances that promote blood clots or other drugs with adverse effects are washed away.

[0074] There are other factors that are important to administer before or during the ischemic event. If this drug is administered after occlusion, there is little or no blood flow in this area, so it is hardly possible to deliver the drug to the involved tissues. See Example III and FIG. 4. For example, AICA riboside is metabolized to AICA ribotide, which is also considered to be the active form of the molecule. This reaction is an energy-requiring reaction that uses ATP. If ATP cannot be utilized due to high metabolic activity and / or an increase in the amount of ATP degradation, AICA riboside or a similar drug will not be in its active form. Furthermore, during rapid ATP degradation, inosine in the cell significantly competes with the drug entering the cell. This is because both compounds are purine nucleotide analogs.

[0075] Furthermore, the compounds of the present invention are considered to be beneficial in combination with certain other modes of treatment, as described below. When the compounds of the present invention are taken prophylactically, the amount of adenosine released during an acute ischemic event is increased, and when patients with a heart attack receive such treatment, sudden death due to arrhythmia before arriving at the hospital can be greatly reduced. Furthermore, the microvascular bed is protected while the patient is being transported to the hospital and until further treatment is administered.

[0076] Since acute ischemic events are often asymptomatic for a period of time, a significant amount of time has already passed by the time the patient realizes what has happened and seeks help. Thrombolytic treatment can be administered not only when medical assistance arrives at the patient's location, but also when the ambulance arrives or when the patient arrives at the hospital. All thrombolytic treatments (e.g., injection of tissue plasminogen activator (t-PA), streptokinase, urokinase, or anticoagulants (e.g., heparin or coumarin)) are useful for expanding occlusions in adjacent areas that occur during a heart attack or stroke, for example. Currently, patients need to receive this treatment within about 1 hour after an acute ischemic event occurs. After several hours, irreversible damage occurs to the tissue (especially the microvascular bed). If the patient has prophylactically ingested AICA riboside or another compound of the present invention, the patient's microvascular bed will be protected for a longer time because the amount of adenosine present has increased.

[0077] Increasing the amount of adenosine released prevents superoxide free radical generation and / or microvascular occlusion and damage. Therefore, the patient is protected for a long period of time after an acute ischemic event. For example, if it is 8 to 16 hours after the occurrence of a cardiovascular occlusion, one of the thrombolytic treatments can also be performed to open the adjacent lesion. Also, opening the adjacent lesion is the only beneficial method when it is possible to perfuse the downstream microvessels.

[0078] The compounds useful in the present invention are beneficial when combined with a thrombolytic agent (e.g., tissue plasminogen activator) and other agents that are free radical scavengers or other agents that prevent the generation of free radicals. Examples of free radical scavengers are superoxide dismutase, a protein that is infused after an ischemic event, or substances whose effectiveness has not been proven, such as catalase, acetylcysteine (mucomyst), vitamin E, glutathione, and selenium. Examples of compounds that are thought to prevent free radical generation are allopurinol by inhibiting xanthine oxidase, and icosopentanoic acid by downregulating prostaglandin metabolites, and finally, an antibody against a specific receptor for activated granulocytes. This antibody prevents microvascular occlusion. The compounds useful in the present invention inhibit the NADPH oxidase free radical generating system of granulocytes by increasing the amount of adenosine and are useful in combination with agents (e.g., allopurinol that inhibits free radical generation from xanthine oxidase).

[0079] Another disorder that is caused or may be caused by blood flow restriction is myocardial arrhythmia. The onset of arrhythmia is initiated by blood flow restriction, but the exact cause is unknown. However, it is known that lipid peroxidation by oxygen radicals is arrhythmogenic. Since oxygen radicals are produced by granulocytes, it is expected that arrhythmia can be controlled by inhibiting superoxide generation by granulocytes by the method of the present invention. Furthermore, the concentration of mast cells is high in atherosclerotic regions. Suppressing the activation of mast cells can result in a decrease in the release of other mediators of arrhythmia. Adenosine has a direct anti-arrhythmic effect on muscle cells. The prophylactic effect of treating arrhythmia with AICA riboside is shown in Examples VI and XIV, and according to these results, it has been shown that the number of premature ventricular contractions and ventricular tachycardia events decreases. During arrhythmia, the amount of ATP catabolism and adenosine release increase by rapidly burning cells.

[0080] Adenosine, which is released from neurons when neurons are stimulated and ATP is broken down during epileptic (epileptogenic) activation, normally feedback inhibits this epileptic (epileptogenic) activation. The presence of compounds useful in the present invention significantly increases the amount of suppression of epileptic events. Example XIII shows that AICA riboside reduces the frequency of seizures induced by pentylenetetrazol and prolongs the latency period.

[0081] Patients with autoimmune diseases, arthritis, or other inflammatory conditions are expected to have ATP catabolism occur during the cellular excitatory state associated with the inflammatory response, so treatment with purine nucleosides or analogs useful in the present invention alleviates the symptoms. Inflammatory diseases are diseases that commonly occur in humans and are thought to involve an immune response against one's own tissues. When the autoimmune response is heightened, different immune cells need to interact to suppress this response. Therefore, chemical substances that interfere with the necessary cell-cell interactions are thought to interfere with the onset of this series of diseases. One immune cell type necessary for the development of the autoimmune response is lymphocytes. Adenosine is well known to suppress the action of lymphocytes, and administration of compounds useful in the present invention (such as AICA riboside or ribavirin) inhibits or significantly reduces the aggregation of immune cells during inflammatory events and has a significant therapeutic effect on patients with inflammatory diseases. Furthermore, as described above, adenosine suppresses the production of oxygen-free radicals in granulocytes and suppresses the adhesion of endothelial cells, and these two phenomena are thought to be important factors in many inflammatory processes (such as autoimmune diseases).

[0082] Conditions that are potentially associated with a chronically low level of adenosine can also be treated with the compounds of the present invention. Such conditions include autism, insomnia, cerebral palsy, schizophrenia, and other neuropsychiatric symptoms. A dosage range of 0.1 mg / kg / day to about 200 mg / kg / day is understood to be beneficial. The results of a clinical trial of administering AICA to patients without adenylosuccinase (autism) are shown in Example X. When 5 mg / kg / day of AICA riboside was orally administered once, the dosage was increased to 5 mg / kg / day orally administered twice, and finally, 10 mg / kg / day was orally administered twice, a clear improvement was seen in one of the two patients, and according to the inventors' description, "good activity and easy handling during treatment" was observed for both patients, and thus the inventors are requesting the continuation of the clinical trial. No clinical or biochemical side effects were observed, suggesting that even higher dosages may result in even more beneficial effects.

[0083] Regarding mast cell degranulation, for example, treatment with AICA riboside or ribavirin is beneficial for patients with various diseases. For example, patients with allergies (especially asthma, hay fever (allergic conjunctivitis and allergic rhinitis), chronic urticaria, pigmented urticaria, and eczema) can be expected to benefit from treatment with purine nucleosides and purine nucleoside analogs. As discussed in the Textbook of Immunology by B. Benacerra and A. Unanue (Williams & Williams Baltimore / London, 1979), the key to suppressing the allergic reaction is to prevent the release of pharmacologically active substrates from mast cells. Mast cells are large basophilic staining cells with numerous granules containing substrates (such as histamine), and this substrate is released from mast cells during an allergic reaction and is necessary to maintain the allergic reaction. The release of these pharmacologically active substrates present in mast cells is called "degranulation". Therefore, a chemical substance that prevents degranulation should have a beneficial effect on reducing the severity of the allergic reaction. For this reason, allergic patients can be successfully treated with these molecules because AICA riboside or ribavirin prevents mast cell degranulation. When mast cells are activated, prostaglandins and leukotrienes (undetermined mediators), which are the delayed reactive substrates of anaphylaxis, are released. The purine nucleosides and analogs useful in the present invention also suppress the release of these mediators of inflammation. As shown in Example XI, when mast cells are prophylactically treated with ribavirin, β-hexosaminidase release is significantly weakened. The results are described in FIGS. 10 and 11. Similarly, from the results of Example XII, it was shown that AICA riboside inhibits mast cell activation (leukotriene C 4 release) and degranulation (β-hexosaminidase release).

[0084] The invention of the present applicants relates to the discovery of a specific therapeutic concentration for preventing tissue damage associated with reduced blood flow in humans with respect to AICA riboside, and an effective dosage while avoiding undesirable side effects. The invention of the present applicants also relates to the discovery of a specific therapeutic concentration and dosage of AICA riboside for preventing or reducing serious side effects (including such events in patients at risk of cardiovascular and / or cerebrovascular adverse events). The applicants have discovered that it is preferable to maintain the AICA riboside concentration in the blood vessel at about 1 μg / ml to about 20 μg / ml in order to obtain the beneficial effects of AICA riboside and prevent side effects that may occur at higher dosages. The applicants have discovered that the ideal range is about 3 to about 6 μg / ml, and particularly about 5 μg / ml.

[0085] Accordingly, in a first aspect, the present invention features a method for preventing tissue damage associated with reduced blood flow in a human, comprising administering to the human an amount of AICA riboside that maintains the plasma concentration of AICA riboside at about 1 μg / ml to about 20 μg / ml, preferably about 3 to about 6 μg / ml, more preferably about 5 μg / ml, for a time sufficient to reduce tissue damage in the human. By setting the AICA riboside in the human body to this concentration, it is desirable to set the serum uric acid to a concentration not exceeding about 16.0 mg / dl, more preferably not exceeding about 9.0 mg / dl.

[0086] "Preventing tissue damage" means reducing the frequency, duration, and / or severity of ischemic events and / or reducing the adverse effects of undesirable blood flow reduction to the tissue. The occurrence, duration, and severity of ischemic events may be measured by methods known in the art. For example, when using AICA riboside during coronary artery bypass graft (CABG) surgery, the following methods may be used: (1) comparison of the amount of ST segment change with respect to the recording results of a continuous Holter electrocardiogram; (2) evaluation of local wall motion by transesophageal echocardiography; (3) continuous measurement of creatine phosphokinase MB; and (4) continuous 12-lead electrocardiogram analysis. Methods for measuring the adverse effects of undesirable blood flow reduction are also known in the art. Adverse effects of tissue damage include adverse events, such as cardiovascular and / or cerebrovascular adverse events including events observed in relation to CABG surgery. Such adverse events include cardiac death (i.e., mainly due to causes related to the heart), transmural and / or non-transmural myocardial infarction, cerebrovascular disorders, congestive heart failure, and life-threatening arrhythmias that may occur during and / or after such surgery. Other adverse events preventable by administration of AICA riboside include liver damage (demonstrated by elevated enzymes), pancreatic damage (demonstrated by elevated enzymes), disseminated intravascular coagulation syndrome (including those caused by intestinal ischemia), and death (due to causes other than the heart). Reducing the risk of tissue damage means reducing the likelihood of tissue damage compared to the likelihood of tissue damage that exists when AICA riboside is not administered. The brain tissue can be protected from damage caused by blood flow reduction by using AICA riboside or its prodrug.

[0087] AICA riboside means 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (also known as acadesine).

[0088] In a second aspect, the invention features a method of preventing tissue damage associated with reduced blood flow in a human, which comprises administering to the human AICA riboside in an amount sufficient to maintain a plasma concentration of AICA riboside of from about 0.01 mg / kg / min to about 2.0 mg / kg / min; preferably from about 0.05 mg / kg / min to about 0.2 mg / kg / min; more preferably about 0.1 mg / kg / min for anesthetized patients and about 0.125 mg / kg / min for non-anesthetized or short-term anesthetized patients, for a time sufficient to reduce tissue damage in the human.

[0089] In certain embodiments, the tissue in which damage is prevented is the myocardium or the microvasculature of the heart. In other embodiments, the tissue in which damage is prevented is the brain tissue or the microvasculature of the brain.

[0090] In certain embodiments, the tissue damage to be prevented is tissue damage resulting from an undesired reduction in blood flow occurring during surgery, e.g., during cardiac surgery (e.g., CABG surgery) or vascular surgery. In these embodiments, depending on factors such as the duration of the surgery, administration of the above compound may begin shortly before induction of anesthesia, continue during the surgery, and for about 1 hour after the end of the surgery, or for at least about 7 hours or more after the end of the surgery.

[0091] In another embodiment, AICA riboside is administered in a perfusion fluid used to perfuse the heart of a patient undergoing cardiac surgery during the surgery. Preferably, the AICA riboside concentration in the perfusion fluid is from about 5 μM to about 100 μM; more preferably about 20 μM.

[0092] In another embodiment, AICA riboside is administered in combination with, or simultaneously with, allopurinol, preferably in an amount of about 100 mg / day to about 1200 mg / day, more preferably about 300 mg / day. Allopurinol reduces uric acid concentration and is administered in combination with, or simultaneously with, AICA riboside (or a prodrug of AICA riboside), so that a higher dosage of AICA riboside or prodrug can be administered while avoiding the occurrence of side effects due to an increase in uric acid concentration. As described above, it is desirable that the uric acid concentration does not exceed about 16 mg / dl, preferably does not exceed about 9 mg / dl.

[0093] In another embodiment, the invention further includes identifying a human in need of preventing a decrease in blood flow prior to administering AICA riboside (or a prodrug thereof). One of ordinary skill in the art will recognize that "identifying" means determining a patient at risk of tissue damage (e.g., a patient undergoing surgery or other procedure). Risk factors for patients undergoing cardiac surgery include advanced age (e.g., age over 70); emergency or urgent surgery that may be complicated by unstable angina; failure of percutaneous transluminal coronary angioplasty; reduced left ventricular function (such as determined by an ejection fraction of less than about 40%); chronic or acute renal insufficiency; arrhythmia (during treatment); or MI within the past several years. See, e.g., Mangano, Anesthesiology 72:153-184 (1990). Risk factors for patients undergoing non-cardiac surgery include advanced age (e.g., age over 65-70); coronary artery disease as evidenced by atherosclerotic heart disease, i.e., peripheral vascular disease or carotid artery disease; diabetes; renal insufficiency; heart failure during treatment; left ventricular hypertrophy and hypertension; hypertension for more than 5 years; emergency or urgent surgery; MI within 6 months to 1 year prior to surgery; angina; arrhythmia or hypercholesterolemia. The invention also includes identifying patients in need of prophylactic administration of AICA riboside due to chronic conditions, genetic conditions or similar conditions, or due to progressive or recent MI or progressive or recent stroke resulting from angina, transient ischemic attack. Thus, patients not undergoing surgery may similarly be at increased risk of tissue damage.

[0094] In another aspect, the invention features a method of preventing tissue damage associated with decreased blood flow in a human, comprising administering AICA riboside at a total dosage of 10 mg / kg to 200 mg / kg; preferably 30 mg / kg to 160 mg / kg. In cardiac surgery, the preferred amount is about 40 mg / kg. In other indications (e.g., non-cardiac surgery), the preferred amount is about 120 mg / kg. One of ordinary skill in the art will recognize that this total dosage can be achieved by varying the concentration of AICA riboside administered, the rate of administration and / or the time of administration.

[0095] In another aspect, the present invention features a method of preventing tissue damage associated with undesirable blood flow reduction in a human by administering a prodrug of AICA riboside in an amount effective to achieve a plasma concentration of AICA riboside of about 1 μg / ml to about 20 μg / ml, preferably about 3 to about 6 μg / ml, more preferably about 5 μg / ml. The amount of prodrug required to achieve this concentration can be readily determined by one of ordinary skill in the art using standard methodologies. The prodrug may be administered in combination with, or concurrently with, allopurinol, which may preferably be administered in an amount of about 100 mg / day to about 1200 mg / day, more preferably about 300 mg / day. This administration avoids side effects due to high uric acid concentrations. The prodrug may be administered as described above for AICA riboside itself.

[0096] In another aspect, the present invention features a method of preventing adverse clinical outcomes (including such events in patients at risk of cardiovascular and / or cerebrovascular adverse events) by administering AICA riboside or a prodrug thereof in an amount effective to achieve a plasma concentration of AICA riboside of about 1 μg / ml to about 20 μg / ml, preferably about 3 to about 6 μg / ml, more preferably about 5 μg / ml. "Adverse clinical outcomes" means events that clinically have an adverse effect on the patient. "Cardiovascular adverse events" means events related to the heart or blood vessels that are disadvantageous to the patient. "Cerebrovascular adverse events" means events related to blood vessels that affect the brain and are disadvantageous to the patient.

[0097] The present invention further includes the identification of patients at risk of adverse clinical outcomes, including cardiovascular and cerebrovascular adverse events. Risk factors for patients undergoing cardiac surgery include advanced age (e.g., age over 70 years); emergency or urgent surgery that may be complicated by unstable angina; failure of percutaneous transluminal coronary angioplasty; reduced left ventricular function (such as determined by an ejection fraction of less than about 40%); chronic or acute renal insufficiency; arrhythmia (during treatment); or MI within the past several years. See, e.g., Mangano, Anesthesiology 72:153-184 (1990). Risk factors for patients undergoing non-cardiac surgery include advanced age (e.g., age over 65-70 years); atherosclerotic heart disease, i.e., coronary artery disease as evidenced by peripheral vascular disease or carotid artery disease; diabetes; renal insufficiency; heart failure during treatment; left ventricular hypertrophy and hypertension; hypertension for more than 5 years; emergency or urgent surgery; MI within 6 months to 1 year prior to surgery; angina; arrhythmia or hypercholesterolemia. The present invention also includes the identification of patients in need of prophylactic administration of AICA riboside due to a chronic, genetic or similar condition, or due to progressive or recent MI resulting from angina, transient ischemic attack, or due to progressive or recent stroke or recent MI. Thus, patients who do not undergo surgery may also be at increased risk of tissue damage.

[0098] In another aspect, the present invention features a method of preventing adverse clinical outcomes (including such events in patients at risk of cardiovascular and / or cerebrovascular adverse events) by administering AICA riboside at a dosage of about 0.01 mg / kg / min to about 2.0 mg / kg / min, preferably about 0.05 mg / kg / min to about 0.2 mg / kg / min; more preferably about 0.1 mg / kg / min or 0.125 mg / kg / min, depending on the anesthesia, for a sufficient period of time to reduce the risk of tissue damage.

[0099] In certain embodiments, the cardiovascular adverse event to be prevented is myocardial infarction. "Myocardial infarction" includes transmural and non-transmural myocardial infarctions. In the case of CABG surgery, transmural MI is demonstrated by the presence of new Q waves during an ECG test and an increase in CK-MB concentration, and non-transmural MI is demonstrated by an increase in CK-MB concentration without the presence of new Q waves. In other embodiments, the cardiovascular event to be prevented is cardiac death. "Cardiac death" means the death of a patient primarily due to the heart (e.g., myocardial infarction, arrhythmia or ventricular dysfunction).

[0100] Another aspect of the invention provides a method of preventing or reducing side effects in a patient with myocardial infarction by administering an effective amount of acadine, or a prodrug, analog or salt thereof. In one embodiment, the myocardial infarction has occurred within the last 24 months, within the last 36 months and within the last 48 months. Another embodiment provides a method wherein the patient is female and / or is between about 65 and about 95 years of age.

[0101] In certain embodiments, the cerebrovascular event to be prevented is cerebrovascular disorder. "Cerebrovascular disorder" means a disorder of the brain associated with reduced blood flow (e.g., stroke).

[0102] In certain embodiments, the risk of cardiovascular adverse events or cerebrovascular adverse events results from symptoms (e.g., angina or transient ischemic attack). In other embodiments, the risk of cardiovascular adverse events or cerebrovascular adverse events results from cardiac surgery (e.g., CABG surgery) or as a result of non-cardiac surgery (e.g., vascular surgery). In the case of surgery, AICA riboside may be administered starting a little before induction of anesthesia, during the continuation of the surgery, and for about 1 hour after the end of the surgery, or about 7 hours after the end of the surgery. Administration may be continued for a longer time, e.g., 24 hours after the surgery or more. In the case of non-cardiac surgery, long-term administration is particularly effective because adverse events tend to occur later. For example, in cardiac surgery, MI tends to occur mainly on the day after the surgery, but in non-cardiac surgery, it has been observed that MI tends to occur mainly 2 or 3 days after the surgery. Therefore, in the case of non-cardiac surgery, AICA riboside (or prodrug) is administered for a long time (e.g., 7 to 48 hours) after the surgery.

[0103] Another aspect of the present invention provides a method for preventing or reducing side effects in a patient undergoing non-vascular surgery by administering to the patient an effective amount of acadine or a prodrug, analog, or salt thereof. Non-vascular surgeries include non-vascular surgeries of the abdomen, nerves, gynecology, orthopedics, urology, and otolaryngology. More specifically, non-vascular surgeries include resection of the small and large intestines, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringodectomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc.

