WO1994017809A1

WO1994017809A1 - Adenosine deaminase inhibitor therapies

Info

Publication number: WO1994017809A1
Application number: PCT/US1994/001184
Authority: WO
Inventors: Harry Edward Gruber; Mark David Erion; Gary Steven Firestein; Mark Alan Young
Original assignee: Gensia, Inc.
Priority date: 1993-02-03
Filing date: 1994-02-02
Publication date: 1994-08-18
Also published as: IL108522A0; AU6297294A

Abstract

Methods of prophylactically and affirmatively treating certain conditions by administering adenosine deaminase inhibitors. Such conditions include thrombotic conditions, conditions characterized by ischemia and conditions characterized by inflammatory responses, including sepsis.

Description

DESCRIPTION

Adenosine Deaminase Inhibitor Therapies

Field of the Invention

The present invention relates to the field of medicine and relates more particularly to methods for pro¬ phylactically and affirmatively treating certain bodily states that respond beneficially to an increase in extra¬ cellular adenosine by providing the patient with an adenosine deaminase inhibitor or a derivative, inter¬ mediate or prodrug thereof .

Background of the Invention Adenosine has been reported to have cardioprotective

(Olafsson et al . , Circulation, 76:1135 (1987)) and neuro- protective properties (Dragunow and Faull, Trends in

Pharmacol . Sci. , 9:193 (1988) ; Marangos, Medical

Hypothesis, 32:45 (1990)) . It is reportedly released from cells in response to alterations in the supply of or demand for oxygen (Schrader, Circulation, 81:389 (1990)) , is said to be a potent vasodilator, and is believed to be involved in the metabolic regulation of blood flow (Berne, Circ. Res . , 47:808 (1980)) . However, adenosine has a short half life (< 1 sec) in human blood (Moser et al . , Am. J. Physiol. , 256:C799 (1989)) and has been reported to exhibit negative inotropic, chronotropic and dromotropic effects (Belardinelli et al . , Prog. in Cardiovasc . Diseases, 32:73 (1989)) . Furthermore, systemic adminis- tration of adenosine causes coronary steal by prefer¬ entially dilating vessels in nonischemic regions. Consequently, high doses of adenosine are toxic and severely limit its therapeutic potential. However, it is believed that by increasing extracellular adenosine concentration locally, i.e., at the target site within the target tissue, the beneficial effects of adenosine can be provided and the toxic systemic effects minimized (Gruber, U.S. Patent No. 4,912,092, issued March 27, 1990, for "Methods for Increasing Extracellular Adenosine and for Stabilizing Mast Cells") .

Extracellular adenosine concentration is determined by many factors, including: factors involved in adenosine production, such as the concentration of adenosine mono- phosphate (AMP) and the presence of enzymes involved in AMP metabolism; adenosine metabolism; adenosine transport across cell membranes; and "wash-out" of adenosine from the site of its production. Figure 1 illustrates pathways by which adenosine is formed and degraded within cells . The metabolism of adenosine may utilize some of these 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 phos¬ phorylase (not established in humans) ; (9) adenosine kinase; (10) 5' nucleotidase and nonspecific phosphatase; (11) adenylate kinase; (12) nucleoside diphosphokinase; (13) adenylate cyclase; (14) AMP deaminase; and (15) adenylosuccinate synthetase and adenylosuccinate lyase.

Initial metabolism of adenosine occurs either by phosphorylation of the 5' hydroxy group of adenosine to form AMP or by deamination of adenosine to form inosine. The enzyme adenosine kinase (AK) catalyzes the conversion of adenosine to AMP, and the enzyme adenosine deaminase (ADA) catalyzes the conversion of adenosine to inosine.

Potent and selective ADA inhibitors have been identified and proposed (Klohs and Kraker, Pharm. Reviews 44:459 (1992) , for study as immunosuppressive drugs. The rationale is based on observations in humans or animals in which ADA activity is inhibited or genetically deficient by about 98% or greater. At this degree of enzyme inhibi¬ tion, there is reported toxicity to both B and T lympho- cytes, resulting in severe combined immunodeficiency (Thompson and Seegmiller, Adv. in Enz . and Related Areas of Mol. Biol. , 51:167 (1980)) .

TUTE SHEET (RULE 26) Thus, either ADA deficiency or a high level of ADA inhibition leads to immunotoxicity and loss of B and T cell function. The mechanism of immunotoxicity is reported to arise from a build-up of 2' -deoxyadenosine triphosphate (dATP) , which results from an increase in the concentration of the ADA substrate 2' -deoxyadenosine (Thompson and Seegmiller, 1980, supra) .

