WO2007140181A2

WO2007140181A2 - Combination therapy comprising an adenosine uptake inhibitor and a non-selective adenosine receptor antagonist

Info

Publication number: WO2007140181A2
Application number: PCT/US2007/069440
Authority: WO
Inventors: Edward Leung
Original assignee: King Pharmaceuticals Research And Development, Inc.
Priority date: 2006-05-26
Filing date: 2007-05-22
Publication date: 2007-12-06
Also published as: WO2007140181A3

Abstract

The present invention relates to a pharmaceutical combination comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome, through stimulation of the adenosine A3 receptor.

Description

COMBINATION THERAPY COMPRISING AN ADENOSINE UPTAKE INHIBITOR AND A NON-SELECTIVE ADENOSINE RECEPTOR ANTAGONIST

Field of the Invention

[001] The present invention relates to a pharmaceutical combination comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a nonselective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome, through stimulation of the adenosine A₃ receptor.

Background of the Invention

[002] Adenosine is known to be an endogenous moduϊator of a number of physiological functions. At the cardiovascular system level, adenosine is a strong vasodilator and a cardiac depressor On the central nervous system, adenosine induces sedative, anxiolytic and antiepileptic effects On the respiratory system, adenosine induces bronchoconstriction. At the kidney levei, it exerts a biphasic action, inducing vasoconstriction at low concentrations and vasodilation at high doses Adenosine acts as a ϋpolysis inhibitor on fat cells and as an antiaggregant on platelets Increased adenosine levels could protect against excessive cellular damage and organ dysfunction However, the protective effect of adenosine itself is insufficient because extracellular adenosine is rapidly taken up into adjacent cells and subsequently metabolized

[003] Three major classes of adenosine receptors, classified as A₁, A₂, and A₃, have been characterized pharmacologically The A₁ receptors are coupled to the inhibition of adenylate cyclase through Gi proteins, and have also been shown to couple to other second messenger systems, including inhibition or stimulation of phosphoinosrtol turnover and activation of ion channels, Adenosine A₂ receptors are further divided into two subtypes, A_2A and A_2B, at which adenosine and adenosine agonists activate adenylate cyclase with high and iow affinity, respectively. Whereas, the A₁ and A₃ receptors inhibit the activity of the same enzyme The adenosine A₃ receptor sequence was first identified in a rat testes cDNA library, and this sequence, later cloned by homology to other G-protein coupled receptors from a rat brain cDNA library, was shown to correspond to a novel, functional adenosine receptor

[004] The discovery of the adenosine A₃ receptor has opened new therapeutic vistas in the purine field in particular, the A₃ receptor is implicated in processes of inflammation, hypotension, and mast cell degranulation, e g _τ the A₃ selective agonist, IB-MECA (CF101), is currently being developed for inflammatory disorders such as rheumatoid arthritis, dry eye syndrome and psoriasis (Can-Fite BioPharma Ltd .). The A₃ receptor apparently also has a role in the central nervous system, e g, IB-MECA induces behavioral depression and upon chronic administration and protects against cerebral ischemia. Selective antagonists for the A₃ receptor are sought as potential anti-inflammatory or possibly anti-ischemic agents in the brain More recently, A₃ antagonists have been under development as antiasthmatic, antidepressant, antiarrhythmic, renal protective, antiparkinson and cognitive enhancing drugs

[005] Inhibitors of adenosine uptake or transport have been used clinically for some time for the treatment of certain cardiovascular diseases, e.g., adenosine uptake inhibitors have been employed as vasodilators and platelet aggregation inhibitors More recently, the adenosine uptake inhibitor, KF24345, has been shown to significantly suppress iipopolysaccharide- induced tumor necrosis factor-α (TNF-α) production and leukopenia in mice (Noji et at , Journal of Pharmacology and Experimental Therapeutics 2002, 300(1), 200-205)

[006] Cancer diseases are characterized by abnormal and unregulated ceil growth Uncontrolled cell division and growth Seads to cell accumulation and tumor growth. Some areas of the body are more commonly involved, but essentially any area of the body can become cancerous Although cancer is often referred to as a single condition, it actually consists of more than 100 different diseases Most cancers are named for the type of cell or organ in which they start, if a cancer spreads (metastasizes), the new tumor bears the same name as the original (primary) tumor