[0104] In another embodiment, AICA riboside is administered to a patient undergoing such a surgery and is administered in a perfusion fluid used to perfuse the patient's heart during the surgery. Preferably, the AICA riboside concentration in the perfusion fluid is from about 5 μM to about 100 μM, more preferably about 20 μM.

[0105] In another embodiment, AICA riboside is administered in combination with, or concurrently with, allopurinol, preferably in an amount from about 100 mg / day to about 1200 mg / day, more preferably about 300 mg / day.

[0106] In another embodiment, the present invention provides a method for preventing or reducing the occurrence of cardiovascular adverse events or cerebrovascular adverse events in a patient undergoing CABG surgery. The method includes the following steps: (a) intravenously administering AICA riboside to the patient at 0.1 mg / kg / min for about 7 hours during the surgery; and (b) perfusing the patient's heart with a perfusion fluid of 20 μM AICA riboside.

[0107] Yet another aspect of the present invention provides a method for preventing stroke in a patient undergoing CABG by administering an effective amount of acadesine, or a prodrug, analog or salt thereof.

[0108] In another aspect, the present invention features a method for preventing or reducing the risk of a human at risk of myocardial infarction from developing severe myocardial infarction. The method includes administering AICA riboside or a prodrug thereof to a human for a time sufficient to reduce the risk of myocardial infarction in an amount such that the plasma concentration of AICA riboside in the human is from about 3 μg / ml to about 6 μg / ml. The risk of myocardial infarction can be increased by surgery, cardiac surgery (e.g., CABG surgery) or non-cardiac surgery (e.g., vascular surgery), or by factors other than surgery (e.g., signs of irreversible ischemia, such as angina or silent ischemia, or progressive MI or recent MI or stroke).

[0109] In another aspect, the present invention features a method of preventing or reducing the risk of a human at risk of cerebrovascular accident from developing a severe cerebrovascular accident. The method includes administering to the human an amount of AICA riboside or a prodrug thereof for a time sufficient to reduce the risk of cerebrovascular accident such that the plasma concentration of AICA riboside in the human is from about 3 μg / ml to about 6 μg / ml. The risk of cerebrovascular accident can be increased by surgery, cardiac surgery (e.g., CABG surgery) or non-cardiac surgery (e.g., vascular surgery), or by risks other than surgery (e.g., transient ischemic attack).

[0110] In another embodiment, the present invention features a method of preventing or reducing the risk of a human from developing a severe cardiac death. The method includes administering to the human an amount of AICA riboside or a prodrug thereof for a time sufficient to reduce the risk of cardiac death such that the plasma concentration of AICA riboside in the human is from about 3 μg / ml to about 6 μg / ml, preferably about 5 μg / ml. The risk of cardiac death can be increased by surgery, cardiac surgery or non-cardiac surgery. For example, this risk can occur with CABG surgery.

[0111] AICA riboside may be administered continuously or in multiple divided doses. To reduce the risk of tissue damage, AICA riboside may be administered for at least about 15 minutes. It may be administered for a time exceeding about 4 hours, preferably exceeding about 7 hours. In other cases, AICA riboside may be administered for a time exceeding about 10 hours, about 12 hours, about 16 hours, about 24 hours, or about 48 hours.

[0112] AICA riboside may be administered intravenously, orally, by injection into a coronary artery or an artery, or by any other method known in the art (including introduction into the patient's blood by extracorporeal circulation, e.g., using a heart-lung machine or dialysis). AICA riboside may be administered prophylactically or in response to a known physical condition.

[0113] In one embodiment, AICA riboside is prepared as a therapeutic solution from a lyophilized form in order to prevent the liquid formulation from discoloring to an observable extent during storage. Preferably, AICA riboside is non-pyrogenic.

[0114] Another aspect provides a pharmaceutical formulation comprising acadesine, or a prodrug, analog or salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. This formulation is provided to a patient in need of having the concentration of acadesine, or a prodrug, analog or salt thereof, in plasma reach 1 μg / ml to 20 μg / ml over a sufficient period of time, and this formulation is lipophilic. In one embodiment, this period is about 7 hours. In another embodiment, this pharmaceutical formulation is in micelle form.

[0115] In another aspect, the present invention features a kit for administering AICA riboside to a patient undergoing cardiac surgery (e.g., CABG surgery), comprising a lyophilized form of AICA riboside used to prepare an AICA riboside solution for perfusion into the vein of a patient undergoing cardiac surgery, and an AICA riboside solution used to prepare a myocardial protection perfusion solution for perfusion into the heart of a patient undergoing cardiac surgery. Preferably, AICA riboside is non-pyrogenic. Preferably, the lyophilized AICA riboside is provided in an amount of 100 mg to 2,000 mg, more preferably in an amount of 500 mg. Preferably, the AICA riboside solution is provided in a volume of 1 ml to 20 ml, more preferably in a volume of 5 ml. Preferably, the concentration of AICA riboside in the solution is about 1 mg / ml.

[0116] The lyophilized AICA riboside may be mixed with a suitable diluent, such as water or saline solution, to form a form suitable for injection into a patient.

[0117] The AICA riboside solution may be an aqueous solution, a saline solution, or a myocardial protective solution. The AICA riboside solution is a concentration suitable for adding a myocardial protective perfusion solution so that the final concentration of AICA riboside in the myocardial protective solution is 5 μM to 100 μM, preferably 20 μM. For example, when 5 ml of 1 mg / ml AICA riboside is added to 1 L of the myocardial protective perfusion solution, the resulting concentration is about 5 μg / ml or 20 μM.

[0118] One of the advantages due to the applicants' discovery of specific useful therapeutic concentrations and dosages of AICA riboside is that side effects due to increased uric acid concentration in serum or urine are reduced, and / or the effect of reduced crystalluria is obtained at this dosage, and even if this effect cannot be exerted, the blood glucose concentration is reduced.

[0119] What the applicants further discovered is that in the case of an anesthetized patient, a lower dosage is required to achieve the desired blood concentration than in a non-anesthetized patient. The dosage required in an anesthetized patient is considered to be about 20 - 50% of that in a non-anesthetized patient. Therefore, the preferred dosage of AICA riboside (or prodrug) for a non-anesthetized patient or a patient anesthetized for a short period is higher than the preferred dosage for an anesthetized patient. Therefore, a dosage of about 0.075 mg / kg / min to about 0.30 mg / kg / min is preferred in this case, more preferably about 0.10 mg / kg / min to about 0.15 mg / kg / min, and most preferably about 0.125 mg / kg / min.

[0120] (Definition) As used herein, unless otherwise indicated to the contrary, the following terms have the meanings set forth below.

[0121] The term "hydrocarbyl" refers to an organic group composed mainly of carbon and hydrogen, including alkyl groups, alkenyl groups, and alkynyl groups, and aromatic groups including aryl groups and aralkyl groups, and groups having a mixture of saturated and unsaturated bonds, alicyclic groups (carbocyclic or cycloalkyl) groups, or these groups substituted with aryl (aromatic) groups or combinations thereof, and may refer to a group including a linear, branched, or cyclic structure or a combination thereof.

[0122] The term "alkyl" refers to a saturated aliphatic group including linear, branched, and carbocyclic groups. The term "lower alkyl" refers to a linear or branched alkyl group having from 1 to 6 carbon atoms in total, including primary, secondary, and tertiary alkyl groups. Typical lower alkyls include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like.

[0123] The term "aryl" refers to an aromatic group having about 6 to 14 carbon atoms and includes cyclic aromatic systems (such as phenyl and naphthyl).

[0124] The term "aralkyl" refers to an alkyl group having about 1 to 4 carbon atoms substituted with an aryl group having 6 to 10 carbon atoms, and examples include benzyl, p-chlorobenzyl, p-methylbenzyl, and 2-phenylethyl.

[0125] The term "alkenyl" refers to an unsaturated alkyl group having at least one double bond (e.g., CH 3 CH=CH(CH 2 ) 2 --) and includes both linear alkenyl groups and branched alkenyl groups.

[0126] The term "alkynyl" refers to an unsaturated group having at least one triple bond (e.g., CH 3 C≡C(CH 2 ) 2 ) and includes both linear and branched groups.

[0127] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0128] The term "acyl" refers to the following group

[0129]

Chem.

[0130] The term "alkylene" refers to bifunctional straight-chain, branched-chain, and cycloalkylene groups, such as ethylene, propylene, 2-methylpropylene (e.g.,

[0131]

Chem.

[0132]

Chem.

[0133] The term "amide" or "amido" refers to the following group

[0134]

Chem.

[0135] The term "carboxamide" refers to the following group

[0136]

Chem.

[0137]

Chem.

[0138] The term "acylamino" refers to the following group

[0139]

Chem.

[0140] The term "carbonate ester" refers to the following group

[0141]

Chem.

[0142] The term "acyl ester" refers to the following group

[0143]

Chem.

[0144] The term "phosphate ester" refers to the following group

[0145]

Chem.

[0146] The term "mixed ester" refers to a compound having at least one carbonate ester group and at least one acyl ester group, or a compound having a combination of different acyl ester or carbonate ester groups.

[0147] The term "carboxylic acid ester" or "carboxy ester" refers to the following group

[0148]

Chem.

[0149] The term "carbocyclic AICA riboside" refers to an analog of AICA riboside in which the oxygen atom of the ribosyl ring is replaced by methylene (--CH 2 --)

[0150] The term "hydrocarbyloxy" refers to the group R'O-- (wherein R' is hydrocarbyl).

[0151] The term "alkoxy" refers to the group R'O-- (wherein R' is alkyl).

[0152] The term "hydrocarbylthio" refers to a group having the formula R'S- (wherein R' is hydrocarbyl).

[0153] The term "hydrocarbylamino" refers to the group --NHR' or --NR' 2 (wherein R' is an independently selected hydrocarbyl group).

[0154] The term "hydrocarbylimidate" refers to the following group

[0155]

Chem.

[0156] The term “carboxamidooxime” refers to the following group

[0157]

Chem.

[0158] The term “hydrocarbyloxyamidine” refers to the following group

[0159]

Chem.

[0160] The term “hydrocarbyloxycarbonyl” refers to the following group

[0161]

Chem.

[0162] The term “hydrocarbyloxycarboxy” refers to the following group

[0163]

Chem.

[0164] The term “thioester” refers to the following group

[0165]

Chem.

[0166] (Preferred AICA riboside analogs) According to the present invention, preferred analogs of AICA riboside include compounds of formula I or pharmaceutically acceptable salts thereof.

[0167] [Chemical formula] In the formula, X is --O-- or --CH 2 --; R 1 is hydrogen, amino, hydrocarbylamino, acylamino or dihydrocarbylaminoalkyleneamino; R 2 is hydrogen, cyano, hydrocarbylimidate, carboxamideoxime, hydrocarbyloxyamidine, carboxamide or carboxylic acid or amide, ester, thioester or a salt thereof; R 3 is hydrogen, hydrocarbyl, amino, hydrocarbylamino, halogen, hydroxy (including the tautomer 2-imidazolone), hydrocarbyloxy, sulfhydryl (including the tautomer 2-imidazolethione), or hydrocarbylthio; R 4 and R 5 are independently hydrogen, alkyl, acyl or hydrocarbyloxycarbonyl; R 6 is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azide, acyloxy or hydrocarbyloxycarboxy or phosphate ester group or a salt thereof; provided that when R 1 is amino, R 2 is unsubstituted carboxamide, R 3 is hydrogen; R 4 and R 5 are hydrogen, acyl or hydrocarboxycarbonyl, R 6 is not hydroxy, acyloxy or hydrocarbyloxycarboxy.

[0168] Or, R 2may be a group having the following formula.

[0169]

Chemical formula

[0170] Preferred compounds include (i) compounds in which R 1 is amino, R 2 is carboxamide, one of the amide hydrogens is substituted with a hydrocarbyl group, more preferably an aralkyl group (e.g., the hydrocarbyl group or aralkyl group may be optionally substituted with suitable substituents as described below), R 3 is hydrogen, R 4 and R 5 are hydrogen or hydrocarbyloxycarbonyl, and more preferably R 6 is hydroxy or amino (series I); (ii) compounds in which R 1 is amino, R 2 is carboxamide, R 3 is halogen or sulfhydryl, R 4 is hydrogen, R 5 is hydrogen, and R 6 is hydroxy (series II); (iii) compounds in which R 1 is amino, R 2 is carboxamide, R 3 , R 4 and R 5 are hydrogen, and R 6Compounds in which (Series III) and (iv) R is amino 1 is amino and R 2 is carboxamide and R 3 is hydrogen and R 4 is alkyl and R 5 is hydrogen and R 6 Compounds in which (Series IV) is hydroxy are exemplified.

[0171] Particularly, from the viewpoint of showing activity in various experimental models, preferred compounds include Compounds No. 10, 23, 25, 29, 47, 52, 53 (Series I), 27, 43 (Series II), 21, 66 (Series III) and 20, 34 (GP-1-250) and 32 (GP-1-262) (Series IV) in Tables XII and XIII.

[0172] (Preferred novel AICA riboside analogs) One preferred group of the compounds of Formula I includes specific novel AICA riboside analogs. In this analog, X is --O-- or --CH 2 --; R 1 is amino, hydrocarbylamino or dihydrocarbylaminoalkyleneamino, and R 2 is carboxamide or cycloalkyl or aryl or aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide, in which one of the amide hydrogens (hydrogens bonded to the nitrogen atom) is optionally replaced by alkyl; or R 2 is carboxamide in which both amide hydrogens are replaced by alkyl or both are replaced by an alkylene group or an aralkylene group to form a ring; or R 2 is --C(O)--S--R 7 (R 7is alkyl, cycloalkyl, aryl or aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide; or R 2 is a group of formula II, where R 1 , R 3 , R 4 , R 5 and R 6 are as defined above together with formula I, alk is alkylene having 2 to 8 carbon atoms; R 3 is hydrogen, amino, hydrocarbylamino, halogen, hydroxy (including the tautomer 2-imidazolone), hydrocarbyl, sulfhydryl (including the tautomer 2-imidazolethione) or hydrocarbylthio; R 4 and R 5 are independently hydrogen, hydrocarbyl (having 1 to about 18 carbon atoms), acyl or hydrocarbyloxycarbonyl; R 6 is hydroxy, hydrogen, hydrocarbyl, halogen, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azide, acyloxy, hydrocarbyloxycarbonyl or phosphate ester or salt; provided that --X-- is --O-- or --CH 2 --, R 1 is amino, R 2 is unsubstituted carboxamide, R 3 is hydrogen, R 4 and R 5 are hydrogen, acyl or hydrocarboxycarbonyl, R 6 is not hydrogen, hydroxy, acyloxy or hydrocarbyloxycarbonyl, or when R 4 and R 5 are both hydrogen, R 6 is not a phosphate ester; X is oxygen, R 1 is amino, R 2is an unsubstituted carboxamide, and R 3 is sulfhydryl, and R 4 and R 5 are both hydrogen, then R 6 is not acetoxy; when X is oxygen and R 1 is amino and R 2 is an unsubstituted carboxamide, and R 3 is chloro, bromo, amino or methoxy, and R 4 and R 5 are both hydrogen, then R 6 is not hydroxy, or when R 4 and R 5 are both acetyl, then R 6 is not acetoxy; provided that when X is oxygen and R 1 is amino and R 2 is benzylcarboxamide or p-iodophenylcarboxamide, and R 3 is hydrogen, then R 4 and R 5 are not both hydrogen, then R 6 is not hydroxy; or when R 2 is p-iodophenylcarboxamide, then R 4 and R 5 are not both acetyl, then R 6 is not acetoxy.

[0173] Preferred compounds include those in which R 1 is amino and R 2 is a carboxamide substituted with an aralkyl group having 1 to 3 ring substituents as described above (more preferably a benzyl group), or a cycloalkyl. Preferred compounds from the viewpoint of showing activity in various experimental models include the compounds of Compound Nos. 23, 25, 29, 47, 52 and 53.

[0174] An example of a particularly preferred compound is one in which X is oxygen, R 1 is amino, R 2 is p-chlorobenzylcarboxamide, and R 3 , R4 and R 5 is hydrogen and R 6 is amino, and salts thereof. One particularly preferred salt is the hydrochloride salt. Other particularly preferred salts are the sodium and potassium salts, especially the disodium and monopotassium salts.

[0175] A preferred AICA riboside analog is a compound represented by formula (Ia).

[0176]

Chemical formula

[0177] In one aspect, the present invention provides a kit for use in administering a compound of formula Ia, a prodrug, analog or salt thereof, to a patient undergoing cardiac surgery, comprising a lyophilized form of a compound of formula Ia, a prodrug, analog or salt thereof for injection into the patient, and a solution of a compound of formula Ia, a prodrug, analog or salt thereof for perfusion of the patient's heart. In another aspect, the present invention provides a kit for use in administering a compound of formula Ia, a prodrug, analog or salt thereof, to a patient, comprising a sterile container of a lyophilized compound of formula Ia, a prodrug, analog or salt thereof.

[0178] In one embodiment, the present invention provides a cardioplegic solution comprising a composition containing a compound of formula Ia.

[0179] In another embodiment, the present invention provides a method for preventing or reducing side effects in a patient undergoing CABG surgery, comprising administering to the patient during surgery a composition containing an effective amount of a compound of formula Ia, a prodrug, analog or salt thereof.

[0180] In another embodiment, the present invention provides a pharmaceutical formulation comprising a compound of formula Ia, its prodrug, analog or salt, and at least one pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, this formulation is provided to a patient who needs to have a plasma concentration of 1 μg / ml to 20 μg / ml over a sufficient period of time. In another embodiment, this period is about 7 hours. In another embodiment, this formulation is applicable for oral administration. In another embodiment, this formulation is applicable for oral administration in a solid dosage form.

[0181] In another aspect, the present invention provides a myocardial protection solution comprising a composition containing a compound represented by formula (IIa) and a pharmaceutically acceptable salt thereof.

[0182] [Chemical formula] In the formula, R 2 is hydrogen, --CN and the following groups

[0183] [Chemical formula] selected from the group consisting of, T is oxygen, sulfur, NOH, NH and NO(CH 2 ) n CH 3 (n is 0 to 2), U is lower alkoxy, amino, a 3- to 6-membered heterocyclic ring optionally condensed with a 3- to 6-membered aryl ring, and the following groups:

[0184] [Chemical formula] [In the formula, A is either NH or S, n is 0 to 3, i is 0 to 2, Q is either hydrogen or hydroxy, E represents a nitro group or a hydroxy group, provided that when U is amino, T is not sulfur, NOH, NH or NOCH 3 ; when T is amino, U is not lower alkoxy; when A is amino and n is 1, Q is not hydroxy; R 3 is selected from hydrogen, halogen and S--W (where W is phenyl or substituted phenyl or hydrogen), and when T is not oxygen, U is not amino; R 4 and R 5 are each independently selected from hydrogen, --COCH 3 and lower alkyl, or together form a cyclic carbonate; R 6 is hydroxy, phosphate ester, --OSO 2 NH 2 , sulfhydryl, halogen, --OCOCH 3 , --SCH 3 , --SOCH 3 , NH 2 and N 3 selected from, provided that when R 2 is CONH 2 , CONH - para - iodophenyl, hydrogen, CN or CONHCH 2 --φ, R 3 is hydrogen or halogen, R 4 and R 5 are hydrogen, acyl, or together form a cyclic carbonate, R 6 is not halogen, phosphate ester, OH or --O - acyl. The concentration of this compound, its prodrug, analog or salt is 5 μM to 100 μM.

[0185] In one embodiment, a kit for use in administering an acadine analog to a patient undergoing heart surgery may include a lyophilized form of a compound of formula IIa, its prodrug, analog or salt for injection into the patient, and / or a solution form of a compound of formula IIa, its prodrug, analog or salt for perfusion into the patient's heart.

[0186] Preferred AICA riboside analogs are 5-amino-1-β-D-(5-benzylamino-5-deoxy-1-β-D-ribofuranosyl)imidazole-4-carboxamide having the chemical structure of formula (IIIa).

[0187]

Chemical Structure

[0188]

Chemical Structure

[0189] In one aspect, the present invention provides a cardioplegic solution containing a compound of formula IIIa or formula IVa at a concentration of about 5 μM to about 100 μM. In another aspect, a lyophilized form of a compound of formula IIIa or formula IVa, its prodrug, analog or salt for injection into a patient, and a solution containing a compound of formula IIIa or formula IVa, its prodrug, analog or salt for perfusion into the patient's heart are provided. A kit for use in administering a compound of formula IIIa or formula IVa to a patient undergoing cardiac surgery is provided.

[0190] In another aspect, the present invention provides a kit for administering a compound of formula IIIa or IVa, its prodrug, analog or salt to a patient, which includes a sterile container of the lyophilized compound of formula IIIa or IVa, its prodrug, analog or salt.