It has been reported that ADA inhibitors have desirable characteristics in various models of ischemia- induced cardiac dysfunction. In models of normothermic global ischemia in isolated, buffer-perfused hearts, ADA inhibition with erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) or 2' -deoxycoformycin has been reported to improve recovery of contractile function following reperfusion (Zhu et al . , PNAS 88(2) :657 (1991) ; Zhu et al., J. Am. Phvsiol . Soc. , 259:H835 (1990) ; Dhasmana et al. , J. Cardiovasc. Pharmacol. 5:1040 (1983)) . Similar protection was reportedly observed with 2'- deoxycoformycin treatment during 2 hours of cold cardio- plegic ischemia and reperfusion (Boiling et al . , J. Thora. Cardiovasc. Surg. 99:469 (1990)) . Others, however, have reported that addition of EHNA alone to perfusion buffer does not improve functional recovery following global ischemia (Humphrey and Seelye, J. Thorac . Cardiovasc. Surg. 84:16 (1982) ; Abd-Elfattah et al . , Circulation 82:5 (1990) ) .

Beneficial effects of ADA inhibition on cardiac function have also been reported in animal models of regional ischemia. u et al . , Cvtobios. 50:7 (1987) , and Dorheim et al . , Surgery 110:2 (1991) reported improved cardiac functional recovery in a model of transient, reversible ischemia (15 minutes of regional ischemia and subsequent reperfusion) , and these effects were purportedly due to enhancement of adenosine levels in the myocardium. In a model of irreversible damage from regional ischemia, McClanahan et al. , Circulation, 86:1-23 (1992) , reported a reduction in the amount of tissue necrosis in animals treated with 2' -deoxycoformycin.

It has also been reported that ADA inhibitors inhibit platelet aggregation under certain in vitro conditions. Dawicki et al . , Biochem. Pharmacol. 34:3965 (1985) , reported that the combination of (but not the individual use of) an adenosine deaminase inhibitor (2' -deoxy¬ coformin) and an adenosine kinase inhibitor (5-iodotu- bercidin) provided comparable inhibition of platelet aggregation in whole blood in vitro as was observed with the erythrocyte nucleoside transport system inhibitor, dilazep. Agawal and Parks, Biochem. Pharmacol. 24:2239

(1975) , reported that the adenosine deaminase inhibitor, coformycin, plus adenosine prolonged the inhibition of platelet aggregation observed compared to treatment with adenosine alone. In this study, coformycin was examined for its ability to inhibit adenosine diphosphate (ADP) - induced platelet aggregation in vitro. Because these experiments involved the exogenous administration of ADP, adenosine is elevated to a high level where the contri¬ bution of ADA (which has a Km of about 50 μM) to adenosine metabolism is increased to an artificially high level relative to the contribution of AK (which has a Km of about 0.5 μM) . Because ADA inhibitors are immunosuppressive (possibly through 2' -deoxyadenosine-mediated toxicity to the immune system) , the use of ADA inhibitors would appear to be contraindicated in a host whose immune system is fighting an infection. Although ADA inhibitors can enhance certain antivirals by altering their metabolism

(Klohs and Kraker, Pharm. Reviews 44:459 (1992)) , ADA inhibitors alone can cause a decrease in viral resistance

(Thompson and Seegmiller, 1980, supra) . ADA inhibitors have been reported to be active against malaria by interfering with purine utilization (Klohs and Kraker, 1992, supra) . However, ADA inhibitors have not been thought to be beneficial in conditions such as sepsis since ADA deficiency has been reported to cause immunosuppression (Thompson and Seegmiller, 1980, supra) . ADA inhibitors have not been commercially developed as adenosine regulating agents for several reasons . First, the value of adenosine regulating agents for the treatment of a variety of human diseases was not appre¬ ciated until the work of Gruber et al . , Circulation 80:1400 (1989) and Gruber U.S. Patent No. 4,912,092 issued March 27, 1990. In addition, as stated in Gruber et al . U.S. Patent No. 5,082,829, issued January 21, 1992, ADA inhibitors are immunotoxic possibly due to their inhibition of 2' -deoxyadenosine metabolism (Thompson and Seegmiller, 1980, supra) .

2' -Deoxycoformycin-treated animals have been reported to show marked immunosuppression (Goodman and Gilman' s The Pharmacological Basis of Therapeutics, p. 1274, Gilman et al . eds . (7th ed. 1985) . This is consistent with reports that children born with severe ADA deficiency lack T cell and B cell function, which improves on introduction of exogenous ADA (Giblett, et al . , Lancet 2:1067 (1972)) . ADA inhibitors have not been seen as useful agents of therapy, except to treat immunologic cancer and possibly immunologic diseases (Klohs and Kraker, 1992, supra) and no such compounds have been developed for medical treatment of patients with ischemia, thrombosis or sepsis.

