NO328251B1 - Use of A1 adenosine receptor antagonist for the preparation of drugs suitable for the treatment of pulmonary disease - Google Patents

Description

Technical field of the invention

The present invention relates to the use of an A1 adenosine receptor antagonist as stated in claim 1 for the production of drugs in cardiology, medicinal chemistry and pharmacology and in particular such A1 adenosine receptor antagonists for reducing pulmonary vasoconstriction or improving pulmonary hemodynamics.

Background for the invention

Pulmonary diseases can be life-threatening. Pulmonary edema and pulmonary hypertension are two such diseases. Pulmonary edema can be caused by a number of physical conditions, for example altered alveolar capillary membrane permeability, acute respiratory distress syndrome, increased pulmonary capillary pressure, decreased oncotic pressure and lymphatic insufficiency. The causes of pulmonary hypertension include, hypoxemia, respiratory system disorders, heart disease, thrombotic disease and embolic disease.

Conventional treatment of these pulmonary diseases involves drugs such as calcium channel blockers, diuretics, morphine sulfate, vasodilators such as nitrates, positive ionotropic agents, prostacyclin and anticoagulants.

Adenosine is an intracellular and extracellular messenger substance formed by all cells in the body. It is also formed extracellularly by enzymatic conversion. Adenosine receptors are divided into four known subtypes (ie, Ai, A2a, A2b, and A3) based on their relative affinity for different adenosine receptor ligands and by sequence analysis of genes encoding these receptors. The activation of each of the subtypes produces unique and sometimes conflicting effects. Adenosine is associated with coronary and systemic vasodilation. The presence of adenosine receptors and the function of these receptors in the pulmonary vasculature has been demonstrated in several species, including humans (see, for example, Kucukhuseyin et al., J. Basic Clin. Physiol. Pharmacol., 8(4), pp. 287-299 (1997); Hong, J.L., et al., J. Physiol., 508(Ptl), pp. 109-118 (1998)). These studies indicate that both A1 and A2 subtype receptors are present in the pulmonary vasculature. Activation of A2 receptors leads to dilation and relaxation of these vessels (see, for example, McCormack et al., Am. J. Physiol., 256(1 Pt 2), pp. H41-H46

(1989); Szentmiklosi et al., Naunyn Schmiedeberg's Arch. Pharmacol., 351(4), pp. 417-425 (1995); Cheng et al., Am. J. Physiol., 270(1 Pt 2), pp. H200-H207 (1996); Neely et al., Am. J. Physiol., 270(2 Pt 2), pp. H610-H619 (1996)). In contrast, these studies have shown that activation of Ai receptors leads to constriction and contraction of these vessels, resulting in increased resistance to blood flow (see Neely et al., J. Pharmacol. Exp. Ther., 258 (3), pp. 753-761 (1991); Broadly et al., J. Auton. Pharmacol., 16(6), pp. 363-366 (1996); see also Szentmiklosi (1995), Cheng (1996)

and Neely (1996), supra).

Despite the availability of a number of drugs to treat pulmonary diseases, such as pulmonary edema and pulmonary hypertension, the median duration of survival after the diagnosis of primary pulmonary hypertension is 2.8 years (D'Alonzo et al., Ann. Intern Med., 115, pp. 343-349

(1991)). Most of the current treatments involve non-specific vasodilatation and a reduction in peripheral (systemic) vascular resistance. These reductions in blood vessel tension in other parts of the body can result in reduced blood pressure that worsens the clinical situation by causing tissue underperfusion. Thus, there remains a need for new, pharmaceutically acceptable compounds and compositions and improved methods for reducing vasoconstriction and improving pulmonary hemodynamics in patients suffering from pulmonary edema and pulmonary hypertension.

Summary of the invention

Applicants have solved the above problem by discovering that Ax-adenosine receptor antagonists having a structure as set forth in claim 1 are capable of reducing pulmonary vasoconstriction and improving pulmonary hemodynamics without an accompanying reduction in peripheral vascular resistance. The compounds useful in the application according to the present invention exhibit their desired effects via specifically antagonizing or blocking the Ai adenosine receptor.

In the application according to the invention, the Ax adenosine receptor antagonist is selected from:

a compound of formula I:

where Ri and R2 are independently selected from the group consisting of alkyl, with from 1-6 C atoms,

R 3 is selected from a polycyclic group selected from 3-oxa-tricyclo[3.2.1.0<2-4>]oct-6-yl and bicyclo [2 . 2. 2 ] oct-1-yl-4-propionic acid,

Xi and X2 are 0, and

R6 is hydrogen:

In certain embodiments of the present invention, the compounds of formula I are selected from: 3-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl) -bicyclo[2.2.2]oct-1-yl]-propionic acid;

In more preferred embodiments, the Ax-adenosine receptor antagonist used according to the present invention is selected from the group consisting of: 3-[4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purine- 8-yl)-bicyclo[2.2.2]oct-1-yl]-propionic acid;

8-(3-Oxa-tricyclo[3.2.1.0 2'4]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione;

In the most preferred embodiments, the A2-adenosine receptor antagonist used in the method according to the present invention is 3-[4-(2,4-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl )-bicyclo[2.2.2]oct-1-yl]-propionic acid.