[0191] In another aspect, the present invention provides a method for preventing or reducing side effects in patients with reduced left ventricular function with an ejection fraction of less than 30% by administering to a patient an effective amount of a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof. In another aspect, the present invention provides a method for preventing or reducing side effects in patients having a history of one myocardial infarction, a history of two myocardial infarctions, a history of three myocardial infarctions or a history of at least three myocardial infarctions by administering to a patient an effective amount of a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof. In one embodiment, the most recent myocardial infarction has occurred within the most recent 24 months, within the most recent 36 months and within the most recent 48 months. In another embodiment of the two methods described above, the patient is female and / or is between about 65 and about 95 years of age. In another embodiment, the compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof is administered at a concentration such that the concentration in the patient's plasma is from about 1 μg / ml to about 20 μg / ml over a sufficient period of time. In one embodiment, the plasma concentration is maintained for about 7 hours. In another embodiment, the compound of formula IIIa or formula IVa, or a prodrug, analog or salt thereof is administered at 0.1 mg / kg / min. In another embodiment, the compound of formula IIIa or formula IVa is administered to the patient for about 7 hours.

[0192] Another aspect of the present invention provides a method for preventing or reducing side effects in a patient undergoing non-vascular surgery by administering to the patient an effective amount of a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof. In one embodiment, the present invention is applicable to a wide range of non-vascular surgeries, such as, but not limited to, non-vascular heart surgery, non-vascular abdominal surgery, non-vascular nerve surgery, non-vascular gynecological surgery, non-vascular orthopedic surgery, non-vascular urological surgery, non-vascular vascular surgery, and non-vascular otolaryngological surgery. More specifically, non-vascular surgeries include resection of the small and large intestines, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringotomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc.

[0193] Another aspect provides a pharmaceutical formulation comprising a compound of formula IIIa or formula IVa, a prodrug, analog or salt thereof, and at least one pharmaceutically acceptable carrier, diluent or excipient, which formulation is provided to a patient in need thereof where the concentration of the compound of formula IIIa or formula IVa, a prodrug, analog or salt thereof in plasma is required to be from about 1 μg / ml to about 20 μg / ml over a sufficient period of time. In another embodiment, this period is about 7 hours. In another embodiment, this pharmaceutical formulation is in micellar form. In one embodiment, this formulation is lipophilic.

[0194] In another aspect, the present invention is used to administer acadine, or a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof, to a patient in need thereof, and provides a pharmaceutical formulation for spray or aerosol.

[0195] In another aspect, there is provided a kit for use in administering to a patient undergoing cardiac surgery, acadesine, or a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof, in a lyophilized form for use in preparing a solution containing acadesine, or a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof for injection, and an aerosol form or sprayable form of acadesine, or a compound of formula Ia, IIa, IIIa or formula IVa, or a prodrug, analog or salt thereof for direct application to the patient's heart. In another embodiment, the solution for injection is for spraying or for aerosol use.

[0196] (Preparation of Preferred Novel AICA Riboside Analogs) The novel substituted imidazole analogs of the present invention can be synthesized by well-known chemical reactions as shown in the following examples. Generally, the compounds of formula (I) are prepared from 4-methyl-5-nitro-1H-imidazole via the route described by Baker et al. (Baker D., J. Org. Chem. 47:3457 (1982)) to prepare 1-benzyl-5-nitro-1H-imidazole-4-carboxylic acid and reducing the nitro group to obtain the desired amino group at R 1 Alternatively, an elegant method for synthesizing AICA riboside has been reported by Ferris et al. (Ferris, J.P., J. Org. Chem. 50:747 (1985)), which is a general synthetic route to 4-substituted 5-imidazoles starting from appropriately protected ribosides and diaminomaleonitrile. This route allows for the introduction of the desired R 3 alkyl, hydrocarbyl and aryl groups by selecting an appropriate orthoester and cyclizing the maleonitrile to obtain an imidazole. Other desired R 3The substituents can be introduced by the method described by Miyoshi et al. (Miyoshi T., Chem. Pharm. Bull. 24(9):2089(1976)) for the preparation of 2-bromo and 5-amino-2-thio-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-4-imidazolecarboxamide or by the method of Ivanovics et al. (Ivanovics, G. A. et al., J. Org. Chem. 25:3631(1974)) for the preparation of 5-aminoimidazole-4-carboxamide substituted with 2-alkoxy, 3-amino, and 2-hydroxy (tautomeric form 2-imidazolone). Desirable R 1 Compounds in which the substituent is acylamino can be prepared by acylating the corresponding appropriately protected R 1 amino compound with the desired acyl anhydride and de-O-acylating with ammonia or sodium methoxide. Compounds in which R 1 is alkylamino or arylamino can be prepared by reductive alkylation of the corresponding appropriately protected R 1 amino compound with the desired hydrocarbylamine as described by Sato et al. (Chem. Pharm. Bull. 37:1604(1989)).

[0197] R 6 Compounds in which R is acyloxy or hydrocarbyloxycarbonyl can be selectively prepared by reacting the appropriate hydrocarbyl acid anhydride or hydrocarbyl chloroformate with the riboside protected with 2’3’-O-isopropylidene as described by Miyoshi et al. (supra) and removing the isopropylidene group with a dilute aqueous solution of acid. Compounds in which R 6 is hydrocarbyloxy can be prepared from the protected 5-substituted pentose using the method of Ferris et al. (supra) (Snyder J. R., Carbonhydr. Res. 163:169(1987)). 6A compound of formula (I) wherein R is sulfhydryl, hydrocarbylthio or hydrocarbylamino can be prepared from 5'-deoxy-5'-iodo-2,3'-isopropylideneimidazole riboside (Srivastava P.C., J. Med. Chem. 18:1237 (1975)) by nucleophilic substitution of the halogen with a desired amine or mercaptan. R 6 A compound of formula (I) wherein R is alkylamide or arylamide can be prepared from the corresponding 5-amino-5'-deoxyimidazole riboside by acylating with a desired alkyl or aryl acid anhydride and de-O-acylating with ammonia or sodium methoxide. R 6 A compound of formula (I) wherein R is hydrocarbyl can be prepared from 1-(2,3-O-isopropylidene-β-D-ribo-pent-1,5-dialdo-1,4-furanosyl)imidazole by a modification of the Wittig reaction of nucleotides described by Montgomery et al. (J. Het. Chem. 11:211 (1974)). R 6 A compound of formula (I) wherein R is phosphate or phosphate ester can be prepared by the general method of Khwaja et al. (Tetrahedron 27:6189 (1971)) for nucleoside phosphates.

[0198] (Utility) The AICA riboside analog compounds of the present invention are particularly useful for reducing or preventing damage (i.e., a condition resulting from restricted blood supply) occurring during events associated with ischemia. Such damage events include heart attacks, or myocardial infarctions, a condition in which one or more of the coronary arteries that supply blood to the heart muscle (i.e., myocardium) become blocked and, if prolonged, additional adverse tissue damage occurs. Compounds such as AICA riboside increase local adenosine concentration, increase blood flow to ischemic myocardium, and reduce tissue damage.

[0199] One of the current treatments for heart attacks is thrombolytic therapy, which involves administering a thrombolytic agent (e.g., streptokinase or tissue plasminogen activator factor (tPA)). However, these drugs must be used within several hours (1 - 3 hours) of a heart attack, and the later they are used, the more significantly their effectiveness decreases. The compounds of the present invention can be administered prophylactically (i.e., before the event) to obtain benefits and are clearly useful.

[0200] Angina pectoris is a condition where there is sufficient blood supply to meet the normal needs of the heart, but the blood supply to the heart is not sufficient when the amount of supply to the heart needs to be increased (e.g., during exercise) and / or when the blood supply is restricted (e.g., during coronary artery spasm). Patients with angina pectoris or patients with a condition related to a transient ischemic event or an ischemic state without subjective symptoms can also benefit from this type of treatment with adenosine.

[0201] Currently, many clinical procedures are used to increase the blood supply to the heart for advanced coronary artery disease or persistent chest pain at rest. These procedures include percutaneous transluminal coronary angioplasty (PTCA) (also known as angioplasty), percutaneous transluminal directional coronary atherectomy, laser atherectomy, intravascular stents, and coronary artery bypass graft surgery. The compounds of the present invention are also useful as adjuvant therapies for these techniques.

[0202] Another factor that causes cardiovascular problems is cardiac arrhythmia (i.e., irregular heartbeat), which results in insufficient ability of the heart to supply blood. The ability of these compounds (e.g., AICA riboside) to reduce arrhythmia is useful for suppressing this condition.

[0203] Stroke and traumatic conditions of the central nervous system (CNS) result from a decrease in blood supply to the CNS and are susceptible to treatments that increase the adenosine concentration in the endangered tissue to facilitate tissue survival. Other indications improved by agents that affect local blood flow include organ transplantation, skin flap grafting in reconstructive surgery, peripheral vascular diseases, endotoxemia, hemorrhagic shock, pulmonary edema, lung injury or sepsis, which are secondary complications of burns (thermal injuries), pulmonary hypertension, microembolization, impotence, glomerulonephritis or progressive glomerulosclerosis, atherosclerosis, myocarditis, vasculitis and cardiomyopathy, and cardiac arrest.

[0204] It is now clear that a prominent component of the neurodegeneration resulting from stroke or CNS trauma is caused by an increased release of excitatory amino acids, which results in neurons being stimulated to death. Adenosine has been reported to inhibit the release of excitatory amino acids (Burke and Nadler I. Neurochem. 51:1541 (1988)). Since the compounds of the present invention raise the adenosine concentration, they are also useful in conditions involving excitatory amino acids such as Huntington's chorea or Alzheimer's disease (Marangos et al. Trends Neurosci. 10:65 (1987)) and Parkinson's disease (Sonsella et al. Science 243:398 (1989)). These studies, together with the results from experimental models of memory (Harris et al. Brain Res. 323:132 (1984)), further suggest that these compounds are useful for treating disorders related to the effects of the aging process on CNS function.

[0205] Adenosine has been reported to be an endogenous regulator of inflammation because it affects the function of stimulated granulocytes (Cronstein et al., J. Clin. Invest. 78:760-770 (1986)) and affects the functions of macrophages, lymphocytes and platelets. Accordingly, the compounds of the present invention are useful in conditions involving inflammatory processes such as arthritis, osteoarthritis, autoimmune diseases, adult respiratory distress syndrome (ARDS), inflammatory bowel disease, necrotizing enterocolitis, chronic obstructive pulmonary disease (COPD) and other inflammatory disorders.

[0206] Adenosine has been shown to serve as a natural anticonvulsant (Lee et al., Brain Res. 321:1650-1654 (1984); Dunwiddie, Int. Rev. Neurobiol. 27:63-139 (1985)). Accordingly, agents that increase adenosine concentration are useful for the treatment of seizure disorders. In recent studies, Marangos et al., Epilepsia 31:239-246 (1990) reported that AICA riboside is an inhibitor of seizures in experimental animal models.

[0207] AICA riboside analogs are also useful for the treatment of patients with chronically low adenosine concentrations or patients for whom high adenosine concentrations are beneficial, such as patients with autism, cerebral palsy, insomnia, anxiety or other neuropsychiatric disorders or patients with irritable bowel syndrome. Indeed, many studies (Komhuber and Fischer Neurosci. Lett. 34:32 (1982); Kim et al. Eur. Neurol. 22:367 (1983)) have related the pathophysiology of schizophrenia to excitatory amino acids.

[0208] The compounds of the present invention are also useful for treating other conditions in which AICA riboside itself exerts beneficial effects. For example, AICA riboside has been reported to have an anti-allergic effect in a bronchospasm model of guinea pigs induced by antigen sensitization (submitted to Bergren et al. (J. of Allergy and Clinical Immunology (1990))), so AICA riboside analogs are also beneficial for the treatment of asthma, hay fever or allergic diseases.

[0209] Accordingly, the AICA riboside analogs of the present invention are useful for treating various clinical conditions where it is beneficial to increase extracellular adenosine concentration and, in some cases, simultaneously scavenge free radicals and / or antioxidant activity is also beneficial.

[0210] The compounds of the present invention are administered to the affected tissue at a rate of 0.01 to 3.0 μmole / min / kg, preferably 0.1 to 1.0 μmol / min / kg. In situations where a longer infusion is desired, the compound may be administered at a lower rate, for example 0.003 to 0.3 μmole / kg / min, preferably 0.01 to 0.1 μmole / kg / min. If these compounds are administered intravenously as discussed below, the rates described above are readily maintained. When other methods (e.g., oral administration) are used, sustained release formulations may be preferred to control the release rate of the active ingredient. These compounds are administered at a dosage of about 0.01 mg / kg / day to about 200 mg / kg / day, preferably about 0.5 mg / kg / day to about 100 mg / kg / day. Examples of preferred oral dosages are 0.3 to 30 mg / kg / day, most preferably 1 to 10 mg / kg / day.

[0211] For the purposes of the present invention, the compounds of the present invention may be administered by various means. Such various means include formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles, and include oral administration, parenteral administration by spray inhalation, topical administration, or rectal administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, and arterial injections using various injection techniques. Arterial injection and intravenous injection, as used herein, include administration by catheter. Preferred for certain indications is an administration method that can rapidly reach the tissue or organ being treated. For example, in the case of myocardial infarction, intravenous injection is preferred. When an organ outside the body is treated, perfusion is preferred.

[0212] The pharmaceutical composition containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. The compositions for oral purposes may be prepared according to any method known in the art of manufacturing pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives in order to obtain a good preparation. Tablets containing the active ingredient together with a non-toxic pharmaceutically acceptable excipient suitable for the manufacture of tablets are also preferred. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricants such as magnesium stearate, stearic acid or talc. The tablets may or may not be coated, or may be coated by known techniques (microencapsulation which delays disintegration, delays absorption into the gastrointestinal tract, and acts continuously over a long period of time). For example, the delaying material may be used, for example, glyceryl monostearate or glyceryl distearate alone or in combination with waxes.

[0213] The pharmaceutical formulation for oral use may be a hard gelatin capsule. In this case, the active ingredient is mixed with an inert solid diluent, which is, for example, calcium phosphate or kaolin. In a soft gelatin capsule, the active ingredient is mixed with a water or oil medium. The oil medium is, for example, peanut oil, liquid paraffin or olive oil.

[0214] The aqueous suspension of the present invention contains the active substance in a state of being mixed with an excipient suitable for producing an aqueous suspension. Such excipients include suspending agents such as carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth gum and acacia gum, and dispersing agents or wetting agents such as natural phosphatides (such as lecithin), condensation products of alkylene oxides and fatty acids (such as polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (such as heptadecaethyleneoxy cetanol), and condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate). The aqueous suspension may contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents such as sucrose or saccharin.

[0215] The oily suspension may be adapted by suspending the active ingredient in a vegetable oil (such as groundnut oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). The oral suspension may contain a thickening agent such as beeswax, solid paraffin or cetyl alcohol. Sweetening agents (such as those described above) and flavoring agents may be added to provide an oral preparation. These compositions may be preserved by adding an antioxidant such as ascorbic acid.

[0216] The dispersible powders and granules of the present invention suitable for the preparation of an aqueous suspension by adding water give a mixture of a dispersant or wetting agent, a suspending agent and one or more preservatives with the active ingredient. Suitable dispersants or wetting agents and suspending agents are those discussed and exemplified above. Further excipients, for example, sweeteners, flavoring agents and coloring agents may be present.

[0217] The pharmaceutical composition of the present invention may be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil (for example, olive oil or peanut oil), a mineral oil (for example, liquid paraffin), or a mixture thereof. Suitable emulsifying agents include natural gums (for example, gum acacia and tragacanth gum), natural phosphatides (for example, soybean lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (for example, sorbitan monooleate), and condensation products of ethylene oxide with these partial esters (for example, polyoxyethylene sorbitan monooleate). The emulsion may contain sweetening agents and flavoring agents.

[0218] Syrups and elixirs may be formulated with sweetening agents such as glycerol, sorbitol or sucrose. Such formulations may contain analgesics, preservatives, flavoring agents or coloring agents.

[0219] The pharmaceutical formulation of the present invention may be in the form of a sterile injectable preparation, for example, a sterile injectable aqueous suspension or an oily suspension. This suspension may be adapted according to known techniques using the appropriate dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may be a sterile injectable solution or suspension (e.g., 1,3 - butanediol solution) of a non - toxic parenterally acceptable diluent or solvent, or it may be prepared as a lyophilized powder. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, a sterile solid oil may be conveniently used as a solvent or suspending medium. For this purpose, any brand of solid oil containing synthetic monoglycerides or diglycerides may be used. Furthermore, fatty acids such as oleic acid may also be used in injectable preparations.

[0220] The amount of active ingredient that can be mixed with a carrier substance to provide a single dosage form will vary depending on the host to be treated and the particular dosage form. For example, a delayed - release formulation for oral administration to humans may contain the active substance in an amount of 20 - 200 μmoles, mixed with an appropriate and convenient amount of carrier substance in an amount varying from about 5% to about 95% of the total composition. It is preferred that a pharmaceutically - measurable amount of the pharmaceutical formulation be prepared for administration. For example, an aqueous solution for intravenous injection may contain about 20 - about 50 μmoles of the active ingredient per liter of solution so that an appropriate volume can be injected at a rate of about 30 ml / hour.

[0221] As will be understood by those skilled in the art, the specific dosage concentration for any particular patient depends on various factors including the activity of the specific compound being used; the age, weight, general health, sex, and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs previously administered; and the severity of the particular disease being treated.

[0222] Examples of the use of the method of the present invention include the following. It is understood that these examples are illustrative and the method of the present invention is not limited to these examples.

[0223] This method may be used for thrombolysis for coronary artery occlusion. This compound may be given as a sterile injectable preparation containing water or isotonic sodium chloride as a solvent. This solution can be administered intravenously or into the coronary artery, or directly into the carotid artery simultaneously with left heart catheterization. The administration rate can be varied, for example, from 0.2 to 1 μmole / min / kg with an infusion volume of 30 ml / hour. The treatment time is typically about 96 hours.

[0224] Angina pectoris and early myocardial infarction can be treated by intravenous injection using a sterile injectable preparation at the rate described above.

[0225] The compounds of the present invention can also be administered intravenously to a patient during cardiac bypass surgery or to a patient during other surgeries at risk of myocardial infarction. This compound can be added directly to the solution administered by membrane oxidation at the rate described above, or can be added directly to the cardioplegic solution.

[0226] Organs can be protected using the method of the present invention by perfusing the organ with a solution containing the compound of the present invention. The dosage administered varies with the perfusion rate of the organ, as will be well understood by those skilled in the art. This method can be applied particularly to organs and tissues used in organ transplantation.

[0227] (Description of the Preferred Embodiment) The inventors of the present application have identified many AICA riboside analogs that enhance the degree of functional recovery after ischemia in an ischemia experimental model. As shown in Table I, the good results treated with the preferred analogs are at least equivalent to AICA riboside ((Compound Nos. 11, 40 (Series I) and 19 (Series III)), and in many cases, equivalent effects were obtained at concentrations lower than AICA riboside (e.g., Compound Nos. 10, 23, 25, 29, 47, 52, 53 (Series I), 27 (Series II), 21 and 66 (Series III)). Preferred compounds include prodrugs, such as carboxylic acid esters of 2'-hydroxyl and 3'-hydroxyl. For example, preferred prodrugs of Series IIII are R 4 and R 5(Formula I) both form cyclic carbonates. In a functional assay, the ability of the compounds to increase extracellular adenosine concentration was specifically evaluated, and many of these preferred analogs showed significantly higher potential than AICA riboside. As a result of evaluating the ability to inhibit stimulated contractions in isolated ileum and the functional responses mediated by adenosine, these preferred series of compounds were shown to have superior efficacy to AICA riboside (Table II). Furthermore, N-4 substituted AICA riboside analogs (Series I) significantly increased the tissue adenosine concentration in the hearts of ischemic rats compared to AICA riboside (Table III) and inhibited adenosine utilization in coronary endothelial cells (Table IV). Many compounds from this preferred series (I) bind with high affinity to the adenosine transport site specific for NBTI (Table V). These data suggest that this series of analogs, which are preferred compared to AICA riboside, have the ability to increase extracellular adenosine concentration at least partially and are functionally superior, and this ability can be explained by inhibiting adenosine transport (see Table V and Figures 22 and 23). C-2 substituted AICA riboside analogs (Series II) are thought to increase adenosine release by Compound No. 13 affecting adenosine production in cell culture (Table VI). Furthermore, these specific compounds are inhibitors of the adenosine metabolism enzyme adenosine kinase (see Table VII). 2'-C substituted AICA riboside analogs (Series IV) significantly modulate adenosine utilization in a cell culture model (Figures 2A, 2B and 2C). In this preferred series (IV), each test compound is an effective inhibitor of another important adenosine metabolism enzyme, adenosine deaminase (Table VII). Therefore, these compounds increase extracellular adenosine concentration more effectively than AICA riboside, and this fact can be explained by their high inhibitory potency against adenosine deaminase.