[007] Cancers of the gastrointestinal tract, including the liver, bile ducts, and pancreas, constitute the largest group of maiignant tumors. Colorectal cancer is one of the most common neoplastic diseases in Western countries and one of the leading causes of caπcer- reiated deaths.. Inactivation of the adenomatous polyposis coli (APC) tumor-suppressor gene during early adenoma formation is thought to be the first genetic event in the process of colorectal carcinogenesis followed by mutations in oncogenes like K-Ras, and tumor- suppressor genes like p53. Identification of the interaction of APC with the proto-oncogene β-catenin has linked colorectal carcinogenesis to the Wnt signal transduction pathway {Kolligs et al. Digestion 2002, 66(3), 131-144)

[008J The three most common types of cancer treatment are surgery, radiotherapy and chemotherapy However, there exists a need for new and better methods of treating cancer diseases, e g,, in the current chemotherapeutic treatment of human cancer, adverse side effects associated with the chemotherapeutic agents are often severe. Another often- encountered challenge with chemotherapeutic treatment is limitations in effectiveness due to the cancerous growth or cells developing muitidrug-resistance (MDR)

[009] One approach that has recently emerged is the use of adenosine A₃ receptor modulators, including A3 agonists, in the treatment of cancer and other related disorders For examples, IB-MECA, has been shown to inhibit HCT- 116 human colon carcinoma cell growth (Ohana et al., British Journal of Cancer 20QZ, 89(8), 1552 -1558), and the activation of the adenosine A₃ receptor has been implicated in the inhibition of melanoma ceil growth by deregulating protein kinase A and key components of the Wnt signaling pathway (Madi et al , Journal of Biological Chemistry 2003, 278(43), 42121-42130) A₃ selective receptor agonists has also been found to induce apoptosis at high concentrations in HL-60 human leukemia cells (Kohno et a}., Biochemical and Biophysical Research Communications 1996, 219(3), 904-910)

[010] Rheumatoid arthritis (RA) is traditionally considered a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints it is a disabling and painful inflammatory condition, which can lead to substantial loss of mobility due to pain and Joint destruction. RA is a systemic disease, often affecting extra-articular tissues throughout the body including the skin, blood vessels, heart, lungs, and muscles. The symptoms that distinguish RA from other forms of arthritis are inflammation and soft-tissue swelling of many joints at the same time (polyarthritis). The joints are usually affected initially asymmetrically and then in a symmetrical fashion as the disease progresses. As the pathology progresses the inflammatory activity leads to erosion and destruction of the joint surface, which impairs their range of movement and leads to deformity There is no known cure for RA, and the major goals of treatment of the arthritis are to reduce pain and discomfort, prevent deformities and loss of joint function

[011) Osteoarthritis (OA) is a condition in which low-grade inflammation results in pain in the joints, caused by wearing of the cartilage that covers and acts as a cushion inside joints. As the bone surfaces become less well protected by cartilage, the patient experiences pain upon weight bearing, including walking and standing. Due to decreased movement because of the pain, regional muscles may atrophy, and ligaments may become more lax OA is the most common form of arthritis The treatment for OA is relatively limited because there are currently no pharmacological agents capable of retarding or preventing the disease The treatment of OA is predominantly focused on relief of pain, and maintenance of quality of jife and functional independence

[012] Psoriasis is believed to be an immune-mediated disease which affects the skin and joints. It commonly causes red scaly plaques to appear on the skin. The scaly patches caused by psoriasis, called psoriatic plaques or lesions, are areas of excessive skin production and inflammation. Skin rapidly accumulates at these sites and takes a silvery - white appearance Plaques frequently occur on the skin of the elbows and knees, but can affect any area including the scalp and genitals The disorder is a chronic recurring condition which varies in severity from minor localized patches to complete body coverage Fingernails and toenails are frequently affected (psoriatic nail dystrophy).. Psoriasis can also cause inflammation of the joints, which is known as psoriatic arthritis. Ten to fifteen percent of people with psoriasis have psoriatic arthritis. Currently, there is no cure for psoriasis. However, there are many treatment options that can clear psoriasis for a period of time Each treatment has advantages and disadvantages, and what works for one patient may not be effective for another