Additionally, ADA inhibitors have not been developed as adenosine regulating agents because the importance of

ADA in adenosine metabolism is unclear. Many factors, such as local adenosine concentration, the kinetic characteristics of the enzymes AK and ADA, and the intracellular specific activity of AK and ADA, dictate which metabolic pathway predominates in adenosine metabolism at a particular time or location.

Adenosine concentrations in blood and tissue under physiological conditions are about 10. nM and rise to about 500 nM during ischemia (Gruber et al. , Circulation 1990, supra and Engler and Gruber in The Heart and Cardio-

ΠESTITUTE SHEET (RULE 26) vascular System, Chapter 6, (1991)) . These concentrations are well below the Km for adenosine for ADA suggesting that ADA plays a minor role in adenosine metabolism.

The relative importance of AK and ADA are not completely established in most tissues . It has been reported that AK is an excellent site for regulating adenosine metabolism (Browne et al. , European Patent

Application No. 92300580.5, published July 29, 1992) .

In fact, ADA is viewed as predominantly regulating deoxyadenosine (dAdo) metabolism. Data reported in the literature appears to support the major role ADA plays in dAdo metabolism in that ADA has a low Km for dAdo. In addition, dAdo levels are elevated to a greater degree in the urine and plasma of ADA deficient patients . Of note, ADA inhibitors have not been commercially developed as adenosine regulating agents, despite the existence of potent and specific inhibitors, such as erythro-9, 2-hydroxy-3-nonyl adenine (EHNA) and 2'- deoxycoformycin. These compounds were reported as ADA inhibitors in the 1970's (Schaeffer and Schwender, J. Med. Chem. 17:6 (1974) ; Agarwal, et al . , Biochem. Pharmacol. 26:359 (1977) ) .

Contrary to the above, however, as further described below, we have determined that ADA inhibitors find unexpected medical utility in the treatment of conditions including inflammation, especially sepsis, thrombosis, and conditions characterized by ischemia.

Summary of the Invention.

Applicants have determined that ADA inhibitors have utility as medicinal agents in ischemia, thrombosis and inflammation, especially sepsis.

In one aspect, the present invention is directed to methods of preventing or treating a condition characterized by ischemia in a mammal . These methods involve administering to the mammal an amount of a compound which inhibits adenosine deaminase but which does

SUBSTITUTE SHEET (RULE 26 not cause clinical immunodeficiency, that is, an amount which does not compromise immune function to a clinically significant degree in said mammal. Clinically significant immunodeficiency refers to immunodeficiency which results in an increased frequency or severity of infection(s) by pathogenic organisms. It can also refer to infections with opportunistic organisms. Conditions characterized by ischemia include myocardial infarction, angina, stroke and transient ischemic attack, among others. Preferably, the amount administered provides less than about 98% inhibition of adenosine deaminase activity, and more preferably provides less than about 95% inhibition of adenosine deaminase activity. Achieving inhibition of adenosine deaminase activity without causing clinically significant immunodeficiency may be accomplished by administering an adenosine deaminase inhibitor over a short period of time, for example, up to about three hours (e.g. , administration before, during and after percutaneous transluminal angioplasty (PTCA) ) or for up to about 24 hours or up to about seven days (e.g. , for an application such as preventing or treating myocardial infarction) .

In another aspect, the present invention is directed to methods of preventing or treating thrombosis by administering a therapeutically effective amount of a compound which inhibits adenosine deaminase. By therapeutically effective amount is meant an amount which reduces adenosine deaminase activity below the level existing before administration of the adenosine deaminase inhibiting compound. Thrombosis refers to the in vivo formation of a blood clot, especially within a vessel and particularly in an artery. Thrombosis may result in myocardial infarction or stroke. Preferably, a therapeu¬ tically effective amount of an adenosine deaminase inhibiting compound is an amount which inhibits adenosine deaminase activity but does not cause clinically significant immunodeficiency. Such therapeutically effective amount may be an amount which provides less than about 98% inhibition, and preferably less than about 95% inhibition of adenosine deaminase activity, and/or may be administered for a short period of time, as described above.