[0228] AICA riboside analogs have also been evaluated for their effects on platelet function. As shown in Table IX, certain compounds inhibit platelet aggregation in human whole blood. Inhibition of platelet aggregation by many of the test compounds is enhanced in the presence of non-inhibitory concentrations of adenosine. Adenosine has been reported to be a potent antiplatelet agent, but has a short half-life in the blood. Thus, the inhibition of platelet aggregation observed in the presence of these AICA riboside analogs is due to the adenosine-modulating activity of these compounds.

[0229] Certain preferred AICA riboside analogs (Compound No. 53 (1-468), Compound No. 21 (1-227)) are orally bioavailable in dogs (see Table X). Further, treatment with AICA riboside analog Compound No. 53 (1-468) confers a functional benefit on a canine model of stable angina (see Table XI). In addition to this cardiovascular benefit, certain AICA riboside analogs (Compound Nos. 10 (1-186) and 11 (1-226) (Series I)) also exhibit a protective effect in a murine model of cerebral ischemia (Figure 21).

[0230] To aid in understanding the present invention, the results of a series of experiments show the benefits of these preferred analogs in an ischemia model, and further provide logical evidence that these analogs exhibit higher potency compared to AICA riboside. A series of examples illustrating the synthesis of these compounds are also shown. These examples, of course, are not to be construed as specifically limiting the present invention, and such variations of the present invention that are within the scope of those skilled in the art, whether currently known or developed in the future, are considered to be within the scope described in this specification and the claims.

[0231] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are given for illustrative purposes only. Many modifications, variations and substitutions can be made by those skilled in the art without departing from the present invention. It should be understood that various modifications of the embodiments of the present invention described herein can be used in the practice of the present invention. The claims that define the scope of the present invention and the methods and structures within the scope of these claims and their equivalents are intended to fall within the scope of the rights.

[0232] (Definition) "Administered" or "administration" refers to introducing a blood coagulation inhibitor into a patient. Administration refers to dosing by a human (e.g., a healthcare provider or the patient himself).

[0233] "Blood coagulation inhibitor" refers to any drug, agent or pharmaceutical composition that suppresses, prevents or inhibits the formation of blood clots and dissolves or destroys blood coagulants. A blood coagulation inhibitor can be any blood coagulation inhibitor currently known to those skilled in the art or developed in the future. A blood coagulation inhibitor can be any type of drug of blood coagulation inhibitors known to those skilled in the art, including but not limited to antiplatelet agents, thrombolytic enzymes, coagulation inhibitors, glycoprotein IIb / IIIa inhibitors, glycosaminoglycans, thrombin inhibitors, anticoagulants, heparin, low molecular weight heparin, coumarin, indandione derivatives, tissue plasminogen activator and combinations thereof. A blood coagulation inhibitor can be in any pharmaceutical dosage form and can be administered by any route known to those skilled in the art.

[0234] "Perioperative period" refers to the period before surgery (preoperative), the period after surgery (postoperative), the period during surgery (intraoperative), and / or any combination of these periods. For example, the blood coagulation inhibitor can be administered for 48 hours during the perioperative period, that is, the blood coagulation inhibitor can be administered for 48 hours before surgery (preoperative), 48 hours after surgery (postoperative), 48 hours during surgery (intraoperative), or for 48 hours during any combination of these periods. Administration during the perioperative period can be a single administration or multiple administrations within the perioperative period. It is understood by those skilled in the art that "preoperative" refers to the period before surgery, "postoperative" refers to the period after surgery, and "intraoperative" refers to the period during surgery.

[0235] "Long term" refers to the time after discharge from the hospital and refers to a period of 6 months or more. For example, the blood coagulation inhibitor can be administered once at the time of discharge and continuously administered for a period of 6 months, 1 year or more, after the perioperative period.

[0236] "Surgery" or "operation" refers to any manual method, operative method or procedure for treating a disease, disorder or deformity or preventing a deformity. Surgery includes methods or procedures performed while the patient is under anesthesia (including local anesthesia or general anesthesia). Surgery can generally be performed by a physician, surgeon or dentist in a hospital or other healthcare facility. A patient undergoing surgery can be admitted to the hospital or visit the hospital for outpatient treatment (e.g., outpatient surgery). Surgery does not include percutaneous intervention (PTI) or percutaneous transluminal coronary angioplasty (PTCA).

[0237] "Coronary artery bypass graft" or "CABG" refers to a heart surgery in which one or more bypass grafts are transplanted between the aorta and coronary artery vessels, usually using the saphenous vein or internal thoracic artery as the graft. "Venous graft CABG" refers to a CABG surgery using the saphenous vein for transplantation. "Arterial graft CABG" refers to a CABG surgery using the internal thoracic artery for transplantation.

[0238] (Administration timing) The blood coagulation inhibitor can be administered during the perioperative period (before surgery, after surgery and / or during surgery or any combination thereof). For example, when the half-life of the drug is long (24 to 48 hours), the blood coagulation inhibitor can be administered once within 48 hours (or 24 hours) before surgery and can be repeatedly administered during or after surgery. Drugs with a short half-life can be given immediately before surgery and administered during or after surgery. In some patients and some circumstances, the treating physician may decide to interrupt the preoperative treatment, close the wound so that bleeding does not occur in the area (non-open blood vessels) before starting anticoagulant therapy, and then start administration after surgery (e.g., 48 hours after surgery). Administering the blood coagulation inhibitor so promptly after surgery is within the scope of the present invention.

[0239] Perioperative administration includes the preoperative period (before surgery), the postoperative period (after surgery), the intraoperative period (during surgery), and / or any combination of these periods. For example, the blood coagulation inhibitor can be administered for a period of 6 months, 3 months, 1 month, 1 week, 96 hours, 48 hours or less during the perioperative period; that is, the blood coagulation inhibitor can be administered for a period of 6 months, 3 months, 1 month, 1 week, 96 hours, 48 hours or less before surgery, and for a period of 6 months, 3 months, 1 month, 1 week, 96 hours, 48 hours or less after surgery, or for a period of 6 months, 3 months, 1 month, 1 week, 96 hours, 48 hours or less before and after surgery. Further, the blood coagulation inhibitor can be administered for a period of, for example, 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour during the perioperative period; that is, the blood coagulation inhibitor can be administered for a period of 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour before surgery, and / or for a period of 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours or 1 hour after surgery and / or during surgery. The blood coagulation inhibitor can be administered for the same time before and after surgery. For example, the blood coagulation inhibitor can be administered for 48 hours before surgery and 48 hours after surgery. The blood coagulation inhibitor can be administered for different times before and after surgery. For example, the blood coagulation inhibitor can be administered for 48 hours before surgery and 24 hours after surgery. For example, the blood coagulation inhibitor can be administered for 36 hours before surgery and 36 hours after surgery. The blood coagulation inhibitor can be administered for 36 hours before surgery and 12 hours after surgery. For example, the blood coagulation inhibitor can be administered for 12 hours before surgery and 12 hours after surgery. For example, the blood coagulation inhibitor can be administered for 8 hours before surgery and 8 hours after surgery. For example, the blood coagulation inhibitor can be administered for 6 hours before surgery and 8 hours after surgery. For example, the blood coagulation inhibitor can be administered for 6 hours before surgery and 6 hours after surgery. For example, the blood coagulation inhibitor can be administered for 8 hours before surgery and 4 hours after surgery. The blood coagulation inhibitor can be administered for 4 hours before surgery and 4 hours after surgery. The blood coagulation inhibitor can be administered for 2 hours before surgery and 8 hours after surgery.The blood coagulation inhibitor can be administered 4 hours before surgery and 1 hour after surgery. For example, the blood coagulation inhibitor can be administered 24 hours before surgery and during surgery. For example, the blood coagulation inhibitor can be administered during surgery and 6 hours after surgery.

[0240] During the perioperative period, the blood coagulation inhibitor can be administered in a single dose or in multiple doses at one time. In certain embodiments, perioperative administration can be carried out without interruption of the blood coagulation inhibitor (e.g., continuous infusion or transdermal delivery). In another embodiment, perioperative administration is one or more separate administrations within the perioperative time frame (e.g., one administration during the perioperative period or multiple administrations during the perioperative period). In one embodiment, the blood coagulation inhibitor can be administered within 6 days, 5 days, 4 days, 3 days, 2 days or 1 day during the perioperative period. In another embodiment, the blood coagulation inhibitor can be administered within 48 hours, 36 hours, 24 hours, 12 hours, 8 hours, 6 hours or 1 hour during the perioperative period.

[0241] The blood coagulation inhibitor can be administered during surgery, for example, simultaneously with the use or interruption of cardiopulmonary bypass, or simultaneously with reperfusion of the ischemic area. Administration can be continued for a long period, for example, after discharge after surgery, and for a period of 6 months, 1 year or more after surgery.

[0242] In certain embodiments, if the patient has been chronically treated with a blood coagulation inhibitor before surgery, in a manner controlled with standard practice, the blood coagulation inhibitor is not interrupted before surgery.

[0243] During the perioperative period, the patient does not need to be conscious when receiving the blood coagulation inhibitor. For example, the blood coagulation inhibitor can be given during surgery while the patient is under anesthesia. In some surgeries for ambulatory or outpatient patients, the patient is conscious, and under such conditions, the blood coagulation inhibitor can be given during surgery when the patient is conscious.

[0244] This treatment can be continued after discharge. During the long-term treatment, as described above, the fitting and dosing can be continued or adjusted, or the type of blood coagulation inhibitor can be changed to another one.

[0245] (Complications of surgery and operation) The present invention provides a method for preventing or reducing postoperative morbidity and mortality. In a particular aspect, this method includes administering a blood coagulation inhibitor perioperatively to prevent or reduce postoperative complications. The blood coagulation inhibitor can be administered perioperatively, that is, before surgery, during surgery and / or after surgery and after discharge. The postoperative morbidity and mortality can also be significantly prevented or reduced after the hospitalization period.

[0246] Surgery refers to any manual, operative, or procedural method for treating a disease, disorder, or deformity or preventing a deformity. Surgery includes methods or procedures that are performed with the patient under anesthesia (including local or general anesthesia). Surgery can generally be performed by a physician, surgeon, or dentist in a hospital or other healthcare facility. A patient undergoing surgery can be admitted to the hospital or can receive outpatient treatment (e.g., outpatient surgery). For the purposes of the present invention, surgery includes, but is not limited to, abdominal surgery (e.g., abdominal visceral surgery), extracorporeal surgery (e.g., removing an organ from the body, operating on it, and then retransplanting it), cardiac surgery (e.g., heart surgery), brain surgery (e.g., brain surgery), cineplastic (e.g., surgery that creates a hole through the muscle adjacent to the severed portion of an amputated limb and enables the muscle to be used to manipulate a prosthesis), cosmetic surgery (e.g., surgery that improves the appearance of a patient by plastic repair, correction, or removal of scars), dentofacial surgery (e.g., surgery related to facial defects and the structure of the mouth), nerve surgery (e.g., surgery related to the peripheral or central nervous system), oral surgery (e.g., surgery related to defects in the structure of the mouth, jaw, and related areas), orthopedic surgery (e.g., surgery that deals with bones and bone tissue), pelvic surgery (e.g., surgery mainly related to the pelvic region of obstetrics and gynecology), reconstructive surgery (e.g., surgery related to restoring, reconstructing, correcting, or improving the shape and appearance of a body structure that is defective, damaged, or deformed due to injury, disease, or growth and progression), or rectal surgery (e.g., rectal surgery), urological surgery (e.g., surgery mainly related to the male urogenital system), vascular surgery (e.g., vascular surgery), and surgery related to otolaryngology (e.g., surgery of the eye, nose, throat, or related structures). Surgery can be a conservative surgery (e.g., surgery that preserves or excises an organ, tissue, or limb with a disease or disorder with minimal risk) or a radical surgery (e.g., surgery designed to remove all of a locally advanced disease and the adjacent area of lymph nodes). In certain embodiments, surgery can be cardiac surgery including heart valve replacement, heart and heart-lung transplantation, and transplantation of artificial heart devices and defibrillators, valve replacement or valve repair, and surgery for congenital deformities.

[0247] In certain embodiments, when the heart surgery is CABG, the surgery can be coronary artery bypass grafting using the saphenous vein or the internal thoracic artery, which are referred to herein as vein graft CABG and arterial graft CABG, respectively. In one embodiment, when the surgery is vein graft CABG, an anticoagulant other than aspirin is administered from 12 hours before the surgery to 7 hours after the surgery. In another embodiment, when the surgery is vein graft CABG, an anticoagulant other than dipyridamole is administered from 48 hours before the surgery to 24 hours after the surgery. See Goldman et al., 1988, Circulation 77:1324-32; Chesebro et al., 1982, NEJM 307:73-8; Chesebro et al., 1984, NEJM 310:209-14. In another embodiment, when the surgery is vein graft CABG, the anticoagulant is not ticlopidine or aprotinin. See Drug Facts and Comparisons, updated monthly, September, 2002, Facts and Comparisons, Wolters Kluwer Company, St. Louis, MO.

[0248] In certain embodiments, when the heart surgery is arterial graft CABG, the anticoagulant is not aprotinin.

[0249] The present invention can be used for a wide range of surgeries. Without limitation, heart surgery, abdominal surgery, nerve surgery, gynecological surgery, orthopedic surgery, urological surgery, vascular surgery, and otolaryngological surgery are included. More specifically, surgeries include resection of the small and large intestines, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, salpingo-oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringotomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, debridement, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc.

[0250] Surgeries for inpatients or outpatients include those that generally do not require hospitalization and / or general anesthesia. Examples of such surgeries include the placement of tympanostomy tubes and hemorrhoidectomy.

[0251] The present invention can prevent or reduce the morbidity and mortality rates during the recovery period due to postoperative hospitalization and after discharge. The postoperative morbidity and mortality rates may be caused by complications from any surgery. Complications of surgery can include cardiac complications (myocardial infarction, congestive heart failure, severe cardiac arrhythmia, ischemia), neurological complications (stroke, brain damage, cognitive impairment, transient ischemic attack, seizure), renal complications (renal failure, renal dysfunction or renal death), gastrointestinal tract complications (infarction, intestinal obstruction, ischemia, mesenteric thrombosis or GI death), pulmonary complications (incompleteness, respiratory distress syndrome, edema), etc.

[0252] (Blood coagulation inhibitor) The present invention provides a method for preventing or reducing postoperative morbidity and mortality rates. In certain aspects, this method includes the perioperative administration of a blood coagulation inhibitor to prevent or reduce postoperative complications. The blood coagulation inhibitor can be administered perioperatively, that is, before surgery, during surgery and / or after surgery and after discharge.

[0253] The blood coagulation inhibitor of the present invention can be any drug, agent or pharmaceutical composition that prevents or inhibits blood coagulation. The inhibitor acts by preventing or inhibiting blood coagulation generation by any of various mechanisms, including reducing blood coagulation factors or platelet activity or aggregation, or reducing the effects of inducing factors such as inflammation or stress. The blood coagulation inhibitor can act by decomposing or dissolving after blood coagulation formation. It is obvious to those skilled in the art that there are various blood coagulation material agents, including antiplatelet agents, thrombolytic enzymes, coagulation inhibitors, glycoprotein IIb / IIIa inhibitors, glycosaminoglycans, thrombin inhibitors, anticoagulants, heparin, low molecular weight heparin, coumarin, indandione derivatives, and tissue plasminogen activators. See The Physicians’ Desk Reference (56th ed., 2002) Medical Economics; Mosby’s Drug Consult, 2002, Elsevier Science; Goodman and Gilman’s The Pharmacologic Basis of Therapeutics, (9th ed. 1996) Pergamon Press; Drug Facts and Comparisons, updated monthly, September, 2002, Facts and Comparisons, Wolters Kluwer Company, St. Louis, MO.

[0254] For the purposes of the present invention, any drug, agent or pharmaceutical composition that prevents or inhibits blood clot formation, or that lyses or destroys blood clots, is suitable for use in the present invention.Such blood coagulation inhibitors can be, for example, cilostazol (PLETAL(R), Otsuka), clopidogrel (PLAVIX(R), Sanofi), ticlopidine (TICLID(R), Syntex), tirofiban (AGGRASTAT(R), Merck), eptifibatide (INTEGRILIN(R), COR Therapeutics), abciximab (REOPRO(R), Eli Lilly), anagrelide (AGRYLIN(R), Roberts), dipyridamole (PERSANTIN(R), Boehringer Ingelheim), aspirin (ECOTR(R) and others), dipyridamole / aspirin (AGGRENOXS(R), Boehringer Ingelheim), dalteparin (FRAGMIN(R), Pharmacia), enoxaparin (LOVENOX(R), Aventis), tinzaparin (INNOHE(R), DuPont), heparin (various products), danaparoid (ORGANON(R), Organon), antithrombin III (THROMBATE(R), Bayer), lepirudin (REFLUDAN(R), Hoechst-Marion Roussel), argatroban (ACOVA(R), SmithKlineBeecham), bivalirudin (ANGIOMAX(R), Medicines Company), warfarin (COUMADIN(R), DuPont), anisidione (MIRADON(R), Schering), alteplase (ACTIVASE(R), Genentech), reteplase (RETAVASE(R), Boehringer Mannheim), tenecteplase (TNKASE(R), Genentech), drotrecogin (XIGRIS(R), Eli Lilly), anisoylated plasminogen streptokinase complex (EMINASE(R), Roberts), streptokinase (STREPTASE(R), Astra), urokinase (ABBOKINASE(R), Abbott) and combinations thereof.

[0255] It is understood by those skilled in the art that the blood coagulation inhibitor is used for the treatment of a blocked catheter and for maintaining the patency of a vascular access device. Heparin, urokinase, streptokinase, and alteplase are commonly used for this purpose. The treatment of a blocked catheter and the use of a blood coagulation inhibitor for maintaining the patency of a vascular access device are not within the scope of the present invention. In certain embodiments where the blood coagulation inhibitor is low molecular weight heparin, the surgery is preferably not a hip replacement, knee replacement, or abdominal surgery. When the drug is dalteparin, the dosage is preferably not 2500 IU subcutaneously once a day starting 1 to 2 hours before surgery and repeated once a day until 5 to 10 hours after surgery, or not 5000 IU subcutaneously once a day repeated before surgery and until 5 to 10 hours after surgery. When the drug is enoxaparin, the dosage is preferably not 40 mg subcutaneously once a day starting 9 to 15 hours before surgery and continuously for 21 days, or not 40 mg subcutaneously once a day starting 2 hours before surgery and continuously for 7 to 10 days (12 hours continuously if tolerated).

[0256] In certain embodiments where the blood coagulation inhibitor is heparin, the surgery is preferably not a surgery related to both the abdomen and chest or heart surgery. When the drug is heparin, the dosage is preferably not 5000 units subcutaneously 2 hours before surgery, and not 5000 units every 8 to 12 hours for 7 days or until the patient can walk completely. When the drug is heparin, the dosage is preferably not 150 units / kg in patients receiving systemic perfusion for open heart surgery. When the drug is heparin, the dosage is preferably not 300 units / kg for a procedure less than 60 minutes or not 400 units / kg for a procedure over 60 minutes.

[0257] In certain embodiments where the blood coagulation inhibitor is danaparoid, the surgery is not an elective total hip replacement. When the drug is danaparoid, the dosage is preferably not 750 anti-Xa units subcutaneously twice a day starting from 1 to 4 hours before surgery until 2 hours short of 7 to 10 days after surgery.

[0258] In certain embodiments where the blood coagulation inhibitor is warfarin, the surgery is preferably not a cardiac valve replacement surgery. When the drug is warfarin, the dosage is preferably not 1 mg per day until 20 days before surgery.

[0259] In certain embodiments, when the cardiac surgery is vein graft and CABG, a blood coagulation inhibitor other than aspirin is administered within the time from 12 hours before surgery to 7 hours after surgery. In certain embodiments, when the cardiac surgery is vein graft CABG, a blood coagulation inhibitor other than dipyridamole is administered within the time from 48 hours before surgery to 24 hours after surgery. See Goldman et al., 1988, Circulation 77:1324-32; Chesebro et al., 1982, NEJM 307:73-8; Chesebro et al., 1984, NEJM 310:209-14. In certain other embodiments, when the cardiac surgery is vein graft CABG, the blood coagulation inhibitor is not ticlopidine or aprotinin. See Drug Facts and Comparisons, updated monthly, September, 2002, Facts and Comparisons, Wolters Kluwer Company, St Louis, MO.