[013] Clearly, there is strong need to develop new and improved therapies for the treatment of inflammatory disorders such as rheumatoid arthritis, osteoarthritis and psoriasis. [014] Recently, the adenosine A₃ receptor agonist IB-MECA has been demonstrated to exert significant antirheumatic effects in different autoimmune arthritis models by suppression of TNF-α production (Baharav et a! , Journal of Rheumatology 2005, 32(3), 469- 476), and as noted before !B-MECA is currently in development for the treatment of rheumatoid arthritis, psoriasis and dry eye syndrome (Can-Fite BioPharma Ltd.). For example, Phase lia pilot study data indicate that IB-MECA has disease modifying antiinflammatory activity in RA patients failing methotrexate (MTX) therapy

[015] These and other findings have made the A₃ receptor a promising therapeutic target.

Summary of the Invention

[016] While existing focus is on subtype selective A₃ agonists, herein for the first time is disclosed a novel way to selectively stimulate the adenosine A₃ receptor by employing an adenosine uptake inhibitor, which will increase adenosine levels, in combination with a nonselective adenosine receptor antagonist, which will block all adenosine receptor subtypes except the A₃ subtype

[017] It has now been discovered that selective stimulation of the adenosine A₃ receptor results in effective treatment of cancer, and thereby adenosine uptake inhibitors may be employed for the treatment of cancer diseases when administered in combination with a nonselective adenosine receptor antagonist

[018] It has also been discovered that selective stimulation of the adenosine A₃ receptor results in effective treatment of inflammatory disorders such as rheumatoid arthritis, psoriasis and dry eye syndrome, and thereby adenosine uptake inhibitors may be employed for the treatment of inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrom© when administered in combination with a non-selective adenosine receptor antagonist.

[019] Accordingly, the present invention provides a pharmaceutical combination, including free and fixed combinations, comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome

[020] In another aspect, the present invention provides a method for the treatment of cancer diseases through stimulation of the adenosine A₃ receptor, wherein the method comprises administering to a warm-blooded animal, including man, in need thereof, a therapeutically effective amount of a pharmaceutical combination comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist or a pharmaceutically acceptable salt thereof

[021 J Yet in another aspect, the present invention provides a method for the treatment of inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome through stimulation of the adenosine A₃ receptor, wherein the method comprises administering to a warm-blooded animal, including man, in need thereof, a therapeutically effective amount of a pharmaceutical combination comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof.

[022} Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

Detailed Description of the Invention

[023] As described herein above, the present invention provides a method to selectively stimulate the adenosine A₃ receptor by employing a combination of an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, in combination with a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof. [024] Accordingly, the present invention provides a pharmaceutical combination, including free and fixed combinations, comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome, through stimulation of the adenosine A₃ receptor

[025] Listed below are some of the definitions of various terms used herein to describe certain aspects of the present invention,. However, the definitions used herein are those generalty known in the art and apply to the terms as they are used throughout the specification unless they are otherwise limited in specific instances

[026] The term "adenosine uptake inhibitor" as used herein refers to any agent which is efficacious in blocking adenosine transport into ceils,

[027] The term "non-selective adenosine receptor antagonist" as used herein refers to all known compounds that will block all adenosine receptor subtypes except the adenosine A₃ subtype

[028] As used throughout the specification and in the claims, the term "treatment"¹ embraces alt the different forms or modes of treatment as known to those of the pertinent art and in particular includes preventive, curative, delay of progression and palliative treatment

[029] The term "combination" or "pharmaceutical combination" of an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, means that the components can be administered together as a pharmaceutical composition or as part of the same, unitary dosage form, i e , a fixed combination, A combination aiso includes administering an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, each separately but as part of the same therapeutic regimen, i e , a free combination. The components, if administered separately, need not necessarily be administered at essentially the same time, although they can if so desired, Thus, a combination also refers, e g , administering an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, as separate dosages or dosage forms, but at the same time. A combination also includes separate administration at different times and in any order

[030] As referred above, the term "fixed combination" as used herein means that the active compounds are all administered to a patient simultaneously in the form of a single entity or dosage As an example, a fixed combination would be one capsule containing two active compounds.

[031] As referred above, the term "free combination" as used herein means that the active compounds are ail administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits

[032] The term "therapeutically effective amount" refers to an amount of a drug or a therapeutic agent that will elicit the desired biological or medical response of a tissue, system or an animal (including man) that is being sought by a researcher or clinician.