In another aspect, the present invention is directed to methods of preventing or treating a condition charac¬ terized by an inflammatory response in a mammal by administering to the mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase. Preferably, a therapeutically effective amount is an amount which inhibits adenosine deaminase but which does not cause clinically significant immunodeficiency. A therapeutically effective amount of an adenosine deaminase inhibitor may be an amount which provides less than about 98% inhibition, and less than about 95% inhibition of adenosine deaminase activity may be particularly preferred, especially in chronic treatment. In certain embodiments, these methods may involve acute or prophylactic treatment (for a short period of time as described above) of a particular condition which involves an inflammatory response, such as sepsis, septicemia, septic shock, endotoxic shock, endotoxemia, meningitis, burns, adult respiratory distress syndrome, and necrotizing enterocolitis. In other embodiments, these methods may involve treatment of a condition involving an inflammatory response, such as arthritis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, iridocyclitis, vasculitis, ischemia, reperfusion injury, peripheral vascular disease, or atherosclerosis.

In certain embodiments, the present invention pertains to methods of preventing or treating sepsis in a mammal by administering to the mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase. Sepsis may result from infection with any one of a number of organisms, including gram negative bacteria, gram positive bacteria, viruses, mycobacteria,

8 fungi, yeasts and worms. In other embodiments, the invention relates to the prevention or treatment of septicemia, for example, endotoxemia, by administering a therapeutically effective amount of an adenosine deaminase inhibitor. The present invention also includes methods of preventing or treating septic shock, including endotoxic shock, by administering a therapeutically effective amount of an adenosine deaminase inhibitor.

Compounds which inhibit adenosine deaminase include coformycin, 2' -deoxycoformycin and erythro-9- (2-hydroxy-3- nonyl) adenine (EHNA) . Of course, other ADA inhibitors, now known or later developed, may be used in the methods of the present invention. Use of derivatives, inter¬ mediates or prodrugs of adenosine deaminase inhibitors in such methods is also within the scope of the invention, as is the use of pharmaceutically acceptable salts of such compounds. Preferred routes of administration and dosages are provided.

Brief Description of the Drawings Figure 1 depicts metabolic pathways of adenosine.

Figure 2 graphically depicts the results of the experiments described in Example 3. Percent survival is plotted against hours after exposure to E. coli LPS and carrier or E. coli LPS and the ADA inhibitor, EHNA.

Detailed Description of the Invention

Despite indications in the art that ADA inhibitors would not be medically useful in the claimed methods, we have determined, surprisingly, that such compounds are useful in methods of preventing or treating certain conditions and disease states, including conditions characterized by ischemia (such as stroke, transient ischemic attack, myocardial infarction and angina) , conditions characterized by thrombosis and conditions characterized by an inflammatory response (especially sepsis ) .

C IT ς ccr RULE 26 Examples of ischemic conditions include diseases that arise from, or are aggravated by, insufficient blood flow through a particular organ or portion thereof. For example, heart attacks or strokes, the microvascular disease of diabetes mellitus (which can affect the brain, the kidney, the heart, the skin, the retina, and the peripheral nerves and their associated microvasculatures) , or events resulting in a less prolonged loss of blood flow, such as angina pectoris, transient ischemic attacks, bowel ischemia, kidney ischemia, intermittent claudication of skeletal muscle, migraine headaches, and Raynaud's phenomenon can be treated by administering compounds which inhibit adenosine deaminase.

Thrombosis involves the in vivo formation of a blood clot, particularly within a blood vessel, for example, an artery. Thrombosis can involve agglutination of red blood cells, white blood cells, platelets and/or fibrin, and can result in partial or total blockage of a blood vessel.

Sepsis is the systemic inflammatory response to infection. Infection is a medical condition characterized by the presence of an abnormal quantity and/or type of microorganisms or the invasion of normally sterile host tissue by microorganisms. Bacteremia refers to the presence of bacteria in the blood. Similarly, viremia and fungemia refer to the presence of viruses and fungi, respectively, in the blood. Thus, sepsis involves systemic inflammatory responses to infection by one or more of several types of organisms, such as bacteria (gram negative or gram positive) , viruses (including retro- viruses) , mycobacteria, yeast or worms.

A dramatic example of sepsis is septic shock or more specifically endotoxic shock which results from a systemic infection with gram negative bacteria. In the case of endotoxic shock, an explosive cytokine response to intra- vascular gram negative bacteria is caused by endotoxin, or lipopolysaccharide (LPS) , a component of the bacterial cell wall (Takada et al, Critical Rev. Microbiol. 16:477 (1989) ; Loppnow et al . , Advances Ex . Med. Biol . 561 (1990) ) .