[0260] Aprotinin can be used for CABG surgery in one of two dosing regimens (Regimen A or B). Regimen A involves an intravenous administration of 2 million KIU (kallikrein inhibitor units); 2 million KIU are administered with the cardiopulmonary bypass machine (pump / volume), and 500,000 KIU / hour are administered as a continuous maintenance intravenous infusion for the duration of the procedure. Regimen B involves an intravenous administration of 1 million KIU, 1 million KIU are administered with the pump / volume, and 250,000 KIU / hour are administered as a continuous maintenance intravenous infusion for the duration of the procedure. Administration of aprotinin is started after induction of anesthesia and before sternotomy and continues until the surgery is completed and the patient leaves the operating room. Drug Facts and Comparisons, updated monthly, September, 2002, Facts and Comparisons, Wolters Kluwer Company, St. Louis, MO. In certain embodiments where the surgery is venous graft CABG or arterial graft CABG, the blood coagulation inhibitor is not aprotinin.

[0261] The blood coagulation inhibitor may be a combination of two or more blood coagulation inhibitors. The combination of blood coagulation inhibitors can include blood coagulation inhibitors from more than one drug of the types described herein. Further, the combination of blood coagulation inhibitors can include different routes of administration for each blood coagulation inhibitor. The combination of blood coagulation inhibitors can be administered simultaneously. Further, the combination of blood coagulation inhibitors can be administered separately.

[0262] (Dosage, Formulation, and Administration) The blood coagulation inhibitor described in this specification can be administered to a patient to reduce the morbidity and mortality rate after surgery by any means of bringing the blood coagulation inhibitor into contact with the site of action of the blood coagulation inhibitor in the patient's body. The blood coagulation inhibitor can be a pharmaceutical composition that can be administered by any available means. It is understood by those skilled in the art that pharmaceutical formulations can generally be administered together with a pharmaceutical carrier. The pharmaceutical composition and / or the pharmaceutical carrier can be selected based on the selected route of administration and standard pharmaceutical practice. The pharmaceutical composition of the present invention can be applied for oral, parenteral or topical administration in a manner well-known to those skilled in the pharmaceutical field and can be in unit dosage form. Parenteral administration includes, but is not limited to, subcutaneous, intravenous, intramuscular, and arterial injections. For example, it is obvious to those skilled in the art that oral dosage forms can be administered by many routes. Such routes include, but are not limited to, rectal and vaginal administration, and administration via any means (such as a nasogastric tube) that delivers the substrate to the gastrointestinal tract.

[0263] Of course, the dosage administered varies depending on known factors. Such factors include, for example, the pharmacological properties and its mode and route of administration of a particular blood coagulation inhibitor; the age, health, height and weight of the patient; the type of combination therapy; the frequency of treatment; and the desired effect. The dosage of the blood coagulation inhibitor does not need to be maintained at a constant amount but can be adjusted according to parameters well-known to those skilled in the art. Furthermore, the dosage of the blood coagulation inhibitor can be below or above the therapeutic amount.

[0264] The single dosage of the active ingredient can be within the normal dosage range suitable for individual patients. For example, aspirin can be used orally at 40 mg to 160 mg per day. Dipyridamole can be used orally at 75 mg to 100 mg four times a day. Aspirin and dipyridamole can be administered in combination with one commercially available product at 25 mg of aspirin / 200 mg of dipyridamole (AGGRENOXO), or the compositions can be administered simultaneously as individual compositions within the dosage ranges described herein. Heparin can be used subcutaneously at an initial dosage of 10,00 to 20,000 units (an intravenous dosage of 5,000 units can be used first), and then can be administered at 8,000 to 10,000 units every 8 hours or 15,000 to 20,000 units every 12 hours, and the partial thromboplastin time (PTT) can be adjusted to about 1.5 to 2 times normal. Warfarin can be used orally or parenterally at 0.5 to 30 mg per day. Cilostazol can be used orally at 50 to 100 mg twice a day. Clopidogrel can be used orally once at 75 mg in the presence or absence of a 300 mg loading dose. Ticlopidine can be used orally at 250 mg twice a day. Tirofiban can be used parenterally at 0.4 mcg / kg / min for 30 minutes and then continued at 0.1 mcg / kg / min. Eptifibatide can be used parenterally using an intravenous bolus at 180 mcg / kg, and a second bolus can be used to perform a continuous infusion at 2 mcg / kg / min 10 minutes after the first intravenous bolus administration. The dosage of the second parenteral bolus can be 180 mcg / kg. Abciximab can be used parenterally by infusing 0.25 mg / kg as an intravenous bolus over 10 to 60 minutes and then performing a continuous infusion at 0.125 mcg / kg / min up to a maximum of 10 mcg / min for 12 hours. Anagrelide can be used orally at 0.5 mg four times a day to 1 mg twice a day, and can be increased up to a maximum of 10 mg per day. Dalteparin can be used subcutaneously at 2,500 to 5,000 IU once to twice a day. Enoxaparin can be used subcutaneously at 1 mg / kg once to twice a day.Chinzaparin can be used subcutaneously once a day at 175 anti-Xa IU / kg. Danaparoid can be used subcutaneously twice a day at 750 anti-Xa units. Antithrombin III can be used parenterally at a dosage based on the pre-treatment plasma antithrombin III (AT) concentration. The dosage can be calculated as follows.

[0265] [Chemical formula] (See Drug Facts and Comparisons, updated monthly, September 2002, Facts and Comparisons, Wolters Kluwer Company, St. Louis, MO).

[0266] Rapilysin can be administered parenterally at a bolus dosage of 0.4 mg / kg. After an intravenous injection of 15 - 20 seconds, it can be continuously inhaled intravenously at 0.15 mg / kg. Argatroban can be continuously infused at 2 mcg / kg / min. Bivalirudin is administered as a 1 mg / kg intravenous bolus and can be intravenously infused for 4 hours at 2.5 mg / kg / hour. Anisodamine can be used orally at 25 - 300 mg / day. Alteplase can be administered intravenously to patients over 67 kg as a 15 mg intravenous bolus, followed by an injection of 50 mg in the next 30 minutes and 35 mg in the next 60 minutes for a total dosage of 100 mg. In patients weighing less than 67 kg, alteplase can be administered as a 15 mg intravenous bolus, followed by an injection of no more than 50 mg at 0.75 mg / kg in the next 30 minutes and 0.5 mg / kg in the next 60 minutes for a total dosage of 100 mg intravenously. Reteplease can be used parenterally by injecting a 10 - unit intravenous bolus over 2 minutes and injecting a second 10 - unit intravenous bolus over 2 minutes 30 minutes later. Tenecteplase can be used parenterally at a dosage of 30 - 50 mg based on the patient's weight and can be administered as a single bolus over 5 minutes. Drotrecogin can be used parenterally at 24 mcg / kg / hour for a total infusion time of 96 hours. Anistreplase can be used parenterally by intravenous administration of 30 units over 2 - 5 minutes. Streptokinase can be used parenterally by injecting a dosage of 250,000 units over 30 minutes. Additionally, streptokinase can be used intravenously by administering a 20,000 IU bolus followed by administration at 2,000 IU / min for 60 minutes. Urokinase can be used parenterally by administering at a dosage of 4400 units / kg for 10 minutes and continuously administering at 4400 units / kg / hour at a rate of 15 ml / hour for 12 hours.

[0267] The active ingredient of the blood coagulation inhibitor can be administered orally in solid or semi-solid dosage forms (e.g., hard gelatin capsules or soft gelatin capsules, tablets, or powders) or liquid dosage forms (e.g., elixirs, syrups, or suspensions). It can also be administered parenterally in sterile liquid dosage forms. Other dosage forms (e.g., patches or ointments or transdermal administration) are also potentially possible.

[0268] Parenteral dosage forms can be, for example, injectable preparations, such as sterile suspensions, solutions or emulsions of the active ingredient in aqueous or oily vehicles. This composition may contain formulating agents, such as suspending agents, stabilizers and / or dispersing agents. Injectable formulations can be, for example, in unit dosage forms of ampoules or multi-dose containers and may contain added preservatives.

[0269] Injectable formulations can be in powder form for reconstruction prior to use with a suitable vehicle (including but not limited to sterile pyrogen-free water, buffers, dextrose solutions, etc.).

[0270] For intraoperative administration, the active ingredient can be administered directly to the cardiopulmonary bypass machine, directly to the pericardium or directly to the blood vessels exposed in the surgical field.

[0271] For long-term delivery, the active ingredient can be formulated as a depot preparation for administration by implantation (e.g., subcutaneous, intradermal or intramuscular injection). Thus, for example, the active ingredient may be formulated with a suitable polymeric material or hydrophobic material (e.g., as an emulsion of an acceptable oil, or an ion exchange resin or a derivative of low solubility).

[0272] Alternatively, a transdermal delivery system manufactured as an adhesive disk or patch that slowly releases the active ingredient for transdermal absorption may be used. To achieve this, a permeation enhancer may be used to enhance the transdermal permeability of the blood coagulation inhibitor.

[0273] For oral administration, the pharmaceutical formulation or blood coagulation inhibitor may be, for example, in the form of tablets or capsules by conventional means using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art.

[0274] Liquid preparations for oral administration may be, for example, in the form of solutions, syrups or suspensions, or may be present as dry products for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).

[0275] The preparations may, where appropriate, contain buffer salts, flavoring agents, coloring agents and sweetening agents. Preparations for oral administration may be suitably adapted to control the release of the active compound.

[0276] For buccal administration, the composition may be in the form of tablets or troches adapted in a conventional manner. For rectal and vaginal routes of administration, the active ingredient may be adapted as a solution (for retention enemas), suppositories or ointments.

[0277] For administration by inhalation, the active ingredient can be conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., made of gelatin) for use in an inhaler or insufflator may contain a powder mixture of the compound and a suitable powder base (e.g., lactose or starch).

[0278] The composition may be in the form of a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise a foil of metal or plastic (e.g., a blister pack). The pack or dispenser device may be accompanied by instructions for administration.

[0279] The blood coagulation inhibitor can be administered by any suitable route known in the art to ensure bioavailability in the circulation. Administration can be achieved by parenteral routes of administration, e.g., but not limited to, injection into a vein (IV), intramuscular (IM), intradermal, subcutaneous (SC) and intraperitoneal (IP). In certain embodiments, it is administered by a bypass machine, a perfusion device, an invasive device or a catheter. In certain embodiments, the blood coagulation inhibitor is administered by injection, subcutaneously by an implantable pump or by a depot preparation in a dosage amount to achieve a therapeutic effect. Suitable dosage forms are further described in Remington’s Pharmaceutical Sciences, 1990, 17th ed., Mack Publishing Company, Easton, PA (a standard reference in this field and which is hereby incorporated by reference in its entirety).

[0280] Administration can be achieved by a variety of different treatment methods. For example, some oral medications can periodically administer a blood coagulation inhibitor during the day in a cumulative dose that does not reach a toxic amount per day. Or, the blood coagulation inhibitor can be administered daily, starting, for example, 48 hours before surgery and continuing, for example, until 48 hours after surgery.

[0281] Intravenous injection can be periodically administered during the day in a cumulative injection volume that does not reach a toxic amount per day. Or, intravenous injection can be administered daily, starting, for example, 48 hours before surgery and continuing, for example, until 48 hours after surgery. The dosage of the blood coagulation inhibitor may vary. For example, the dosage can be increased stepwise. Depending on the patient's needs, the administration can be slowly infused over a time longer than 1 hour, rapidly infused in less than 1 hour, or infused by a single bolus injection.

[0282] Other administration routes may be used. For example, absorption from the gastrointestinal tract can be achieved by the oral administration route (including, but not limited to, digestion via a nasogastric tube, oral, and sublingual routes). Or, administration via mucosal tissue (e.g., vaginal and rectal modes of administration) can be used. In yet another alternative, the formulations of the present invention can be administered transdermally (e.g., percutaneous) or by inhalation. It is understood that the preferred route may vary with the condition and age of the recipient.

[0283] The actual dosage of the blood coagulation inhibitor varies with the administration route. The blood coagulation inhibitor is generally used in an amount effective to achieve the intended purpose. Of course, it is understood that the amount used depends on the particular application.

[0284] The effective amount may vary, for example, depending on the type of surgery, the patient's condition, the patient's age, the patient's weight, the patient's medical history, the mode of administration, and the judgment of the doctor. It is understood by those skilled in the art that the degree of anticoagulation can be monitored by experimental values (for example, prothrombin time (PT) and partial thromboplastin time (PIT)). The determination of the effective amount is well within the ability of those skilled in the art, particularly from the perspective of the detailed disclosure provided herein.

[0285] The administration of the blood coagulation inhibitor may be repeated intermittently. The blood coagulation inhibitor can be administered alone or in combination with other drugs, for example, other preoperative drugs (such as antibiotics or anesthetics).

[0286] (Combination of blood coagulation inhibitor and acadine) In another aspect of the present invention, a method for preventing or reducing side effects in a patient undergoing surgery is provided, which comprises first administering acadine or its prodrug, analog or salt, and then administering a blood coagulation inhibitor. In one embodiment, the blood coagulation inhibitor is administered during the administration of acadine or its prodrug, analog or salt. In one embodiment, acadine or its prodrug, analog or salt is administered at a total dosage of about 10 mg / kg to about 200 mg / kg. Another aspect provides a method for preventing or reducing side effects in a patient undergoing non-vascular surgery, which comprises administering acadine or its prodrug, analog or salt, and then administering a blood coagulation inhibitor. The present invention can be used for a wide range of non-vascular surgeries. Examples of non-vascular surgeries include non-vascular surgeries of the abdomen, nerves, gynecology, orthopedics, urology, and otolaryngology. More specifically, non-vascular surgeries include resection of the small intestine and large intestine, appendectomy, laparoscopy, puncture, transurethral resection of the prostate (TURP), hysterectomy, tubal ligation, vasectomy, oophorectomy, cesarean section, hemorrhoidectomy, tonsillectomy, myringotomy, placement of tympanostomy tubes, removal of polyps from the colon and rectum, repair of rectal prolapse, removal and treatment of intestinal neoplasms, curettage, thoracentesis, thoracotomy, rhinoplasty, liposuction, etc.

[0287] In one embodiment, the blood coagulation inhibitor is aspirin. In one embodiment, a patient undergoing surgery or non-vascular surgery has had a myocardial infarction in the past. In another embodiment, the past myocardial infarction occurred within 24 months, 36 months or 48 months from the surgery.

[0288] Another aspect of the present invention provides a method for preventing or reducing side effects in a patient undergoing CABG surgery, which comprises first administering acadine or its prodrug, analog or salt, and then administering a blood coagulation inhibitor. In one embodiment, the administration of the blood coagulation inhibitor is carried out during the administration of acadine or its prodrug, analog or salt. In another embodiment, acadine or its prodrug, analog or salt is administered at a total dosage of about 10 mg / kg to about 200 mg / kg. Another embodiment is to administer acadine or its prodrug, analog or salt at 0.1 mg / kg / min. Another embodiment is to administer acadine or its prodrug, analog or salt at 0.1 mg / kg / min over about 7 hours. Another embodiment is to administer aspirin at a dosage of about 400 mg to about 5 g. Another embodiment is to administer aspirin at least once within 48 hours after the surgery. Another aspect of the present invention provides a pharmaceutical formulation comprising acadine or its prodrug, analog or salt, aspirin and a pharmaceutically acceptable carrier, diluent or excipient. This formulation is provided such that the concentration in the patient's plasma is 1 μg / ml to 20 μg / ml over about 7 hours and the dosage of aspirin is about 40 mg to about 5 g. In another embodiment, the present invention provides a method for preventing or reducing side effects in a patient undergoing surgery by administering the above pharmaceutical formulation within 48 hours after the surgery. In another embodiment, the surgery is CABG surgery.

Examples

[0289] The following examples describe specific embodiments of the present invention for purposes of illustration and to provide descriptions of the methods, compositions, and formulations of the present invention. The examples are provided merely to provide specific methodologies useful for understanding and practicing the present invention and should not be construed as limiting the present invention.

[0290] Example I Enhancement of Adenosine Release by AICA Riboside in Lymphoblasts Regarding the enhancement of in vitro release of adenosine by the method recited in the claims, the effect of AICA riboside on adenosine release was demonstrated using the human spleen lymphoblast cell line (WI-L2). The history and characteristics of the cell line are described in Hershfield et al., Science, Vol. 197, p. 1284, 1977. The cell line was maintained in RPMI 1640 cell culture medium supplemented with 20% fetal bovine serum and 2 mM glutamine and various concentrations of AICA riboside and cultured for 48 hours in an atmosphere of 5% carbon dioxide in air. Fetal bovine serum contains purines and purine metabolic enzymes, but to establish the effect of AICA riboside upon 2-deoxyglucose exposure, WI-L2 cells were incubated in RPMI 1640 medium supplemented with 10% heat-inactivated dialyzed fetal bovine serum, 2 mM glutamine, and 1 μM deoxychoformycin.

[0291] The catabolism of intracellular ATP storage was stimulated by adding either 2-deoxyglucose or a calcium ionophore. At various time points, the amount of adenosine released into the supernatant by the cells, or the amount of nucleotides remaining in the cells, was measured by mixing 30 μl of cooled 4.4 N perchloric acid with 300 μl of the supernatant, or by adding 300 μl of cooled 0.4 N perchloric acid to the cells recovered as a pellet and centrifuging the mixture at 500×G for 10 minutes at 4°C. Each of the resulting supernatants was neutralized with 660 μl of a solution containing 2.4 grams of tri-n-octylamine (Alamine 336) (General Mills) in 12.5 ml of 1,1,2-trichloro-1,2,2-trifluoroethane (Freon-113) solvent (described in Khym, Clinical Chemistry, Vol. 21, p. 1245, 1975). After centrifugation at 1500×G for 3 minutes at 4°C, the aqueous phase was removed and frozen at -20°C until assayed for adenosine, inosine, or nucleotides. Adenosine was evaluated isocratically on a C-18 microBondapak reverse-phase column equilibrated with 4 millimolar potassium phosphate (pH 3.4): 60% acetonitrile in water (95:5 v / v) buffer. Adenosine was eluted at 8 - 10 minutes and its identity was confirmed by its sensitivity to adenosine deaminase and by spiking with an adenosine standard. Extraction samples from cell pellets were analyzed for nucleotides by high-performance liquid chromatography on a Whatman Partisil-10 (SAX) column equilibrated with 10 millimolar potassium phosphate (pH 3.78) and eluted with a linear gradient to 0.25 molar potassium phosphate, 0.5 molar KCl (pH 3.45). Continuous monitoring was performed by absorbance at 254 and 280 nm. Peaks were quantified by comparison with appropriate standard high-performance liquid chromatography analyses.

[0292] Figure 2 shows that adenosine release from lymphoblasts is enhanced by pre-treatment with AICA riboside (in the range of 100 - 500 micromoles) for 48 hours. Adenosine of approximately 1.4 nanomoles / 10 6 WI-L2 cells are excreted in the absence of the drug of the present invention, and this number increased to approximately 2.3 nanomoles with 500 micromoles of AICA riboside. When cells are pre-incubated with AICA riboside for 18 hours before 2-deoxyglucose exposure, enhancement of adenosine release occurs, as seen in Figure 7. Also, 3-hour pre-incubation and 4-hour incubation (during 2-deoxyglucose treatment) with either AICA riboside (Figure 8) or ribivirin (Figure 9) result in an increase in adenosine release. Cells were cultured to approximately O.5×10 6 cells / ml (mid-logarithmic phase) (Figure 2) and to approximately 1.0×l0 6 cells / ml (early stationary phase) (Figures 7 - 9).

[0293] Example II In vitro effect of AICA riboside on adenosine release in neuroblastoma cells Neuromuscular diseases in which an increase in adenosine release may be beneficial include cerebral palsy, autism, schizophrenia, and insomnia, etc. Neuroblastoma cell lines were cultured in medium under the conditions described in Example I. The medium was supplemented with 0 or 50 μM AICA riboside. To induce ATP catabolism, the culture medium was exchanged with a medium containing micromolar amounts of calcium ionophore A23187 and 1.0 μM deoxycholomycin. Under such conditions, the treated cells were shown to secrete at least twice as much adenosine as the control cells. Hypoxanthine phosphoribosyltransferase secretes twice less adenosine than cells with normal enzyme and can be corrected by pre-treatment with AICA riboside or ribavirin. The results are shown in Table I below.

[0294]

Chemical formula

[0295] Figures 3 and 4 show the results of a series of second experiments conducted to show the effect of AICA riboside on blood adenosine levels and to correlate the increase in adenosine with the increase in blood flow. Thirteen mongrel dogs were anesthetized with phenobarbital. A cannula was inserted into the anterior cardiac coronary vein, and blood samples were collected in 2N perchloric acid. Physiological saline or physiological saline containing 100 mM AICA riboside was randomly selected for a 45-minute infusion into the femoral vein before coronary artery occlusion at a rate of 1 ml / min. Coronary venous blood was collected and assayed for adenosine in the same manner as the assay described in Example I at 5 minutes before occlusion of the left anterior descending branch of the coronary artery and at 1, 10, 20, 30, and 50 minutes after occlusion, and at 1 minute after reperfusion. Regional myocardial blood flow was measured with radioactive-labeled spheres within 15 μm injected into the left atrium during the ischemic period at 5 and 60 minutes as described by Heymann et al. (supra) CV. Dis. 20;55 (1977). Electrocardiogram and arterial pressure were monitored throughout the ischemic period. Six AICA riboside-treated dogs and five saline-treated dogs survived the treatment. Fine fibrillation was seen in 2 of the surviving saline-treated animals. The concentration of AICA riboside in AICA riboside-treated dogs immediately before occlusion was 57.4 + / - 40.2 μM. The range was 4.4 to 100 μM.