[033] The term "warm-blooded animal or patient" are used interchangeably herein and include, but are not limited to, humans, dogs, cats, horses, pigs, cows, monkeys, rabbits, mice and laboratory animals. The preferred mammals are humans.

[034] The term "pharmaceutically acceptable salt" refers to a non-toxic salt commonly used in the pharmaceutical industry which may be prepared according to methods well-known in the art

[035] Suitable adenosine uptake inhibitors include, but are not limited to, cilostazol, dilazep, dipyridamole, midazolam, nimodipine, nrtrobenzylthioinosine, propentofylline, KF24345 and R75231, the structures of which may be found, e g ., in Noji et ai ., Eur. J Pharmacol 2004, 495, 1-16. [036] Suitable adenosine uptake inhibitors also include those disclosed in EP 1698635, EP 582164, US 2005054637, US 6,649,616, US 5,624,926, US 5,382,584, the entire contents of which are incorporated herein by reference

[037] Preferred adenosine uptake inhibitors to be employed in the methods and the pharmaceutical combinations of the present invention include cilostazol, dipyridamole or KF24345 or, in each case, a pharmaceutically acceptable salt thereof More preferably, the adenosine uptake inhibitor is cilostazol or dipyridamole Most preferably, the adenosine uptake inhibitor is dipyridamole

[038] The adenosine uptake inhibitors described herein above are known, and may be prepared and administered using methods well known in the art, e g , one skilled in art would be able to determine and optimize the dosages of the individual adenosine uptake inhibitors of the instant invention, e.g., by employing techniques described in Goodman and Gilman's "The Pharmacological Basis of Therapeutics, 10* Edition",. It should be noted that cilostazo! is a medication used in the alleviation of the symptoms of intermittent claudication in individuals with peripheral vascular disease, and is manufactured by Otsuka Pharmaceutical Co. under the trade name Pletal® Dipyridamole is used on its own, or in combination with aspirin, to prevent stroke by reducing the ability of platelet to aggregate, and a modified release dipyridamole and the combination with aspirin are currently marketed as Persantine® and Aggrenox®, respectively {Boehringer lngelheim). Dipyridamole is also used in nuclear cardiac stress testing as a coronary vasodilator

[039] Suitable non-selective adenosine receptor antagonist include, but are not limited to, methylxanthines which are widely known to be antagonists for the A₁, A_2n, and A_2B receptors, but not for the adenosine A₃ receptor Among methylxanthines, caffeine, paraxanthine, theobromine, theophylline and aminophylline are preferred More preferably, the nonselective adenosine receptor antagonist is caffeine

[040] As described herein above, the non-selective adenosine receptor antagonists are known, and may be administered using methods well known in the art, e.g., by employing techniques described in Goodman and Gilman's "The Pharmacological Basis of Therapeutics, 10^th Edition" [041] It should be noted that caffeine is a central nervous system and metabolic stimulant, and is used both recreationally and medically to reduce physical fatigue and restore mental alertness when unusual weakness or drowsiness occurs. Caffeine stimulates the central nervous system resulting in increased alertness and wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination, and later at the spinal cord level at higher doses. Once inside the body, it has a complex chemistry, and acts through several different mechanisms

[042] Caffeine is metabolized in the liver by the cytochrome P₄S₀ oxidase enzyme into three metabolic dimethylxanthines, which each have their own effects on the body:

1) Paraxanthine (84%) has the effect of increasing iipolysis, leading to elevated glycerol and free fatty acid plasma levels;

2) Theobromine (12%) dilates blood vessels and increases urine volume Theobromine is also the principal alkaloid in cocoa, and therefore chocolate; and

3) Theophylline (4%) relaxes smooth muscles of the bronchi, and is used to treat asthma The therapeutic dose of theophylline, however, is many times greater than the levels attained from caffeine metabolism .

[043] Aminophylline is a drug that contains theophylline and ethylenediamine in 2:1 ratio It is more soluble in water than theophylline White or slightly yellowish granules or powder, having a slight ammoniaca) odor and a bitter taste Upon exposure to air, it gradually loses ethylenediamine and absorbs carbon dioxide with the liberation of free theophylline Aminophylline is fess potent and shorter-acting than theophylline Its most common use is in the treatment of bronchial asthma.