Acute gram negative bacterial infections evoke characteristic pathophysiological responses, including changes in white blood cell counts, fever, hemodynamic disorders, and various coagulatory disturbances, which may result in respiratory distress syndrome, multi-organ failure, irreversible shock or death. In these path¬ ophysiological events, endotoxin stimulates macrophages and other cells to elaborate various biologically active mediators which induce the phenomena of endotoxemia and bacterial sepsis (Zabel et al, Lancet 30:1474 (1989)) . In animals, intravenous infusion with endotoxin or gram negative bacteria leads to circulatory collapse and death. ADA inhibitors may also be used to prevent or treat conditions caused, in part, by superantigens, such as toxic shock syndrome.

Furthermore, it is surprising that the beneficial effects of ADA inhibitors can occur using doses that do not compromise immune function to a clinically significant degree. In conditions where it is important to inhibit ADA activity up to a level of about 98%, or preferably up to about 95%, one can measure ADA activity by removing a blood sample from an animal treated with the ADA inhibitor and determining the ADA activity in the blood or plasma by known methods (Thompson and Seegmiller, 1980, supra) . ADA activity can also be assessed by measuring 2' -deoxya¬ denosine in blood or urine, 2' -deoxyadenosine triphosphate in blood, T or B cell function or numbers, or antibody levels to assure immune competence.

It is particularly surprising that ADA inhibitors would be useful in the treatment of conditions characterized by an infection, for example, sepsis (as demonstrated in Example 3 herein) , given that a reported side effect of adenosine deaminase inhibitors is the ability to down regulate the immune system resulting in an inability to fight the infection. It is envisioned that

11

S the ADA inhibitor is preferably administered prophy¬ lactically to patients at risk of developing sepsis, such as to patients receiving chemotherapy or those with burns, open wounds (including gunshot wounds and trauma) , leukopenia, perforated bowel and those undergoing surgery especially abdominal (particularly where the bowel is opened) and urologic procedures. Co-treatment with anti- infectious disease agents, such as antibiotics, is also part of the present invention. As explained above, compounds useful in the present invention include inhibitors of ADA, preferably, such compounds are selective inhibitors of adenosine deaminase, that is, their effect or inhibition of adenosine deaminase is much greater than their effects on other adenosine- utilizing enzymes or on adenosine receptors. Examples of selective adenosine deaminase inhibitors include coformycin, 2' -deoxycoformycin and erythro-9- (2-hydroxy-3- nonyl) adenine (EHNA) . Other ADA inhibitors, now known or later developed, may also be used in the methods of the present invention. In addition, a derivative, an inter¬ mediate or a prodrug of an adenosine deaminase inhibitor may be used in methods of preventing or treating ischemic conditions, thrombotic conditions or inflammatory conditions (including sepsis) . The methods of this invention can be understood further by the following examples. These examples should not however be construed as specifically limiting the invention. Variations of the invention, now known or later developed, are considered to fall within the scope of the present invention as hereinafter claimed.

Example 1

In Vitro Antiplatelet Activity of Adenosine Deaminase

Inhibitors

Whole blood was drawn from healthy donors and added to 0.1 vol of sodium citrate (3.8%) to prevent coagula¬ tion. Platelet aggregation was measured by the impedance technique using a Chronolog Whole Blood Aggregometer

(model 500) . Aggregation was induced by addition of ADP

(6-25 μM) , collagen (2-5 μg/ml) or arachidonic acid (0.5-1 mM) at the minimal concentration inducing full aggregation in untreated controls. Either of the adenosine deaminase inhibitors, 2' -deoxycoformycin (DCF) or erythro-9- [2- hydroxy-3-nonyl] adenosine (EHNA) , or the adenosine transport inhibitors, dipyridamole or 6- [4-nitro- benzyl) thio] -9-3-D-ribofuranosylpurine (NBTI) , was incubated in whole blood for 10 or 60 minutes at 37°C prior to addition of one of various aggregation-inducing agents (i.e. , adenosine diphosphate (ADP) , collagen, or arachidonic acid (Arach. A.)) . In some experiments, adenosine (100 μM) was added to samples 5 minutes before eliciting aggregation. DCF and EHNA were shown to be potent inhibitors of platelet aggregation in human whole blood in the presence of 10 μM adenosine (Ado) , but not in the absence of adenosine. Adenosine (100 μM) alone, the adenosine deaminase inhibitors, DCF (1 mM) and EHNA (1 M) , or the adenosine transport inhibitors dipyridamole (100 μM) and NBTI (100 μM) alone did not inhibit or weakly inhibited platelet aggregation in human whole blood.