[0296] Figure 3 shows that adenosine levels in the draining ischemic region increase dramatically in AICA riboside-perfused dogs. Before ischemia, none of the dogs had measurable intravenous adenosine (<0.01 μM) before and during the injection of AICA riboside or saline. Saline-treated animals had peak adenosine levels (0.22 ± 0.08 μM) 10 minutes after occlusion, which decreased to undetectable levels by 60 minutes. In contrast, AICA riboside-treated animals had peak adenosine levels (1.79 ± 0.35 μM) with 1 minute of ischemia, which remained high at 60 minutes (0.18 ± 0.15). Reperfusion resulted in no detectable adenosine washout in saline-treated animals, but a significant increase in AICA riboside-treated animals. Blood collected from the right atrium (systemic blood sampling) had no detectable adenosine in saline and AICA riboside-treated dogs.

[0297] Figure 4 shows that regional myocardial blood flow to the ischemic myocardium was significantly greater in AICA riboside than in saline-treated animals. Similar differences in flow were seen in the endocardium and epicardium, with no change between 5 and 60 minutes of ischemia. AICA riboside did not change flow to normal myocardium because the flow rates in non-ischemic tissue were remarkably similar between the two groups. Systemic arterial pressure and heart rate at 5 and 60 minutes showed no significant differences between the two groups of dogs. Arterial blood gas content and systemic intravenous granulocyte count did not differ significantly between the two groups. Thus, AICA riboside is thought to enhance collateral coronary blood flow to the ischemic myocardium, as described above, by increasing localized adenosine release and thus vasodilating blood vessels within the ischemic region and / or suppressing granulocyte free radical production and subsequent capillary damage and / or occlusion.

[0298] Example IV Effect of AICA Riboside Treatment on Inosine Levels in Dogs This increase in adenosine levels is at least in part due to a decrease in the amount of ATP converted to inosine, as demonstrated by analyzing the venous blood from dogs in Example III for inosine levels. Figure 5 shows a greater than two-fold decrease in inosine levels during a 60-minute assay period in AICA riboside-treated dogs. This data indicates that the compounds of the invention increase adenosine release by redirecting the catabolism of inosine-derived ATP, which is the more dominant end product under normal conditions, to adenosine.

[0299] Example V Effect of AICA riboside treatment on myocardial infarct size The effect of AICA riboside treatment on myocardial infarct size was examined in rats that were given a bolus of either AICA riboside-containing saline or saline alone and then induced to have restricted blood flow by ligation of the left anterior descending coronary artery. The animals were continuously exposed to an infusion of either AICA riboside-containing saline or saline using an osmotic minipump well known to those skilled in the art. After 3 weeks, the rats were sacrificed and the infarct size was quantified by planimarizing the stained area of the fixed heart. The results showed a 33% reduction in infarct size (p < 0.05) in AICA riboside-treated hearts compared to saline-treated controls.

[0300] Example VI Effect of AICA riboside treatment on arrhythmia One example of the consequences of myocardial ischemia is arrhythmia, and the frequency of arrhythmia is related to the degree of reduction in blood flow. Since adenosine is known to act as an anti-arrhythmic agent and to suppress granulocyte free radical generation (which can cause arrhythmia through lipid peroxidation), the preventive effect of AICA riboside treatment on arrhythmia was investigated. The electrocardiograms recorded during ischemia in Example III were analyzed for the number of premature ventricular depolarizations (PVD) and ventricular tachycardia (VTAC) episodes. Table 2 shows that saline-treated dogs had 112.2 PVDs and 18.2 VTAC episodes during ischemia, compared to 37.8 PVDs and 4.7 VTAC episodes in AICA riboside-treated animals (p < 0.01). One AICA riboside-treated dog (#3, which had frequent arrhythmias) had much lower collateral blood flow velocity and adenosine concentration compared to the other AICA riboside-treated dogs (however, a blood concentration of 27.2 μM AICA riboside).

[0301]

Chem.

[0302]

Chem.

[0303] Example VIII Effect of adenosine on granulocyte / endothelial cell interaction A test was conducted to determine whether adenosine decreases the adhesive affinity of granulocytes for endothelial cells, i.e., "stickiness" (an event that should increase blood flow within the microvessels). The parameter measured was the breaking stress between the two cell types.

[0304] Adenosine caused a two - fold increase in the rolling velocity of granulocytes within microvessels exposed to adenosine by surface under - perfusion of a 20 μM solution, resulting in a two - fold decrease in the breaking stress between granulocytes and endothelial cells (which line the inside of the blood vessel wall), and bringing about a concentration of approximately 2 μM in the blood vessel. This test was performed by intravital microscopy of granulocytes in rat mesenteric microvessels. The rolling velocity of granulocytes relative to the flow velocity of red blood cells was calculated before and after adenosine administration.

[0305] Example IX Effect of AICA riboside on granulocyte accumulation in ischemic myocardium AICA riboside reduces 111 the accumulation of indium - labeled granulocytes in ischemic myocardium. In the series of dogs described in Example III, granulocytes were removed, 111 labeled with indium, and reinjected. After 1 hour of ischemia, the animals were sacrificed, and within the myocardial biopsy material 111Granulocytes in myocardial tissue were quantified by measuring indium content using a gamma counter. The granulocyte content in the ischemic endocardium was lower significantly in AICA riboside-treated dogs (1.03 + / - 0.21×10 6 cells / gram) than in saline-treated animals (1.55 + / - 0.24×10 6 cells / gram). By measuring radiolabeled microspheres of collateral blood flow, results essentially identical to those shown in Example III were obtained. That is, blood flow in ACA riboside-treated dogs was significantly greater than in saline-treated animals.

[0306] Example X Treatment of autistic patients with AICA riboside A test was conducted to examine the beneficial effects of treating individuals with autism with AICA riboside.

[0307] In accordance with approval, a trial of treatment with AICA riboside was initiated in two patients (with autism) having adenylosuccinase deficiency. This trial of treatment was started by oral administration of a single dose of AICA riboside at 5 mg / kg / day on the first day. On the same day, in each patient, blood and urine samples were collected at various time intervals, and lumbar puncture was performed once 2 hours and 3 hours after AICA riboside administration, respectively. In view of the absence of clinical side effects, the same dose of AICA riboside was given on subsequent days, during which the patients stayed in the hospital and urine collection was continued. Since no harmful effects of the nucleoside administration were observed, the dosage of AICA riboside was increased to 2×5 mg / kg / day, and the patients were discharged in the treatment state on the 8th day. On the 55th day, both patients were readmitted for a short period for clinical, biochemical, and psychiatric evaluations. With no clinical side effects, the dosage of AICA riboside was increased from the 46th day to 2×10 mg / kg / day. The treatment was maintained until the 71st day and ended on that day.

[0308] On the 119th day, an intravenous loading test was conducted at a dose of 20 mg / kg / day, and lumbar puncture was performed 1 hour later for the specific purpose of evaluating the penetration of AICA riboside in cerebrospinal fluid (CSF).

[0309] At all doses used, with the available methodology, AICA riboside could not be detected in plasma and CSF. Nevertheless, the nucleoside was reabsorbed in the intestinal tract as shown by the finding that its triphosphate derivative, AICA riboside triphosphate, was present in erythrocytes during long-term oral administration. Even 1 hour after intravenous administration, AICA riboside could not be detected in plasma, but AICA riboside triphosphate was similarly accumulated in erythrocytes, indicating rapid cellular uptake and metabolism of AICA riboside. An accurate assessment of the renal decline of the nucleoside was not obtained.

[0310] Administration of AICA riboside was maintained without significant effect on the urinary excretion of two abnormal compounds, succinyladenosine and SAICA riboside, excreted by these patients, as well as on the urinary excretion of uric acid. Also, the administration did not significantly affect the concentrations of ATP and GTP in erythrocytes. The achieved concentration of AICA riboside triphosphate was of the same magnitude as GTP after oral and intravenous administration of AICA riboside.

[0311] In the assessment of the mental development of both patients immediately before the start of the trial with AICA riboside, significant psychomotor retardation (mental development of about 3 months according to the Bayley method) was shown, accompanied by the following autistic features: persistent asocial motor disability, unresponsiveness to auditory and tactile stimuli, and inadequate response to visual stimuli.

[0312] In the reassessment of these features after 2 months of continuous AICA riboside administration, no change was shown in the older patient. However, in his sister, a clear improvement was shown, with a decrease in the frequency of persistent movements, an improvement in the response to visual stimuli, and most notably, a response to auditory and tactile stimuli could now be recorded. Two months later, after a 6-week interruption of AICA riboside treatment, both patients were evaluated by their father as "more cheerful and easier to handle during treatment", thus prompting his intention to continue the trial.

[0313] The following parameters: erythrocyte count, leukocyte count, platelets and reticulocyte count; leukocyte differentiation; hematocrit, ionogram, Ca, phosphate, urea, creatinine, uric acid, cholesterol, lipids, SGOT, SGPT, CPK, glucose, lactate and ammonia were found to be normal before and during the trial treatment with riboside.

[0314] Example XI Effect of ribavirin on mast cell degranulation By suppressing mast cell degranulation, it is possible to prevent or control the allergic response of patients. Bone marrow collected from the femurs of Balb / C mice was cultured in a 1:1 mixture of Razin medium and conditioning medium as described by Razin et al., Proc. Natl. Acad. Sci. USA 28:2559-2561, 1981, and generated by co-culturing splenocytes from C57B1 / 6J mice and C3H mice in the presence of concanavalin A. After subculturing weekly and culturing in tissue culture for at least 15 days, the resulting cells were 90% pure mast cells and 95% viable as evaluated by trypan blue exclusion. Cells exposed to ribavirin during culture were washed three times before use in the experiment. Parallel cultures of cells cultured in single medium were used as controls for pharmacologically manipulated mast cells. Cell cultures were evaluated by counting the cells at specific time points and comparing the actual number of ribavirin-treated cells to the number of cells cultured in single medium.

[0315] β-Hexosaminidase is easily quantifiable and its release is comparable to that of histamine, so it was selected as a representative granule-associated, preformed mast cell mediator. Mouse bone marrow-derived mast cells were centrifuged at 200×g for 5 minutes, washed three times in Tyrode buffer without divalent cations, and incubated with anti-DNP (dinitrophenyl phosphate) IgE (1 μg / 10 6Sensitize with (cells), and in 400 μl of complete Tyrode buffer, DNP-BSA antigen (175 ng / 3×10 5 cells) or A23187 (10 μg / ml / 3×10 5 cells) and antigen-stimulate at 37 °C for 10 minutes. Centrifuge the reaction mixture at 200×g for 10 minutes, and assay the β-hexosaminidase concentration in the supernatant and pellet by hydrolysis of p-nitrophenyl-β-D-glucosamide as described by Schwartz et al., J. Immunol. 123, 1445 (1979). Spontaneous β-hexosaminidase release was measured in non-antigen-stimulated cells. The percentage of the net released β-hexosaminidase is defined as follows: ##EQU1## (where [β-hex] is β-hexosaminidase and super is the supernatant). Exogenous adenosine was present in the reaction mixture, which was added simultaneously with the secretagogue.

[0316] Mouse bone marrow-derived mast cells antigen-stimulated with A23187 or DNP-BSA antigen released 8 - 15% of the total cell β-hexosaminidase (granule-associated mediator). Ribavirin (10 μM) added at the time of mast cell stimulation had no effect on β-hexosaminidase release. However, mast cells incubated in 10 μM ribavirin for 3 - 7 days, washed, and antigen-stimulated with A23187 showed a significant attenuation of β-hexosaminidase release compared to cells parallel-cultured in single medium (Figure 10). Asterisks (*) identify that the control cell data are significantly different (p < 0.05). Ribavirin exposure did not modify the mast cell mediator content (i.e., the total cell β-hexosaminidase concentration) nor cell viability, and the spontaneous release of β-hexosaminidase was similar in the two cell populations. The dose-response relationship between ribavirin exposure and mediator release is shown in Figure 11. 1 μM of ribavirin for 6 days significantly inhibits mediator release, but the maximum inhibition is evident at 10 μM - 20 μM.

[0317] Example XII Regulation of Mast Cell Activation and Degranulation by AICA Riboside Mast cell activation and degranulation play a major role in allergic diseases such as asthma. Therefore, means to suppress activation and degranulation provide a way to control the disease.

[0318] A. Isolation of Mast Cells. To demonstrate the suppression of degranulation and activation by the method described in the claims, cells were first isolated and cultured as described in Example XI.

[0319] B. Effect of AICA Riboside on Degranulation. The inhibition of degranulation by AICA riboside was demonstrated by showing that AICA riboside inhibits degranulation induced by calcium ionophore A23187 (reflected by the release of the acid exoglycosidase β - hexosaminidase). 1 μg / ml of A23187 was added to mast cells with or without AICA riboside at 37 °C on Tyrode buffer, and the amount of mast cell β - hexosaminidase released was measured. In the presence of 100 μmol of AICA riboside, only 17.6% of hexosaminidase was released, while in its absence, 28.8% was released. Therefore, AICA riboside inhibits mast cell degranulation. The release rate of β - hexosaminidase and the assay method for the enzyme were performed as described by Schwartz et al., J. of Immun., Vol. 123, October 1979, p. 1445. 6 C. Effect of AICA Riboside on the Release of Leukotriene C

[0320] Cells cultured for 6 days in either single medium or medium containing 100 μM AICA riboside were washed and antigen - stimulated with A23187 for 20 minutes. When the concentration of leukotriene C 4 in the supernatant was measured by radioimmunoassay, it was 51 and 13 nanograms / 10 for control and AICA - riboside - treated cells, respectively. 4 in the supernatant was measured by radioimmunoassay, it was 51 and 13 nanograms / 10 6It was shown to be a cell. Leukotriene C 4 Release was significantly reduced by 75% (p < 0.01) by AICA riboside pretreatment. Similar results were also obtained in pretreatment with 10 μM ribavirin for 4 - 6 days, at which time mast cell activation was performed using an antigen that binds to IgE on the mast cell surface.

[0321] Example XIII Suppression of pentylenetetrazol - induced seizures To test the ability of AICA riboside to suppress pentylenetetrazol - induced seizures, rats (10 per condition) were pretreated (randomly and blindly) 30 minutes and 5 minutes before injection of 60 mg / kg of pentylenetetrazol with 1000 mg / kg or 100 mg / kg of intraperitoneal AICA - riboside - containing saline (0.9%), or an equal volume of saline. Two separate seizure specialists observed the animals for 1 hour.

[0322] A 40% reduction in the number of animals having seizures was seen in the group receiving 2000 mg / kg (total dose), and in this group, a dramatic prolongation of the seizure latency was observed (Figure 12).

[0323] Example XIV Suppression of catecholamine - induced arrhythmia To examine whether AICA riboside can protect the heart from isoproterenol (Isuprel) - induced arrhythmia, 9 pairs of rats were tested. One animal of each pair was intraperitoneally injected with 1000 mg / kg of AICA riboside (in water). The other animal of each pair served as a control and was similarly injected with an equal volume of saline (0.9%) as the AICA riboside solution.

[0324] After 5 minutes, both animals were anesthetized by intraperitoneal injection of 330 mg / kg of chloral hydrate. Then, one EKG lead wire was attached to each rat, and the electrocardiograms of the paired rats were recorded simultaneously. To induce arrhythmia, each rat was subcutaneously injected with Isuprel (1000 mg / kg).

[0325] It was started 30 minutes after the introduction of ispradol, carried out for 10 minutes with the recording paper speed of the electrocardiograph set to (5 cm / second), and the arrhythmic beats of both animals were counted.

[0326] In AICA riboside-treated rats, a 39% reduction in paroxysmal ventricular contractions and complete suppression of ventricular fibrillation were observed (Figure 13).

[0327] By the following experiments, the applicant determined the concentration and dosage of AICA riboside that reduce the frequency, duration, and severity of ischemic events, reduce tissue damage, and avoid side effects such as a clinically significant increase in serum and urinary uric acid levels and crystalluria. The applicant also determined the concentration and dosage of AICA riboside that suppress or reduce the severity of adverse clinical outcomes (such as adverse cardiovascular and cerebrovascular events, etc.).

[0328] The following examples do not limit the present invention. Those skilled in the art will recognize that administration of the described amount of AICA riboside will similarly reduce tissue damage caused by reduced blood flow and reduce the occurrence of adverse clinical outcomes (such as adverse cardiovascular and cerebrovascular events, etc.) even in cases other than CABG surgery.

[0329] Example 1 Effect of AICA riboside in patients undergoing coronary artery bypass graft (CABG) surgery: Phase 2 clinical trial This experiment was conducted to evaluate the effect of AICA riboside on the frequency, duration, and severity of myocardial ischemic events and left ventricular function during and after CABG surgery. The effect of AICA riboside treatment on the difficulty of weaning from cardiopulmonary bypass was also evaluated. Furthermore, the effect of AICA riboside on the occurrence of certain adverse clinical outcomes was evaluated.

[0330] Test plan This trial was a multi-center, randomized, double-blind, repeated-dose, placebo-controlled comparative trial, in which 118 patients were evaluated at four centers. Patients scheduled for elective CABG surgery were randomly assigned to treatment with one of two doses of AICA riboside or placebo (continuous infusion throughout the treatment). Clinical outcomes, hemodynamics, and the occurrence and severity of ischemia (by continuous electrocardiogram recording (ECG) and transesophageal echocardiography (TEE)) were recorded and compared between treatment groups.

[0331] Patient This trial included women with no possibility of pregnancy and men at least 30 years old who were scheduled for elective CABG surgery for the treatment of coronary artery disease confirmed by typical changes (stenosis of at least 50% in at least two major blood vessels) shown by coronary angiography performed within 6 months of the surgery. Patients with unstable angina were included if they had been stable for at least 24 hours and had not had a myocardial infarction in the previous two weeks. Patients who had undergone emergency CABG or repeated CABG; patients with a resting ejection fraction of less than 30%, a cardiac index of less than 1.5 L / min / m 2 Patients with a cardiac index less than, or patients with idiopathic cardiomyopathy, significant valvular heart disease, severe left ventricular hypertrophy, or major intracardiac abnormalities were excluded from this trial. Also excluded were patients with insulin-dependent diabetes mellitus or hypoglycemic states, liver or kidney diseases, untreated gout, or a recent history of alcohol or other drug abuse. Thrombolytic therapy was prohibited for two weeks before the surgery, and amiodarone was prohibited for 60 days before the test, and dipyridamole, theophylline, and aminophylline were prohibited for 24 hours before the test. Both smoking and the intake of foods or beverages containing methylxanthines were prohibited from 12 hours before drug administration until discharge from the intensive care unit.

[0332] Treatment and Methods Patients scheduled for CABG surgery and selected as described above were randomly assigned to receive AICA riboside infusion (initially at 0.19 mg / kg / min or 0.38 mg / kg / min; thereafter, at 0.05 mg / kg / min or 0.1 mg / kg / min for the first 6 patients) or placebo, which was started immediately before induction of anesthesia and continued for 7 hours (in all cases, this means that the infusion did not end until the surgery was completed and the patient recovered in the intensive care unit). Also, AICA riboside (final concentration 20 μM) or placebo was added to the crystalloid cardioplegic solution used to perfuse the coronary circulation during the bypass period. No other drugs were added to the crystalloid cardioplegic solution.

[0333] During the preoperative period, routine history, physical examination, study measurements, electrocardiogram (ECG), and chest x-ray were obtained. Continuous ECG (Holter) recordings for at least 8 hours were obtained before intubation. Routine cardiovascular drug applications were continued until the morning of the indicated surgery. Immediately before the start of surgery, a catheter was placed in the radial artery for blood pressure measurement and arterial blood sampling. For hemodynamic measurements, a triple-lumen thermodilution catheter was introduced into the pulmonary artery. After tracheal intubation, an echocardiographic transducer was placed at the midpapillary muscle level using a transesophageal approach.