[044] In one embodiment of the present invention, the pharmaceutical combination comprises the adenosine uptake inhibitor dipyridamole, or a pharmaceutically acceptable salt thereof, and the non-selective adenosine receptor antagonist caffeine

[045] The structure of the active agents identified by code numbers, generic or trade names may be taken from the actual edition of the standard compendium "The Merck Index", or the Physician's Desk Reference, or from databases, e.g., Patents international (e g , IMS World Publications) or Current Drugs Accordingly, any person skilled in the art is fully enabled to identify the active agents of the present invention Likewise, a person skilled in the art is fully enabled to manufacture and test the pharmaceutical indications and properties in standard test models known in the art, both in vitro and in vivo

[046] As indicated herein above, the compounds to be combined may be present as their pharmaceutically acceptable salts. Compounds having, e.g., at least one basic group such as an amino group, may form acid addition salts with acids such as mineral acids, organic carboxylic acids and organic sulfonic acids, e.g., hydrochloric acid, maleic acid and methanesulfonic acid, respectively.

[047] Similarly, compounds having at least one acidic group (for example COOH) may form salts with bases such as alkati and alkafine earth metal salts, e g , sodium, lithium, potassium, calcium and magnesium, as well as ammonium salts, e g._r ammonium, trimethytammonium, diethylammonium and tris(hydroxymethyl)-methyl-ammonium salts and salts with amino acids

[048] The corresponding active ingredients, or a pharmaceutically acceptable salts, may also be used in form of a solvate, such as a hydrate or including other solvents used, e g , in their crystallization.

[049] As described herein above, the present invention also provides pharmaceutical compositions comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[050] A pharmaceutical composition according to the present invention may be employed for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome.

[051] Accordingly, an adenosine uptake inhibitor, e.g., dipyridamole, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, e g,, caffeine, may be co-administered as a pharmaceutical composition. The components may be administered together in any conventional dosage form, usually also together with a pharmaceutically acceptable carrier or diluent. [052] The pharmaceutical compositions according to the invention are those suitable for enteral, such as oral or rectal, transdermal and parenteral administration to mammals, including man For oral administration the pharmaceutical composition comprising an adenosine uptake inhibitor, in particular, dipyridamole, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof, e g , caffeine, can take the form of solutions, suspensions, tablets, pills, capsules, powders, microemulsions, unit dose packets and the like Preferred are tablets and gelatin capsules comprising the active ingredient(s) together with: a) diluents, e g , lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e g , silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e g , magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylceilutose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbants, colorants, flavors and sweeteners.

[053] Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.

[054] Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-90%, preferably about 1-80%, of the active ingredient(s), conveniently from 30-95% for tablets and capsules, and 3-50% for liquid preparations.

[055] The dosage of the active ingredients to be therapeutically effective in stimulating the adenosine A₃ receptor will, of course, depend on a variety of factors, such as mode of administration, horneothermic species, age and/or severity of individual condition, and the disease or disorder to be treated

[056] Preferred dosages for the active ingredients of the pharmaceutical combinations according to the present invention are therapeutically effective dosages, especially those which are commercially available. In general, however, doses employed for adult human treatment will typically be in the range of 5-5000 mg/day, preferably 25-1000 mg/day, e g , for a patfent of approximately 75 kg in weight The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, e g . , as two, three, four or more sub-doses per day The dosage or dosages which will result in optimal effects is achieved by coordinating the pharmacokinetic properties, e g , volume of distribution and time to maximum plasma concentration (T_max), of the therapeutic agents of the present invention so that the therapeutic windows of each agent overlap to the maximum extent possible. Such dosages are readily determined by one skilled in the art

[057] For example, the doses of dipyridamole to be administered to warm-blooded animals, including man, of approximately 75 kg body weight, especially the doses effective to block adenosine uptake to cells, are from about 25 mg to about 1000 mg, preferably from about 100 mg to about 600 mg, e.g., from 200 to 400 mg/person/day, divided preferably into 1 to 4 siπgfe doses, which may, e g , be of the same size Usually, children receive about half of the adult dose. The dose necessary for each individual can be monitored and adjusted to an optimum level. Single doses comprise, e g , 25 mg, 50 mg, 75 mg or 100 mg per adult patient. Likewise, the preferred doses of cifostazol range from about 20 to about 300 mg/person/day More preferably, 100 mg of cilostazof is administered twice a day Single doses comprise, e g , 50 mg or 100 mg per adult patient