13

SUBS Table 1

Antiplatelet Activities (IC₅₀ in μM) of

Various Agents in Human Whole Blood

Agent Preincubation ADP Collagen Arach A. (Time) (10' ) (60') (60' ) (60' )

Deoxyco- formycin -Ado >>11000000 >>11000000 >>11000000 >1000 +Ado 00..6633 00..0055 00..0099 0.05

EHNA -Ado >1000 >1000 +Ado 0.40 0.42

Dipyrid¬ amole -Ado >100 +Ado 2.4

NBTI -Ado >100 +Ado 0.30

Adenosine >100

Example 2

Effect of Adenosine Deaminase Inhibitor on Inhibition of Clot Formation in in vivo Thrombosis Model

The experiments described in this example show the antithrombotic effect of the adenosine deaminase inhibitor, EHNA, in a canine model of arterial thrombosis designed to simulate unstable angina. Four dogs were anesthetized with pentobarbital sodium and instrumented to induce platelet-mediated thrombi in the left circumflex coronary artery. Briefly, this method entails exposure of the heart and coronary artery via a left thoracotomy, and mechanical damage and stenosis of the artery. The pro- cedure is described in more detail in Folts, Circulation

83 (Suppl. IV) :3 1990) . This procedure induces inter¬ mittent clot formation at the site of stenosis, and corresponding intermittent cyclic coronary flow reductions (CFRs) . The CFRs are characterized by the frequency (#CFRs/30 min) , and severity (nadir of flow during peak flow restriction measured in ml/min) , and can be resolved by compounds with antiplatelet activity such as aspirin, and antibodies to GP Ilb/IIIa receptor. In the present

14

SUBSTITUTE S studies, CFRs were established for a 30 minute control period, followed by 30 minutes i.v. infusion of the adenosine deaminase (ADA) inhibitor, EHNA, at 1 mg/kg/hr, and 30 minutes of EHNA at 5 mg/kg/hr. Following termin- ation of infusion, CFRs were followed for an additional 60 minutes . Results are shown in the following Table 2 :

Table 2

Effects of EHNA in a Model of Thrombosis

30' of 5 30' post 60' post Control mg/kg/hr EHNA EHNA

Mean Arterial

Pressure

(mmHg) 90 ± 6 91 ± 6 90 ± 4 87 ± 3

Heart Rate (bts/min) 133 ± 8 135 ± 3 137 ± 3 143 ± 8

Freq. of CFRs (#/30 min) 7 ± 0.7 4.8 ± 1.4 2.3 ± 0.9 1.5 ± 1.5

Nadir of

Flow (ml/min) 2.9 ± 0.8 6.4 ± 2.5 17.1 ± 2.1 27.5 ± 3.3 Number abolished 1 1 1

Total number abolished = 3/4

During the control period, frequency of CFRs was 7 + 0.7 CFRs/30 min and nadir of flow was 2.9 ± 0.8 ml/min. EHNA (5 mg/kg/hr) abolished CFRs in one dog, reduced the average frequency and increased the nadir of flow. Following termination of infusion, CFRs progressively lessened in frequency, the nadir of flow increased, and CFRs were abolished in one animal within 30 minutes and another animal within 60 minutes. Thus, at 60 minutes post EHNA, only weak CFRs remained in one out of the four animals tested. There were no effects on mean arterial pressure or heart rate at any time point. These results demonstrate that inhibition of adenosine deaminase with EHNA results in inhibition of clot formation in an animal model of arterial thrombosis. In contrast, in the same model of coronary thrombosis, neither the adenosine transport inhibitor dipyridamole, nor adenosine itself, imparted antithrom- botic activity. Table 3 compares the effects of dipyridamole, adenosine, and the adenosine-regulating- agent, AICA-riboside (5-amino-4-imidazolecarboxamide riboside) on the frequency and nadir of flow of CFRs in anesthetized dogs . Control values for frequency and nadir of flow were not different for any of the groups . Treatment with AICA-riboside (0.5 mg/kg/min) significantly reduced the frequency of CFRs from 7 ± 0.7 to 0.9 ± 0.6 CFRs/30 minutes and improved the nadir of flow from 2.4 ± 0.7 ml/min to 32 ± 6.0 ml/min. In a separate group of animals (n=6) the effect of acadesine, but not aspirin, was prevented (P<.001) by intracoronary infusion of the adenosine receptor antagonist 8- (p-sulfophenyl) theo- phylline demonstrating the involvement of adenosine in the effect of AICA riboside. In contrast to these results, dipyridamole (25 μg/kg/min) treatment did not change frequency (7 ± 0.9 vs. 8 ± 0.5) or nadir of flow (1.1 ± 0.3 vs. 0.5 ± 0.2) . Neither did adenosine (0.5 mg/kg/min) change frequency (7 ± 0.7 vs. 7 ± 0.8) or the nadir of flow (1.3 ± 0.3 vs. 1.4 ± 0.4) . These data from an in vivo model of thrombosis and unstable angina demonstrate that data obtained from in vitro models of platelet inhibition (as in Example 1) are not necessarily predictive of results observed in intact animal models of thrombosis.