[0334] During the operation, anesthesia was maintained by continuous infusion of fentanyl and midazolam. Routine clinical parameters were recorded using standard operating room monitoring equipment. Continuous two-lead Holter ECG and TEE data were recorded. Standard surgical procedures (e.g., aortic cross-clamping, crystalloid cardioplegia, cardiopulmonary bypass, hypothermia) were used. Anastomoses were constructed, and the patient's bypass was discontinued when the body temperature reached 37°C, except for aortic cross-clamping. The amount of anastomosis was determined by the surgeon. The difficulty in weaning from bypass was determined by the need for one or more of the following: pacemaker, return to bypass, balloon pump, or vasopressor administration. Hemodynamic measurements (including heart rate, arterial blood pressure, pulmonary capillary wedge pressure, and cardiac output) were recorded before sternotomy, 15 and 30 minutes after bypass, and at the time of chest closure. Radial artery and pulmonary artery pressures, myocardial temperature, and systemic body temperature, O 2Saturation, end-tidal CO 2 Saturation, end-tidal CO, and arterial blood gases were measured and ECG recordings were obtained as clinically indicated. Hemodynamic variables (blood pressure, heart rate, pulmonary capillary wedge pressure) were controlled within 20% of baseline using a predefined regimen.

[0335] During the postoperative period on the first postoperative day, morphine and midazolam were used for sedation and analgesia. The required drug applications to the painful cardiovascular system were recorded. During this period, continuous ECG monitoring (Holter) was performed up to 48 hours. Hemodynamic measurements (pulmonary artery pressure and cardiac output) were obtained at 2, 4, 8, and 12 hours (optionally at 24 and 48 hours) and whenever clinically indicated.

[0336] A 12-lead ECG was obtained on admission to the intensive care unit and on the 1st, 2nd, and 3rd postoperative days and at discharge. Creatinine phosphokinase MB band (CK-MB) was obtained every 8 hours for 48 hours and whenever indicated. Radionuclide ventriculography for ejection fraction and wall motion score was performed approximately 14 days postoperatively and as close to discharge as possible. Other tests and measurements (e.g., chest x-ray, pulmonary capillary wedge pressure (PCWP)) were performed whenever clinically indicated for the diagnosis or evaluation of myocardial infarction or congestive heart failure. The timing and dosage of all drug applications to the painful cardiovascular system within 48 hours postoperatively were recorded. The total dosage of all analgesics during the first 24 hours postoperatively was recorded. Fluid intake and output (e.g., blood transfusion and urine output) were recorded for 48 hours. The type and duration of required inotropic therapy and antiarrhythmic interventions were recorded for the first 24 hours postoperatively. A 2-channel Holter recording was obtained during three periods: 8 hours before intubation, from intubation until the end of surgery, and an additional 24 - 48 hours postoperatively.

[0337] Safety assessment In addition to the above hemodynamic monitoring, the following tests were performed at screening, on the first postoperative day, and at discharge.

[0338] 1. Blood tests included hemoglobin, total white blood cell count, and hematocrit and platelet fraction.

[0339] 2. Biochemical tests included serum sodium, potassium, chloride, phosphorus, magnesium, urea, creatinine, serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), total bilirubin, albumin, total protein, uric acid, alkaline phosphatase, creatinine phosphokinase and CPK-MB. Also, CPK-MB was measured every 8 hours for 48 hours after chest closure. Blood glucose and uric acid levels were measured before injection, during cardiopulmonary bypass (CPB), after CPB, upon admission to the intensive care unit (ICU) and 4 and 8 hours after that. Also, these levels were measured 24 hours after CPB and at the time of discharge.

[0340] 3. Urine tests included pH, protein, glucose, ketones, red blood cells, white blood cell casts and crystals in the blood. Also, urine was collected before treatment, at the end of injection, 4 and 8 hours after the end of injection, and the uric acid content was measured.

[0341] 4. Throughout the study, the researchers recorded any adverse events and evaluated their severity and the relationship between these adverse events and the treatment.

[0342] Evaluation of Efficacy One measure of efficacy was the degree to which AICA riboside reduced the occurrence, duration and / or severity of ischemic events (by comparing changes in the S-T segment in continuous Holter recordings before treatment, during treatment and 48 hours after treatment). The efficacy of AICA riboside in reducing the harmful effects of ischemia on myocardial structure was also measured (by evaluating regional wall motion in TEE before and after the bypass period, as well as by measuring the ejection fraction before and after surgery). Primarily, the evaluation of the Holter tape and echocardiogram video tape was performed blindly by two independent observers. In case of disagreement between the two, a third observer was employed to "resolve the issue". The same observer was employed throughout the study.

[0343] The occurrence of adverse clinical outcomes such as cardiac death (mainly, death of patients due to the heart), non-fatal transmural MI (appearance of new Q waves on a 12-lead ECG + CK-MB value of 50 units or more (≥) as measured), non-transmural MI (CK-MB value of 50 units or more), congestive heart failure (low cardiac output requiring an intra-aortic balloon pump or left ventricular assist device) or life-threatening arrhythmias (ventricular fibrillation or electrical defibrillation or ventricular tachycardia requiring drug treatment) was compared between the placebo group and the treatment group. For the diagnosis of myocardial infarction, ECG and CK-MB values were evaluated mainly by observers unaware of the treatment details. When difficulty in weaning from bypass was recognized in a patient, comparison was made between the placebo group and the treatment group by indicating the need for one or more of the following: pacemaker, bypass return, balloon pump or pressor use.

[0344] Statistical Analysis The results reported in this specification do not include all the measured parameters, but the following measurements were made using the indicated methods.

[0345] 1. Group comparability. To evaluate the comparability among the three treatment groups, the following baseline and intraoperative measurements were evaluated using one-way analysis of variance for continuous variables and chi-square test in contingency tables for discrete variables.

[0346] 2. Baseline. Age, gender, cardiovascular disease history (angina, hypertension, MI history, CHF, arrhythmia), ejection fraction, catheterization data (number of stenosed vessels), number of pre-bypass ischemic events and minutes of ischemia per hour (measured by Holter ECG).

[0347] 3. Intraoperative. Number of graft vessels, aortic cross-clamp time, operation time, bypass time.

[0348] 4. Clinical outcomes. The outcomes of cardiac death, MI, CHF, and life-threatening arrhythmias were compared. The specific endpoints of the analysis were grouped into dichotomous endpoints (i.e., when at least one of the above four events occurred vs. when nothing occurred). Fisher's exact test for small samples was used to compare the clinical outcome rates among the three treatment groups. The same comparison of combined active treatment vs. placebo was performed.

[0349] 5. Ischemic events - TEE. Ischemic event data were evaluated using the following analyses during two periods, namely before bypass and after bypass.

[0350] a) The number of patients with ischemic events was compared between groups using Fisher's exact test. This analysis also included changes before and after, as well as before and after combination.

[0351] b) For patients with the event, analyses of mean duration and severity of ischemia were performed. Only patients with a reduced number of ischemic events included in the analysis were used, but when the event occurred, it was possible to examine whether the drug was effective in reducing its magnitude. The distribution of ischemic duration was found to be asymmetric, so a log lO transformation was used to derive a normal component, and one-way ANOVA was used to compare between groups. For severity (ordinary variable, 0 - 4 grades) and the number of events, the Kruskal - Wallis nonparametric test was used. The same comparison was performed for the combined active treatment group.

[0352] 6. Ischemic events - ECG. The analysis of ischemic events shown by ECG was performed using the same method as for echo events. The analysis times were (a) baseline (from Holter start to injection start), (b) before bypass (from injection start to bypass start), (c) after bypass (from side clamp off to injection end), and (d) after treatment (from injection end to Holter end). For patients with the event, the following variables were analyzed: mean duration, maximum ST change, and area under the curve of significant ST segment deviation. Analysis of variance (ANOVA) was used.

[0353] The same comparison was made in the combined aggressive treatment group.

[0354] 7. Ischemia and outcome. The relationship between ischemia (TEE and ECG) detected during the period outlined above and clinical outcome was analyzed using Fisher's exact test (see J. Leung et al.: Prognostic Importance of Postbypass Regional Wall-Motion Abnormalities in Patients Undergoing Coronary Artery Bypass Graft Surgery. Anesthesiology 71:16 - 25, 1989).

[0355] 8. Difficulty in weaning. Patients were considered to have difficulty in weaning if they required one or more of the following interventions: pacemaker, return to bypass, balloon pump, or use of pressor agents. The number of patients who received each of the above interventions was analyzed using X 2 and Fisher's exact test and this was taken as the number of classified patients with difficulty in weaning. Also, the time to weaning in patients with difficulty (defined as the time from removal of the cross-clamp to the end of bypass) was compared by one-way analysis of variance.

[0356] 9. Ejection fraction. Pre- and post-operative ejection fractions were measured using different methodologies. Therefore, changes could not be statistically analyzed. Group mean injection fractions and before and after CABG were presented, showing apparent differences between groups.

[0357] 10. Plasma levels. AICA riboside plasma levels were measured, confirmed to be consistent with random treatment, shown to be dose-proportional, and the achieved plasma levels were evaluated. Individual values and group means were tabulated and drug clearance was calculated. Dose-proportionality was evaluated.

[0358] 11. Adverse effects. When adverse effects occurred, their occurrence, severity, and drug relatedness were tabulated by the reduction in the occurrence of such effects in the high-dose group in the treatment group. No statistical analysis was performed.

[0359] 12. Study data. For the subject selection parameters, the individual values, mean changes, and percentage of change from baseline were tabulated and plotted over time in the treatment group. The following parameters were addressed.

[0360] Urine - uric acid, creatinine, uric acid / creatinine ratio, pH, volume, crystals. All values were tabulated as mean ± S.E.M.

[0361] Blood chemistry - CPK, CK - MB, uric acid, glucose.

[0362] The AU values were tabulated as mean ± S.E.M.

[0363] Results The tests described herein represent the first exposure to continuous long - term infusion of AICA riboside in patients undergoing anesthesia, cardiopulmonary bypass surgery, and hypothermia. When evaluating the dose levels using healthy volunteer subjects or conscious normothermic patient results, uncertainties were seen regarding the applicability of pharmacokinetic tests using short - term infusions in healthy volunteer subjects or conscious normothermic patients.

[0364] The first six patients included in this trial were given doses of 0.19 mg / kg / min and 0.38 mg / kg / min. When the plasma levels of AICA riboside were measured, contrary to expectations, they were found to be approximately 2 - 4 times higher than predicted. The applicant does not wish to be bound by any particular theory, but the high AICA riboside levels may have been caused by reduced drug metabolism due to low liver blood flow and hypothermia. Also, this high level may have been due to the effect of prolonged infusion on the clearance rate. The dose levels were reduced from 0.19 and 0.38 mg / kg / min to 0.05 and 0.1 mg / kg / min. This then resulted in steady-state plasma concentrations of approximately 2.5 and 5.0 μg / ml being achieved in subsequent patients.

[0365] The results of these first six patients, who were initially tested at high doses, are summarized below. This was included in the overall safety analysis. Except for patient A4 who received a placebo, these results were not included in the assessment of efficacy unless otherwise noted with respect to such results.

[0366] In the first high-dose group, four patients received the low dose (0.19 mg / kg / min), one patient received the high dose (0.38 mg / kg / min), and one patient was a placebo patient. Generally, the drug was well tolerated and no serious adverse events were seen. As shown in Table 1, blood glucose levels increased at most time points in all patients. There were no values below the normal lower limit. An increase in urinary uric acid levels with significant hyperuricemia and crystalluria was seen, and urethral catheter irrigation was required in five of the drug-treated patients (Table 1). In these patients, the urine had a clear green coloration, presumably due to high concentrations of AICA riboside and / or its degradation products. Also, as shown by the data in Table 1, blood glucose levels did not decrease.

[0367] Apart from these effects on plasma and urinary uric acid levels and crystalluria, no adverse events were seen that were considered to be related to treatment with AICA riboside. At the low dose (0.19 mg / kg / min), 2 of 4 patients (A3 and A6) had no other adverse events, and 1 patient (A1) had premature ventricular contractions, labile blood pressure, and low blood PO 2 occurrence (which were trivially corrected). This patient also had a sigmoid colon cancer of the rectum that was clearly not related to drug treatment, and appropriate treatment was therefore planned. The fourth low-dose patient (A2) had the occurrence of complete heart block after bypass and then had hypertension approximately 4 hours later. Patient A5, who received 0.38 mg / kg / min, had no events other than ST segment elevation on the ECG after bypass.

[0368]

Chem.

[0369]

Chem.

[0370]

Chem.

[0371]

Chem.

[0372] In almost all cases in all treatment groups, these events were of mild or moderate severity and did not require any other special drug application. Five other events were classified as severe, two were acute myocardial infarctions (patients A26 and A39), one of which also had CHF requiring intra-aortic balloon pump assistance, one was a pulmonary embolism (patient A12) and one had an arterial embolism in the right foot requiring amputation (patient A14).

[0373] One death has been seen in this trial so far. Patient A36 was a 67-year-old male in the placebo group who had unstable angina, inadequately controlled hypertension and high-grade left main disease preoperatively. After a simple surgical procedure, he showed dyspnea in the intensive care unit and the ventilator was found to be malfunctioning. External pacing with other resuscitation means and a final open-chest cardiac massage were not successful.

[0374] In virtually all cases (including all the severe events above), the researchers thought the event was unrelated to the drug or unlikely to be due to the drug, with the following exceptions. Patient A2, who received 0.19 mg / kg / min, had hyperuricemia and orange granules in the urine, and the urine of patient A14 at a dose of 0.05 mg / kg / min showed the same green coloration as described for the first high-dose patient.

[0375] Serum uric acid and glucose levels After the first six patients, the dose of AICA riboside was reduced, but no further clinical evaluation of serum uric acid was performed. As shown in Table 4a, the mean change showed a clear tendency for a dose-related increase in serum uric acid in the treatment group. However, clinically significant hyperuricemia or crystalluria was not seen. During treatment, infusion solutions containing glucose were given. As shown in Table 4b, plasma glucose levels increased in all groups.

[0376]

Chem.

[0377]

Chem.

[0378]

Chem.

[0379] 2. Non-transmural myocardial infarction. Clinically significant elevation of creatine phosphokinase MB band (CK-MB) level, more than 50 I.U., with or without S-T segment elevation, with or without appearance of new Q waves in 12-lead ECG, was observed in 17 (47%) placebo patients, 13 (13.7%) patients receiving low-dose AICA riboside, and 8 (23.5%) patients in the high-dose group (p = 0.10, X 2 test) (Table 6). The results were statistically significant for the combination treatment group vs. placebo (p = 0.046).

[0380]

Chem.

[0381]

Chem.

[0382]

Chem.

[0383] The occurrence of ECG ischemia tended to be lower in the high-dose group (11%) compared to the placebo or low-dose groups (18% and 22%, respectively) (p = 0.42). As shown in Table 8, in patients in whom ischemic events occurred, the severity of postoperative ECG ischemic occurrence was lower in the high-dose group compared to the low-dose or placebo groups, as judged from the described mean duration, mean S-T curve area under the curve (AUC), millimeter-minutes (mm-min), and ischemia minutes per hour (Isch min / h).

[0384]

Chemical formula

[0385] Difficulty in weaning from bypass As described, the patient was determined to have difficulty weaning from cardiopulmonary bypass if one or more of the following interventions were required: pacemaker insertion, bypass reinstatement, use of an intra-aortic balloon pump or administration of vasopressors, or another intervention determined by the investigator to be of difficult withdrawal. There was no significant difference between groups with respect to the need for a pacemaker, bypass reinstatement or intra-aortic balloon pump support. Both the low-dose and high-dose groups showed a strong tendency towards a decrease in the need for vasopressor support (p = 0.19). See Table 9a. When the high-dose and low-dose groups were combined and compared to placebo (Table 9b), the decrease in the need for vasopressor support approached statistical significance (p = 0.08). As a result of the decrease in the need for vasopressor support in the high-drug and low-drug treatment groups, combined with a slight decrease in the need for other support in these patients, there was a strong tendency towards a decrease in weaning difficulty in the drug treatment groups (p = 0.17 when compared individually and p = 0.06 when the combined dose groups were compared to placebo). No difference was seen in any of the above weaning difficulty parameters between the high-dose and low-dose groups (Table 9c).

[0386] There was no statistically significant difference between groups with respect to weaning time measured as the time from cross-clamp removal to bypass termination. However, when only patients with difficulty weaning from bypass (above) were evaluated, a strong tendency towards a decrease in time was seen in the drug-treated patients (Table 9d).

[0387]

Chemical formula

[0388]

Chemical formula

[0389]

Chemical formula

[0390] Pharmacokinetics Table 11 below shows the mean plasma AICA riboside concentrations for 40 patients receiving 0.05 mg / kg / min and 31 patients receiving a dose of 0.1 mg / kg / min before bypass, after bypass, at the end of infusion, and 60 minutes after infusion. This data is also shown graphically in Figure 18.

[0391] [Chemical formula] The desired steady-state plasma concentrations of 2.5 and 5.0 μg / ml at low and high doses, respectively, were approximately similar before and after bypass over time, indicating good dose proportionality. The mean estimated values of the total plasma clearance (CL p ) were approximately the same at low and high doses at both of these times, close to 1.2 L / h / kg (in the range of 1.1 - 1.2 L / h / kg). This indicates that AICA riboside exhibits linear kinetics in patients undergoing CABG surgery at the infusion rates used in this study. These clearance rates are approximately 40 - 50% of those previously seen in conscious healthy male subjects. Dixon, R. et al., J. Clin. Pharm. 31:342 - 347 (1991). The applicant does not wish to be bound by any particular theory, but this difference in drug clearance may be the result of hypothermia, decreased tissue uptake, reduced hepatic blood flow during CABG surgery, or decreased metabolism due to changes in metabolism associated with prolonged infusion. During the period after bypass, an increase in CL p was observed, along with changes in physical condition associated with increased body temperature and hepatic blood flow and a tendency to discontinue anesthesia at this point. After the infusion was terminated, the plasma AICA riboside concentration rapidly decreased to 10% of the steady-state level after 1 hour.

[0392] Discussion Perioperative myocardial infarction (MI) is a not infrequent complication of CABG surgery, and depending on the criteria used for diagnosis, incidences of 10 to 50 percent have been reported. Recent studies have reported adverse effects of perioperative MI on direct mortality, long-term survival, or both (see H. Schaff et al., J. Thorac. Cardiovasc. Surg. 88:972-981 (1984); P. Val. et al., J. Thorac. Cardiovasc. Surg. 86:878-886 (1983); W. Fennell et al., J. Thorac. Cardiovasc. Surg. 78:244-253 (1979); R. Seitelberger et al., Circulation 83:460-468 (1991)).

[0393] The tests of the present invention show that AICA riboside defends against the adverse effects of tissue ischemia, suppresses irreversible myocardial cell necrosis, and reduces the degree of cardiac dysfunction due to ischemic injury throughout the perioperative period (including immediately after surgery (postoperative reperfusion)) (and by mixing with a myocardial protection solution).

[0394] The results of the experiments presented herein show a tendency for a decrease in the incidence of transmural MI when evaluated using both new Q waves and changes in serum enzyme levels (placebo (13.5%) and low and high doses of AICA riboside (4.9% and 5.7%, respectively)). This tendency is even clearer for non-transmural MI (i.e., CK-MB levels greater than 50 I.U. in the absence of ECG changes) (placebo 47.2%, low dose 31.7%, high dose 23.5%).

[0395] When comparing the three groups, a strong tendency (p = 0.10) for a decrease in perioperative MI was seen, but these results did not reach significance. However, when comparing all patients treated with the drug (including the first 5 who received the high dose) to placebo, a statistically significant decrease (p < 0.05) in the rate of perioperative infarction was seen in the drug-treated group.

[0396] The applicant also showed that AICA riboside modifies the duration and severity of ischemic events. The mean duration of post-bypass ischemic events in placebo patients was 175 minutes (±156 minutes). Treatment with AICA riboside resulted in a decrease in the mean duration of post-bypass ischemic events to 125 minutes (±80 minutes) in the low-dose group and 36 minutes (±20 minutes) in the high-dose group (p = 0.04). Also, the fraction of post-bypass ischemia per hour was lower in the high-dose group (27 ± 20) than in the placebo (35 ± 14) and low-dose (40 ± 15) groups.

[0397] Also, the severity of post-bypass ischemia was reduced by administration of high-dose AICA riboside. The mean S-T segment inferior area was 35 ± 14 and 40 ± 15 in placebo and low-dose patients, respectively. However, administration of high-dose resulted in a value of 27 ± 20.

[0398] Also, AICA riboside appeared to have an effect on pre-bypass ischemia, at least on the occurrence of TEE ischemia (6% for high-dose vs. 19% for placebo and 15% for low-dose).

[0399] Also, the results of this trial showed an improvement in the patient's ability to wean from bypass. Patients who received AICA riboside tended to receive less pressor support to recover post-bypass function. In fact, a nearly statistically significant improvement was seen in the reduction of pressor use in the drug-treated group. This indicates that patients who received the drug were less impaired than those in the placebo group.