[058] According to the present invention, the amount of a non -selective adenosine receptor antagonist, e g , caffeine, or a pharmaceutically acceptable salt thereof, administered in combination with an adenosine uptake inhibitor, e g , dipyridamole, or a pharmaceutically acceptable saft thereof, to a patient is a dose effective to treat cancer diseases or inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome in a patient. It is an objective of the present invention to administer a dose of a non-selective adenosine receptor antagonist, e g,, caffeine, or a pharmaceutically acceptable salt thereof, in combination with an adenosine uptake inhibitor, e g , dipyridamole, or a pharmaceutically acceptable sait thereof, which will treat the conditions underlying said diseases or disorders, while minimizing, attenuating or reducing adverse side effects commonly associated with agents currently employed to treat such conditions. Preferably, the dose of a non-selective adenosine receptor antagonist is such that said adverse side effects are eliminated.

[059] For example, caffeine may be administered in combination with an adenosine uptake inhibitor, e.g., dipyridamole, to a patient of approximately 75 kg body weight at a dose between about 40 mg/day and about 400 rng/day However, the actual dose will depend on the particular factors of each case, including the patient's weight and the severity of the patient's condition Preferably, caffeine is administered at a dose between about 40 mg/day and about 400 mg/day More preferably the doge of caffeine ranges from about 100 mg/day and about 200 mg/day

[060] The preferred dose of theophylline ranges from about 400 mg to about 800 mg/day Likewise, the preferred dosage of aminophyiline ranges from about 400 mg to about 800 mg/day More preferably the doge of both theophylline and aminophylline ranges from about 400 mg to about 600 mg/day.

[061] The pharmaceutical compositions according to the present invention as described hereinbefore and hereinafter may be used for simultaneous use or sequential use in any order, for separate use or as a fixed combination

[062] Preferred are combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol, dilazep, dipyridamole, midazolam, nimodipine, nitrobenzylthioinosine, propentofyltine, KF24345 and R75231, or in each case, a pharmaceutically acceptable salt thereof

[063] More preferred are combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol and dipyridamole, or in each case, a pharmaceutically acceptable salt thereof

[064] Most preferred are combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the adenosine uptake inhibitor is dipyridamole, or a pharmaceutically acceptable salt thereof. [065] Preferred are also combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the non-selective adenosine receptor antagonist is selected from the group consisting of caffeine, paraxanthine, theobromine, theophylline and aminophylline, or in each case, a pharmaceutically acceptable salt thereof

[066] More preferred are combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the non-selective adenosine receptor antagonist is caffeine, or a pharmaceutically acceptable salt thereof

[067] Most preferred are combinations, such as a combined preparations or pharmaceutical compositions, respectively, wherein the adenosine uptake inhibitor is dipyridamole, or a pharmaceutically accepted salt thereof, and the non-selective adenosine receptor antagonist is caffeine, or a pharmaceutically acceptable salt thereof

[068] In view of the above, the present invention further relates to a method for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome, through stimulation of the adenosine A₃ receptor, comprising administering to a warm-blooded animal, including man, in need thereof, a therapeutically effective amount of a combination comprising an adenosine uptake inhibitor, in particular dipyrimadole, or a pharmaceutically acceptable salt thereof, and a nonselective adenosine receptor antagonist, e.g., caffeine, or a pharmaceutically acceptable salt thereof

[069] In a further aspect, the present invention provides use of a combination of an adenosine uptake inhibitor in combination with a non-selective adenosine receptor antagonist to selectively stimulate the adenosine A₃ receptor

[070] Accordingly, the present invention relates to the use of a combination comprising an adenosine uptake inhibitor, in particular, dipyrimadole, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, e g , caffeine, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for the manufacture of a medicament for the treatment of cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome [071] As the present invention relates to methods of treatment with a combination of compounds which may be administered separately, the present invention also relates to combining separate pharmaceutical compositions in a kit form The kit may comprise, e g , two separate pharmaceutical compositions: (1) a composition comprising an adenosine uptake inhibitor, in particular dipyridamole, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent; and (2) a composition comprising a nonselective adenosine receptor antagonist, e g , caffeine, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent The amounts of (1) and (2) are such that a beneficial therapeutic effect is achieved. The kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet, wherein each compartment contains a plurality of dosage forms (e g , tablets) comprising, e g , (1) or (2) Alternatively, rather than separating the active ingredient-containing dosage forms, the kit may contain separate compartments each of which contains a whole dosage which in turn comprises separate dosage forms An example of this type of kit is a blister pack wherein each individual blister contains two (or more) tablets, one (or more) tablet(s) comprising a pharmaceutical composition (1), and the second (or more) tablet(s) comprising a pharmaceuticai composition (2) Typically the kit comprises directions for the administration of the separate components The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e g , oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician

[072] The pharmaceutical activities as effected by administration of an adenosine uptake inhibitor, in particular dipyridamole, in a combination with a non-selective adenosine receptor antagonist, e g , caffeine, or a pharmaceutically acceptable salt thereof, can be demonstrated, e g , by using corresponding pharmacological models known in the pertinent art The person skilled in the pertinent art is fully enabled to seiect a reievani animal test model to prove the hereinbefore and hereinafter indicated therapeutic indications and beneficial effects

[073] An adenosine uptake inhibitor, e g , dipyridamole, or a pharmaceutical salt thereof, or the combination partners thereof, can be administered by various routes of administration Each agent can be tested over a wide-range of dosages to determine the optima! drug level for each therapeutic agent alone, or in the specific combination thereof, to elicit the maximal response For these studies, ft fs preferred to use treatment groups consisting of at least 6 animals per group. Each study is best performed in away wherein the effects of the combination treatment group are determined at the same time as the individual components are evaluated Although drug effects may be observed with acute administration, it may be preferable to observe certain responses in a chronic setting A long-term study, when appropriate, is of sufficient duration to allow for the full development of compensatory responses to occur and, therefore, the observed effect will most likely depict the actual responses of the test system representing sustained or persistent effect.

[074] It has been found that a combination of an adenosine uptake inhibitor, e g,, dipyridamole, or a pharmaceutically acceptable salt thereof, with a non-selective adenosine receptor antagonist, e g , caffeine, or a pharmaceutically acceptable salt thereof, achieves greater therapeutic effect in the treatment of conditions associated with cancer diseases, and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, psoriasis and dry eye syndrome, than the administration of an adenosine uptake inhibitor alone Greater efficacy can also be documented as a prolonged duration of action The duration of action can be monitored as either the time to return to baseline prior to the next dose or as the area under the curve (AUC)

[075] Further benefits include the use of lower doses of the individual drugs to be combined according to the present invention, when compared to the doses used when administered aione, e g , the dosages could not only be lower but are often also given less frequently, thereby reducing the incidence of adverse side effects This is in accordance with the desires and requirements of the patients to be treated . For example, in view of the reduced dose of the adenosine uptake inhibitor dipyrimadole, there is a considerable improvement in safety profile and therapeutic window when employing the combination of the present invention

[076] The following Example illustrates the effect of a combination of an adenosine uptake inhibitor, e.g., dipyridamole, and a non-selective adenosine receptor antagonist, e g , caffeine, on cyclic AMP (cAMP) levels in hA₃CHO cells. As the data show, the combination of dipyridamole and caffeine clearly activates the adenosine A₃ receptor as indicated by decreased cAMP levels in the presence of the drug combination:

cAMP Assay in hAsCHO CelJs

Ce// Culture Preparation

[077] CHO cells transfected with A₃ receptors are pelleted by centrifugation at 250 g for 5 min, washed once in Dulbecco's Modified Eagle Medium (DMEM/F12) and re-suspended in the same buffer in a cell density of 1 10⁶ celts/mL and used in the cyclic AMP (cAMP) assays CHO cells are kept under a humidified atmosphere of air/CO₂ at 37°C until the use in cAMP experiments

Measurement of cyclic AMP levels

[078] CHO cells expressing !1A₃ are suspended in 0 5 mL incubation mixture containing NaCI 150 mM, KCI 2 7 mM, NaH₂PO₄0.37 mM, MgSO₄ 1 mM, CaCl₂ 1 mM, glucose 5 mM, Hepes 5 mM, MgCI₂ 10 mM, pH 74 at 37°C Then 0 5 mM Ro 20-1724 as phosphodiesterase inhibitor and caffeine (100 μM) are added and incubated for 5 min in a shaking bath at 37°C Dipyridamole (10 μM) and adenosine {10 μM) are also incubated for 20 min in a shaking bath at 37°C Finally, forskolin {1 μM) was incubated for 5 min at 37°C The reaction is terminated by the addition of cold 6% trichloroacetic acid (TCA) The final aqueous solution is tested for cycfic AMP levels by a competition protein binding assay At the end of the incubation time (150 min at 4°C) and after the addition of charcoal the samples are centrifuged at 2,000 g for 10 min The clear supernatant is mixed with 4 mL of scintillator (Atomlight) and counted in a Packard scintillation counter