16

SUBS Table 3

Effects of AICA Riboside, Dipyridamole and Adenosine in a

Model of Thrombosis

30' of 30' Post

Control Infusion Infusion

AICA riboside (0.5 mg/kg/min) (n=7)

CFRs/30' 7 ± 0.7 1.4 ± 1.2 0.9 ± 0.6 ml/min 2.4 + 0.7 1.7 + 3.3 32 + 6.0

Dipyridamole (25 μg/kg/min) (n=4)

CFRs/30' 7 ± 0.9 8.3 ± 0.5 8 ± 0.5 ml/min 1.1 + 0.3 0.5 + 0.2 0.5 + 0.2

Adenosine (0.5 mg/kg/min) (n=4)

CFRs/30' 7 ± 0.7 7.3 ± 0.8 7 ± 0.8 ml/min 1.3 + 0.3 1.4 + 0.4 1.4 + 0.4

Example 3

Effect of Adenosine Deaminase Inhibitor on Survival in

Endotoxic Shock Model

Ether anesthetized 6-12 week old male Balb/C mice (25-30 grams) were injected intravenously with 700 μg of E. coli 011:B4 LPS (Sigma Chemical Co., St. Louis, MO) dissolved in 100 μl of normal saline. This was followed within 2 minutes by a second intravenous injection with either 100 μl of normal saline or the ADA inhibitor EHNA (erythro-9- [2-hydroxy-3-nonyl] adenosine) dissolved in 100 μl normal saline. The final dose of EHNA was 10 mg/kg for each animal in the treated group. Survival was recorded after 24, 48, and 72 hours.

As shown in Figure 2, the ADA inhibitor, EHNA, protected animals from death. While all control animals were dead by 48 hours after injection with LPS, only about fifty percent of the EHNA-treated mice died (monitored to 72 hours) .

17 Formulations

Compounds useful in the methods of the present inven¬ tion may be administered to the affected tissue at the rate of from 0.01 to 200 nmol/min/kg, preferably from 0.1 to 10 nmol/min/kg. Such rates are easily maintained when these compounds are intravenously administered, as dis¬ cussed below. When other routes of administration are used (e.g., oral administration) , use of time-release preparations to control the rate of release of the active ingredient may be preferred. These compounds may be administered in a dose of about 0.01 mg/kg/day to about 100 mg/kg/day, preferably from about 0.1 mg/kg/day to about 10 mg/kg/day.

For the purposes of the presently claimed methods, ADA-inhibiting compounds may be administered by a variety of means including orally, parenterally, by inhalation spray, topically, or rectally in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intra¬ muscular, and intraarterial injections with a variety of infusion techniques . Intraarterial and intravenous injection as used herein includes administration through catheters . Preferred for certain indications are methods of administration which allow rapid access to the tissue or organ being treated, such as intravenous injections for the treatment of myocardial infarction. When an organ outside a body is being treated, perfusion is preferred. Pharmaceutical compositions 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 oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Pharmaceutical compositions useful in the presently claimed methods may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. Such a suspension may be formu- lated according to the known art using those suitable dispersing or wetting agents and suspending agents.

The amount of active ingredient that may be combined with carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain 2 to 200 μmoles of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5% to about 95% of the total compositions. It is preferred that pharmaceutical composition be prepared which provides easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion should contain from about 2 to about 50 μmoles of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 ml/hr can occur.

As noted above, formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be sorted in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of an adenosine deaminase inhibitor compound.

It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those skilled in the art.

Angina and early myocardial infarcts and other acute conditions can be treated by intravenous administration using a sterile injectable preparation using the rates discussed above.

Capsules comprising adenosine deaminase inhibitors suitable for oral administration according to the methods of the present invention may be prepared as follows: (1) for a 10,000 capsule preparation: 1500g of adenosine deaminase inhibitor is blended with other ingredients (as described above) and filled into capsules which are suitable for administration depending on dose, from about 1 capsule per day to about 8 capsules per day, to an adult human.

Claims

1. A method of preventing or treating a condition which is characterized by ischemia in a mammal comprising administering to said mammal an amount of a compound which inhibits adenosine deaminase activity in said mammal but which does not cause clinically significant immunodeficiency in said mammal.

2. A method according to claim 1 wherein said amount provides less than about 95% inhibition of adenosine deaminase activity.

3. A method according to claim 1 wherein said amount provides less than about 98% inhibition of adenosine deaminase activity.