[0400] Changes in hemodynamics are difficult to interpret in the context of CABG surgery. Heart rate and blood pressure are usually controlled by a variety of pharmacological factors and adjustment of circulating volume, and the effects of treatment with AICA riboside were not seen in these parameters. However, a tendency towards a higher ejection rate was observed in the high-dose AICA riboside group immediately before discharge from the intensive care unit compared to the placebo and low-dose groups. Such improvement in the functional performance of the heart could be consistent with the effects on the level of ischemia and the occurrence of myocardial infarction.

[0401] All these results indicate the beneficial effects of AICA riboside administration (especially at a dosage of about 0.1 mg / kg / min). When combined with the problems of hyperuricemia and crystalluria associated with AICA riboside administration at dosages of 0.19 mg / kg / min, and especially 0.38 mg / kg / min, these results indicate that the therapeutic dosage of AICA riboside is preferably about 0.1 mg / kg / min.

[0402] Conclusion Those skilled in the art, upon considering the above examples, will recognize that the data indicate that AICA riboside administration is safe and effective in suppressing tissue damage caused by an undesirable decrease in blood flow at the described dosages. When administered at the dosages described herein, undesirable clinical hyperuricemia and / or crystalluria can be avoided while maintaining effectiveness.

[0403] Example 2 Effect of AICA riboside in patients undergoing coronary artery bypass graft (CABG) surgery: A phase 3 clinical trial Similar to the experiment described in Example 1, the following experiment was conducted to evaluate the effect of AICA riboside administered to patients undergoing CABG surgery and to determine the effective dosage and concentration of AICA riboside. The applicant has found that AICA riboside has effective concentrations and dosages for the prevention of adverse clinical outcomes (such as adverse cardiovascular events, for example, myocardial infarction and cardiac death) compared to placebo. The applicant has also found that AICA riboside is effective in preventing adverse cerebrovascular events (such as cerebrovascular injury, etc.) compared to placebo. In addition, the applicant has found the concentrations and dosages of AICA riboside that are particularly effective in reducing the co-occurrence of adverse cardiovascular and cerebrovascular events. Also, these concentrations and dosages of AICA riboside are considered to be effective in preventing or reducing the occurrence of congestive heart failure and life-threatening arrhythmias.

[0404] The trial described in this Example 2 was a multicenter placebo-controlled double-blind trial conducted in approximately 600 patients at 20 facilities in the United States. Patients received either the same dosing regimen as that administered in the trial described in Example 1, placebo, or one of two AICA riboside dosages (0.05 or 0.1 mg / kg / min for 7 hours). In all cases, AICA riboside was also administered to patients receiving AICA riboside treatment at a concentration of 5 μg / ml in the cardioplegic solution.

[0405] The trial described in this Example 2 differed from the trial described in Example 1 in patient selection criteria. In the trial described in Example 1, patients considered to be at high surgical and medical risk during CABG surgery, i.e., repeat CABG patients, emergency patients, and patients with inadequate left ventricular function, were excluded. In the trial described in this Example 2, all patients undergoing CABG surgery were considered eligible for enrollment in this trial, except that patients with recent or evolving myocardial infarction were excluded because a new myocardial infarction could be diagnosed. Also, in Example 2, a wider selection of cardioplegic solutions was allowed to reflect typical surgical use patterns.

[0406] Table 12 below shows the statistical analysis of the occurrence of myocardial infarction (defined by ECG and CK-MB levels, i.e., transmural MI), cerebrovascular injury, cardiac death, congestive heart failure, and arrhythmias related to life and death. As shown in Example 1, the low dose of AICA riboside is 0.05 mg / kg / min, and the high dose is 0.1 mg / kg / min.

[0407]

Chemical formula

[0408] 1. An increase in CK-MB concentration up to 100 ng / ml or more at any time point after surgery, and accompanied by a CK-MB sample of 50% or more of this peak value before and after; 2. An increase in CK-MB concentration up to 70 ng / ml or more at any time point 12 hours after surgery, and accompanied by a CK-MB sample of 50% or more of this peak value before and after; or 3. After 24 hours after surgery, if a new increase in CK-MB release to a peak of 12 ng / ml or more by another measurement at least 10 ng / ml occurs immediately before or after the peak, and the level had risen previously, the level must have dropped below 10 ng / ml before this second increase occurred.

[0409] The diagnosis of cerebrovascular injury (CVA) was determined by signs and / or symptoms of significant neurological deficits that persisted for more than 24 hours. If a focal neurological lesion that persisted for more than 24 hours was seen, CVA was considered an endpoint of the study. If a neurologist diagnosed CVA, or if a CT or MRI scan reported a match with a new cerebral infarction or cerebral hemorrhage, patients with non-focal lesions were considered endpoints.

[0410] Cardiac death is defined as the death of a patient mainly due to causes in the cardiac system, such as myocardial infarction, arrhythmia or ventricular dysfunction. All death cases were examined by three independent cardiologists who were not informed about the treatment group.

[0411] The diagnosis of congestive heart failure (CHF) was made by either (1) a severe deterioration of left ventricular function requiring an intra-aortic balloon pump or a left ventricular assist device with a CI < 1.5 l / min / m 2 ; or (2) cardiogenic shock with a CI < 1.5 l / min / m 2 and a PCWP > 20 cm for more than 1 hour.

[0412] The diagnosis of life-threatening arrhythmia was made by either (1) a ventricular arrhythmia requiring electrical defibrillation; or (2) an arrhythmia requiring insertion of a pacemaker at the time of discharge.

[0413] Combining the outcome results in Table 12 shows the occurrence of the following adverse cardiovascular events: combined MI, CVA, cardiac death, CHF and life-threatening arrhythmia. In patients treated with low-dose AICA riboside, there seems to be a tendency for a decrease in the occurrence of adverse events, but the low dose does not seem to show statistically significant efficacy. Therefore, the p-value shown in Table 12 reflects the comparison of high dose (0.1 mg / kg / min) versus placebo.

[0414] Table 12 shows a 61% decrease in the occurrence of combined outcomes in the high-dose group compared to the placebo group (5.3% versus 13%), and the p-value is < 0.05.

[0415] This data showed a 68% reduction in the occurrence of MI in the high-dose group compared to the placebo group (1.5% vs. 4.7%), with a p-value < 0.05, and an 88% reduction in the occurrence of cerebrovascular injury in the high-dose group compared to the placebo group (0.5% vs. 4.2%) (p-value < 0.05). This data also showed a strong tendency towards a reduction in cardiac death in the high-dose group compared to the placebo group (0 vs. 1.4%), although the p-value was not significant.

[0416] In terms of the harmful outcome effects of congestive heart failure and life-threatening arrhythmias, there seems to be a tendency towards a reduction in occurrence in the high-dose group compared to the placebo group (CHF: 2.9% vs. 3.8%; arrhythmias: 1.4% vs. 1.9%).

[0417] The occurrence of all deaths tended to be lower in the high-dose group than in the placebo group (0.5% in the high-dose group vs. 3.3% in the placebo group). The occurrence of myocardial infarction measured by either ECG or CK-MB tended to be lower in the high-dose group compared to the placebo group (20.8% in the high-dose group vs. 24.1% in the placebo group).

[0418] In this study, the uric acid concentrations of all patients were monitored. In treated patients, a clear dose-related increase in uric acid concentration was observed, but plasma uric acid concentrations were generally maintained within or near the normal range. Clinically significant crystalluria was not observed (data not shown).

[0419] Table 13 below shows the occurrence of myocardial infarction and combined clinical outcomes (MI, CVA, cardiac death, CHF, and life-threatening arrhythmias) according to the plasma levels of AICA riboside. From these data, it is clear that the most effective plasma levels of AICA riboside are in the range of 3 - 6 μg / ml. The data in Table 13 reflect the diagnosis of MI by both ECG and CK-MB (as described for Table 12).

[0420]

Chemical formula

[0421] AICA riboside and its prodrugs (the "AICA riboside compounds") can be administered in any standard manner using pharmaceutically acceptable buffers. To deliver the AICA riboside compounds to a patient, it is contemplated that they can be administered by intravenous, intracoronary or intraarterial infusion, direct intramuscular injection, subcutaneously, orally, transdermally, transrectally or by inhalation to the skin or mucosa. The AICA riboside compounds can also be introduced from the outside into the patient's blood, for example, using a cardiopulmonary device or dialysis. Compounds acceptable for pharmaceutical use are well known.

[0422] Preferably, the AICA riboside compound is administered prophylactically. If such a compound is present prior to an ischemic event, the breakdown of net ATP can be beneficially directed to adenosine rather than inosine, and thus tissue damage can be prevented. When the drug is introduced into a patient during or after an event that is inducing ischemia and reaches the ischemic region, the ability to direct ATP to adenosine at that site is reduced because the target ATP pool is depleted relatively rapidly. Also, it is possible that if the drug is present as a prophylactic, the process can be retarded early enough to prevent the event or any permanent damage.

[0423] Other factors make it important to administer the drug before and / or during an ischemic event. When the drug is administered after occlusion, the ischemic region has little or no blood flow and thus reduced ability to reach the tissues involved, unless it undergoes modified reperfusion, for example, by tPA administration, angioplasty or bypass surgery. Also, for example, it is thought that AICA riboside is metabolized to AICA ribotide and that this is one of the active forms of the molecule. This metabolism is an energy-requiring reaction that utilizes ATP. If ATP is not available due to high metabolic activity and / or increased ATP breakdown, AICA riboside cannot become this active form.

[0424] Example 1 Improvement of functional recovery in isolated hearts The ability of several preferred AICA riboside analogs to improve the recovery of heart function after ischemia was examined in an isolated rat heart model.

[0425] An isolated rat heart was cannulated via the ascending aorta and attached to a perfusion apparatus according to the Langendorff method. The heart was perfused at 37 °C with modified Krebs-Henseleit buffer (pH 7.4) at a constant pressure of 100 cm / H 2 O. Left ventricular developed pressure (LVDP) was continuously monitored as a measure of heart function. After 30 minutes of heart equilibration, the pressure was 10 cm / H 2By reducing it to O for 30 minutes, the heart was subjected to reduced flow, i.e., ischemia. Subsequently, the pressure was returned to its original level (100 cm / H 2 O) for an additional 30 minutes to restore the flow. Each AICA riboside analog, together with AICA riboside itself (for comparison), was added to the perfusion buffer to a final concentration of 5 μM or 20 μM. The results are shown in Table I.

[0426]

Chemical formula

[0427] A strip (about 1 cm) of longitudinal muscle was taken from guinea pig ileum, connected to an isometric force transducer, and suspended in a jacketed tissue bath containing Krebs-Ringer solution bubbled with 95% O 2 / 5% CO 2 . Using parallel platinum electrodes, current was passed at 1-minute intervals at a pressure sufficient to induce maximal contraction. The test compound was added to the tissue bath, and the concentration (IC 50 ) that inhibited contraction by 50% was measured. These are shown in detail in Table II.

[0428]

Chemical formula

[0429] Male rats were intraperitoneally injected with either an AICA riboside analog, AICA riboside, or saline as a control. After 60 minutes, the hearts were excised and incubated at 37°C for an additional 60 minutes. Tissue extracts were prepared and analyzed for adenosine by high performance liquid chromatography (HPLC). The ability of this preferred set of AICA riboside analogs to increase tissue adenosine levels is shown in Table III in comparison to AICA riboside. A more detailed comparison of the dose-dependent effect of the AICA riboside analog (compound number 10) selected in this preferred set on tissue adenosine levels is shown in Figure 19 in comparison to AICA riboside (compound number 1).

[0430]

Chem.

[0431]

Chem.

[0432]

Chem.

[0433]

Chem.

[0434] A 200 μl aliquot of a WI-L2 lymphoblast cell suspension (0.5×106) was overlaid on 100 μl and on top of a silicone oil:mineral oil mixture (8:2 by volume). Compounds No. 53 (1-468) at concentrations of 5.0, 50.0 and 500.0 μM were added to the cells, respectively, and the resulting mixtures were incubated either for 1 minute or for 1 hour. Then, 5 μl of radiolabeled adenosine (initial concentration 1 μM, 2.5 μCi) was added to the cell suspension and the mixture was incubated for 10 seconds. The cells were then centrifuged at 13,000 rpm for 15 seconds and the radioactivity of the cell pellet was measured.

[0435] Figure 22 shows the inhibition of adenosine transport by 1-minute preincubation with compound No. 53 (1-468), and Figure 23 shows the inhibition of adenosine transport by 1-hour preincubation with compound No. 53 (1-468).

[0436] Example 6 Effect of AICA Riboside Analogs (Group III) on Adenosine Release from Isolated Cells Group II (C-2)-substituted AICA riboside analogs were compared with AICA riboside itself for their ability to affect adenosine release from coronary endothelial cells. In this experimental model, cells were treated with 50 μM of the test compound and incubated at 37 °C for 16 h. The cells were then washed with phosphate-buffered saline and resuspended in a standard culture medium containing no glucose (to inhibit glycolysis), 50 μM antimycin A (to inhibit oxidative phosphorylation), and 20 μM deoxycoformycin (to inhibit adenosine utilization by adenosine deaminase). This treatment was designed to stimulate an ischemic-like state by inducing net ATP breakdown. The medium was then processed by HPLC. Adenosine values are shown in Table VI.

[0437]

Chemical Structure

[0438] The results are shown in Table VII.

[0439]

Chemical formula

[0440] The effects of these AICA riboside analogs on hypoxanthine and inosine levels (shown in Figures 20B and 20C, respectively) reflect the effects on adenosine levels and show an increase in the effect on adenosine utilization mediated by inhibition of adenosine deaminase. This interpretation is supported by direct measurement of the ability of the analogs to inhibit isolated adenosine deaminase.

[0441] Inhibition of adenosine deaminase activity was measured by spectrophotometry using 1 ml of an assay mixture containing 50 mM potassium phosphate, pH 7.0, 1 mM α-ketoglutarate, 15 units of glutamate dehydrogenase, 0.125 mM NADH, 80 μM adenosine and 0.002 units of bovine intestinal mucosa adenosine deaminase. Different concentrations of the test compound were incubated in the assay mixture for 10 minutes at 37 °C. The reaction was continuously monitored for the oxidation of NADH from the change in absorbance at 340 nm.

[0442] The results are shown in Table VIII.

[0443]

Chemical formula

[0444] The results are shown in Table IX.

[0445]

Chemical formula

[0446] The results are shown in Table X.

[0447]

Chemical formula

[0448]

Chemical formula

[0449] (Example A) (Preparation of 5-amino-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)imidazole-4-carboxamide (Compound No. 2(1-111))) AICA riboside (50 g) was dissolved in pyridine (450 ml) and cooled in an ice bath. Acetic anhydride (80 ml) was added and the ice bath was removed. The reaction mixture was stirred for 3 hours. Elution with methylene chloride:methanol 9:1 on silica gel TLC confirmed the completion of the reaction. Methanol (5 ml) was added to neutralize the unreacted acetic anhydride. The solvent was evaporated by evaporation under high vacuum (bath temperature less than 40 °C). The residue was azeotroped with dimethylformamide and evaporated (3 times with 150 ml). The residue was crystallized from ethanol using a seed crystal. 62 g of white crystalline triacetate was obtained. Melting point 128 °C - 129 °C.

[0450] [Chemical formula] The preparation method of this compound is also described in U.S. Patent No. 3,450,693 (K. Suzuki and I. Kumoshiro (1969)), see also Chem. Abs. 71:816982 (1969).

[0451] (Example B) (N 5 -dimethylaminomethyleneamino-β-D-ribofuranosylimidazole-4-carboxamide (Compound No. 7(1-164)) preparation) 2’,3’,5’-Tri-O-acetyl AICA riboside (10 g) was dissolved in dimethylformamide (30 ml) and dimethylformamide dimethyl acetal (20 ml). The reaction mixture was stirred overnight. By TLC on silica gel, eluting with methylene chloride:methanol 9:1, it was confirmed that the reaction was complete since the starting material had disappeared. The solvent was evaporated by evaporation under high vacuum (bath temperature less than 40 °C). The residue was dissolved in cyclohexylamine and stirred overnight. The solvent was evaporated by evaporation under high vacuum and the residue was crystallized from ethanol. The yield of white crystals was 4.6 g. Melting point 173 °C to 175 °C.

[0452] [Chemical formula] (Example C) (Preparation of 5-amino-1-β-D-ribofuranosylimidazole-4-N-(cyclopentyl)carboxamide (Compound No. 10(1-186))) N-Succinimidyl-5-amino-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)imidazole-4-carboxylate ("Intermediate No. 4") was synthesized according to the procedure of P.C. Srivastava, R.W. Mancuso, R.J. Rousseau and R.K. Robins, J. Med. Chem. 17(11), 1207(1977). Intermediate No. 4 (3.9 g) was dissolved in methylene chloride (60 ml). Cyclopentylamine (0.8 ml) was added and the solution was stirred overnight. Elution with methylene chloride:methanol 9:1 on silica gel TLC confirmed the completion of the reaction as the starting material had disappeared. The solvent mixture was extracted with 5% hydrochloric acid solution (100 ml), saturated sodium bicarbonate solution (100 ml) and water (200 ml). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give 3.1 g of a yellow foam. 3.1 g of the foam was dissolved in methanol (70 ml) and the acetyl groups were removed by cooling in an ice bath. Ammonium hydroxide (60 ml) was added and the ice bath was removed. After stirring for two and a half hours, elution with methylene chloride:methanol 9:1 on silica gel TLC confirmed the disappearance of the starting material. The solvent was evaporated under reduced pressure to give a residue which was purified by elution on a silica column with methylene chloride:methanol 9:1 and 6:1. The same fractions as in the TLC were collected and evaporated under reduced pressure to give 1.1 g of a white foam which was crystallized from methanol-ethyl acetate. Melting point 158 °C - 160 °C.

[0453] [Chemical formula] (Example D) (Preparation of 5-amino-1-β-D-ribofuranosylimidazole-4-N-(cyclopropyl)carboxamide (Compound No. 12(1-232))) This compound was prepared according to the procedure of Example C by changing cyclopentylamine (0.8 ml) to cyclopropylamine (0.5 ml). Starting from 6.2 g of Intermediate No. 4 (succinate ester), the yield was 2.3 g.

[0454] [Chemical formula] (Example E) (Preparation of 5-amino-1-β-D-ribofuranosylimidazole-4-N-(benzyl)carboxamide (Compound No. 11(1-226))) Inosine (10 g) was suspended in dimethylformamide (100 ml) and dimethylformamide dibenzyl acetal (25 ml). The resulting mixture was stirred at 70 °C overnight. TLC on silica gel was eluted with methylene chloride:methanol 6:1 to confirm that the reaction was complete. The solvent was evaporated under reduced pressure to obtain a residue. The residue was dissolved in ammonium hydroxide (130 ml). The mixture was stirred overnight and evaporated under reduced pressure. Ethanol (80 ml) was added to this residue and the resulting mixture was warmed. The solid was collected by filtration. The yield of 1-benzylinosine was 10.5 g, which was characterized by NMR.

[0455] The intermediate 1-benzylinosine (10.5 g) was dissolved in ethanol (1.0 L) and 3M sodium hydroxide solution (140 ml). This solution was refluxed for 3 hours. TLC on silica gel was used to confirm that the reaction was complete. The solvent was removed by evaporation under reduced pressure. The residue was separated by elution with methylene chloride:methanol 6:1 on a silica gel column. The fractions were collected by TLC in the same manner until crystals appeared. The yield of the compound identified as above was 7.4 g as a white solid. Melting point 178 °C - 179 °C.

[0456] [Chemical formula] (Example F) (Preparation of 5-amino-1-β-D-ribofuranosylimidazole-4-carboxylic acid methyl ester (Compound No. 14(1-260))) 5-Amino-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-imidazole-4-carboxylic acid (3.85 g, 10 mmol) was dissolved in 40 ml of tetrahydrofuran and cooled to 0 °C. An excess of diazomethane in ether was added and the mixture was allowed to return to room temperature. Acetic acid was added to decompose the excess diazomethane and the mixture was evaporated to dryness. The residue was purified by silica gel chromatography eluting with ethyl acetate:hexane 7:3. The main product fraction was confirmed by silica gel thin layer chromatography (TLC) using the above system, mixed and evaporated to give 1.2 g of a white foam. This was dissolved in 40 ml of methanol containing 20 mg of sodium methoxide and stirred for 30 minutes. TLC on silica gel eluting with methylene chloride:methanol 6:1 confirmed that no starting material remained and that there was a new product spot with a shorter migration distance. The reaction mixture was neutralized with Dowex 50(H + ) resin and evaporated to give 0.64 g of the desired product as a white foam. IR (KBr): 1725 cm -1 (--CO--OCH 3 ).

[0457]

Chemical formula

Claims

[Claim 1] The invention as depicted in the drawings of this application.

Images