Effect of different stimuli on cAMP assays in hA₃CHO cells

Claims

What is claimed is

1 A pharmaceutical combination for selective stimulation of the adenosine A₃ receptor comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof

2 A pharmaceutical combination according to Claim 1 , wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of cancer diseases

3 A pharmaceutical combination according to Claim 1 , wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of inflammatory disorders

4 A pharmaceutical combination according to any one of Claims 1 to 3, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol, diϊazep, dipyridamole, midazolam, nimodipine, nitrobenzylthioinostne, propentofylline, KF24345 and R75231 , or in each case, a pharmaceutically acceptable salt thereof

5 A pharmaceutical combination according to any one of Claims 1 to 3, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol and dipyridamole

6 A pharmaceutical combination according to any one of Claims 1 to 3, wherein the adenosine uptake inhibitor is dipyridamole

7 A pharmaceutical combination according to any one of Claims 1 to 6, wherein the nonselective adenosine receptor antagonist is selected from the group consisting of caffeine, paraxanthine, theobromine, theophylline and aminophyltine, or a pharmaceutically acceptable salt thereof

8 A pharmaceutical combination according to any one of Claims 1 to 6, wherein the nonselective adenosine receptor antagonist is caffeine

9 A pharmaceutical composition for selective stimulation of the adenosine A₃ receptor comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof

10 A pharmaceutical composition according to Claim 9, wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of cancer diseases

11 A pharmaceutical composition according to Claim 9, wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of inflammatory disorders

12 A pharmaceutical composition according to any one of Claims 9 to 11 , wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol, dilazep, dipyridamole, midazolam, nimodipine, nitrobenzylthioinosine, propentofylline, KF24345 and R75231 , or in each case, a pharmaceutically acceptable salt thereof.

13 A pharmaceutical composition according to any one of Claims 9 to 11 , wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol and dipyridamole

14 A pharmaceutical composition according to any one of Claims 9 to 11, wherein the adenosine uptake inhibitor is dipyridamole

15 A pharmaceutical composition according to any one of Claims 9 to 14, wherein the non-selective adenosine receptor antagonist is selected from the group consisting of caffeine, paraxanthine, theobromine, theophylline and aminophylline, or a pharmaceutically acceptable salt thereof

16 A pharmaceutical composition according to any one of Claims 9 to 14, wherein the non-selective adenosine receptor antagonist is caffeine

17 A method for selective stimulation of the adenosine A₃ receptor comprising administering to a warm-blooded animal, in need thereof, a therapeutically effective amount of a pharmaceutical combination comprising an adenosine uptake inhibitor, or a pharmaceutically acceptable salt thereof, and a non-selective adenosine receptor antagonist, or a pharmaceutically acceptable salt thereof

18 A method according to Claim 17, wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of cancer diseases

19 A method according to Claim 17, wherein the selective stimulation of the adenosine A₃ receptor is employed for the treatment of inflammatory disorders

20 A method according to any one of Claims 17 to 19, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazol, dilazep, dipyridamole, midazolam, nimodipine, nitrobenzylthϊoinosine, propentofylline, KF24345 and R75231 , or in each case, a pharmaceutically acceptable salt thereof.

21. A method according to any one of Claims 17 to 19, wherein the adenosine uptake inhibitor is selected from the group consisting of cilostazoJ and dipyridamole

22 A method according to any one of Claims 17 to 19, wherein the adenosine uptake inhibitor is dipyridamole

23 A method according to any one of Claims 17 to 22, wherein the non-selective adenosine receptor antagonist is selected from the group consisting of caffeine, paraxanthine, theobromine, theophylline and aminophylline, or a pharmaceutically acceptable salt thereof

24 A method according to any one of Claims 17 to 22, wherein the non-selective adenosine receptor antagonist is caffeine