4. A method according to claim 1 wherein said compound is administered over a time period of less than about 3 hours.

5. A method according to claim 1 wherein said compound is administered over a time period of less than about 24 hours.

6. A method according to claim 1 wherein said compound is administered over a time period of less than about 7 days .

7. A method according to claim 1 wherein said condition is myocardial infarction.

8. A method according to claim 1 wherein said condition is stroke.

9. A method according to claim 1 wherein said condition is angina.

21

t T RULE 2$)

10. A method according to claim 1 wherein said condition is transient ischemic attack.

11. A method according to claim 1 wherein said compound comprises coformycin.

12. A method according to claim 1 wherein said compound comprises 2' -deoxycoformycin.

13. A method according to claim 1 wherein said compound comprises erythro-9- (2-hydroxy-3-nonyl) adenine.

14. A method of preventing or treating thrombosis in a mammal comprising administering to said mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase.

15. A method according to claim 14 wherein said compound comprises coformycin.

16. A method according to claim 14 wherein said compound comprises 2' -deoxycoformycin.

17. A method according to claim 14 wherein said compound comprises erythro-9- (2-hydroxy-3-nonyl) adenine.

18. A method according to claim 14 wherein said therapeutically effective amount is an amount which inhibits adenosine deaminase activity but which does not cause clinically significant immunodeficiency.

19. A method according to claim 14 wherein said therapeutically effective amount is an amount which provides less than about 95% inhibition of adenosine deaminase activity.

20. A method according to claim 14 wherein said therapeutically effective amount is an amount which provides less than about 98% inhibition of adenosine deaminase activity.

21. A method according to claim 14 wherein said compound is administered over a time period of less than about 3 hours.

22. A method according to claim 14 wherein said compound is administered over a time period of less than about 24 hours.

23. A method according to claim 14 wherein said compound is administered over a time period of less than about 7 days .

24. A method of preventing or treating a condition characterized by an inflammatory response in a mammal comprising administering to said mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase.

25. A method according to claim 24 wherein said condition is selected from the group consisting of sepsis, septicemia, septic shock, endotoxemia, endotoxic shock, toxic shock, meningitis, burns, adult respiratory distress syndrome and necrotizing enterocolitis.

26. A method according to claim 24 wherein said condition is selected from the group consisting of arthritis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, iridocyclitis, vasculitis, ischemia, reperfusion injury, peripheral vascular disease, and atherosclerosis.

23

C^ ϊaiD irll --- 5

27. A method of preventing or treating sepsis in a mammal comprising administering to said mammal a thera¬ peutically effective amount of a compound which inhibits adenosine deaminase.

28. A method according to claim 27 wherein said sepsis results from infection with an organism selected from the group consisting of a gram negative bacterium, a gram positive bacterium, a virus, a mycobacterium, a fungus, a yeast and a worm.

29. A method of preventing or treating septicemia in a mammal comprising administering to said mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase.

30. A method according to claim 27 wherein said sepsis is from endotoxemia.

31. A method of preventing or treating septic shock in a mammal comprising administering to said mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase.

32. A method according to claim 31 wherein said septic shock is from endotoxic shock.

33. A method according to claim 24 or claim 27 wherein said compound comprises coformycin.

34. A method according to claim 24 or claim 27 wherein said compound comprises 2' -deoxycoformycin.

35. A method according to claim 24 or claim 27 wherein said compound comprises erythro-9- (2-hydroxy-3- nonyl) adenine.

24

&U£b s i I ύ I L Sritt i (BULL 2D)

36. A method according to claim 24 or claim 27 wherein said therapeutically effective amount is an amount which inhibits adenosine deaminase but which does not cause clinically significant immunodeficiency.

37. A method according to claim 24 wherein said therapeutically effective amount is an amount which provides up to about 95% inhibition of adenosine deaminase activity.

38. A method according to claim 24 wherein said therapeutically effective amount is an amount which provides up to about 98% inhibition of adenosine deaminase activity.

39. A method according to claim 24 or claim 27 wherein said compound is administered over a time period of less than about 3 hours.

40. A method according to claim 24 or claim 27 wherein said compound is administered over a time period of less than about 24 hours .

41. A method according to claim 24 or claim 27 wherein said compound is administered over a time period of less than about 7 days.

42. A method of treating a mammal at risk for sepsis comprising administering to said mammal a therapeutically effective amount of a compound which inhibits adenosine deaminase.

43. A method according to claim 42 wherein said risk for sepsis results from a condition selected from the group consisting of a burn, gunshot wound, perforated bowel, chemotherapy treatment, leukopenia, abdominal surgery, and a urological procedure.

25

^■ " E 25