In some embodiments, the Ai adenosine receptor is used in the preparation of medicaments suitable for administration to a human.

In some embodiments, the A1 adenosine receptor antagonist used according to the present invention is formulated together with a pharmaceutically suitable carrier into a pharmaceutically acceptable composition.

The drugs in question are applicable in the treatment of patients who show signs or symptoms of pulmonary diseases. Examples of pulmonary diseases that can be treated include pulmonary edema, pulmonary hypertension and a combination thereof.

In some embodiments, the relevant drugs are used in the treatment of pulmonary edema followed by a condition selected from the group consisting of an imbalance of Starling forces altered by veolar-capillary membrane permeability, lymphatic insufficiency.

In some embodiments, the relevant drugs are used in the treatment of pulmonary hypertension followed by a condition selected from the group consisting of pulmonary arterial hypertension, pulmonary hypertension associated with diseases of the respiratory system or hypoxemia, pulmonary venous hypertension, pulmonary hypertension originating from chronic, thrombotic or embolic disease, pulmonary hypertension arising from disorders directly affecting the pulmonary vasculature.

In some embodiments, the relevant drugs are used in the treatment of a patient who exhibits signs or symptoms of pulmonary disease characterized by at least one of the following conditions: global pulmonary hypoxia, regional pulmonary hypoxia, pulmonary edema, increased pulmonary arterial pressure, increased pulmonary vascular resistance, increased central venous pressure, minimal arterial oxygen saturation, shortness of breath "rattles" and "cackles".

Brief description of the drawings

Figure 1 shows the effect of an A1 adenosine receptor antagonist B9719, 1 mg/kg) on mean arterial pressure (MAP) and heart rate (HR). No change in heart rate or mean arterial pressure was found after treatment with BG9719. Figure 2 shows the effect of an Ax adenosine receptor antagonist (BG9719, 1 mg/kg) on cardiac output (CO), pulmonary artery pressure (PAP) and pulmonary capillary wedge pressure (PCWP). No change in cardiac output was found after treatment with BG9719. Pulmonary artery pressure decreased 30 minutes after treatment with BG9719 and remained low. Pulmonary capillary wedge pressure decreased 90 minutes after treatment with BG9719. Figure 3 shows the measurement of pulmonary vascular resistance (PVC) in the following heart failure preparations after intravenous infusion of an A1 adenosine receptor antagonist (BG9719, 1 mg/kg). PVR decreases by 38% from baseline and returns to baseline levels (+p<0.05 vs. baseline). Figure 4. Systemic vascular resistance (SVR) and pulmonary vascular resistance were measured in the following HV preparations at baseline and after intravenous infusion of an Ax adenosine receptor antagonist (3-[4-(2,6-dioxo-1,3-dipropyl- 2,3,6,7-tetrahydro-1H-purin-8-yl)-bicyclo[2.2.2]oct-1-yl]-propionic acid (BG9928), 1 mg/kg). Results expressed as % change from baseline. At 10 minutes post-treatment with BG9928, PVR decreased 18% from baseline while there was no change in SVR (+ p<0.05 vs. baseline).

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is usually understood by the professional to which the invention belongs.

Although methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below.

To further define the invention, the following terms and definitions are given herein.

As used herein, an "alkyl" group means a saturated aliphatic hydrocarbon group. An alkyl group can be straight or branched and can, for example, have from 1 to 6 carbon atoms in a chain. Examples of straight chain alkyl groups include, but are not limited to, ethyl and butyl. Examples of branched alkyl groups include, but are not limited to, isopropyl and t-butyl.

As used herein, an "antagonist" means a molecule that binds to a receptor without activating the receptor or initiating signal transduction. An antagonist competes with the endogenous ligand for binding sites, thereby interfering with the stimulation or activation of the receptor by the endogenous ligand. Antagonists include antibodies raised against the A1 adenosine receptor that block the adenosine binding site or prevent adenosine from binding to the receptor.

As used herein, "selective antagonist" means an antagonist that binds to a specific subtype of adenosine receptor with higher affinity than to other subtypes. For example, a selective Ai receptor antagonist has high affinity for Ai receptors and has a) nanomolar binding affinity for the Ai receptor subtype and b) at least 10-fold, more preferably 50-fold and most preferably 100-fold greater affinity for the Ai receptor subtype than for another subtype.

As used herein, "antibody" means a polypeptide encoded by an immunoglobulin gene, genes or fragments thereof. The immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, as well as a large number of immunoglobulin variable regions. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE respectively.

Antibodies exist, for example, as intact immunoglobulins (consisting of two heavy chains and two light chains) or as a number of well-characterized fragments thereof. Such fragments include those produced by cleavage with various proteases, those produced by chemical cleavage and/or chemical dissociation, and those produced recombinantly, as long as the fragment remains capable of specific binding to an antigen. Among these fragments are Fab, Fab', F(ab')2/ and single-chain Fv(scFv) fragments. See Fundamental Immunology, Third Edition, W.E. Paul, ed. Raven Press, N.Y. (1993) for a detailed description of epitopes, antibody and antibody fragments. Such Fab' fragments can be readily prepared using conventional chemical synthesis or recombinant DNA technology. Thus, as used herein, the term antibody includes anti-substance fragments produced by the modification of whole antibody or such synthesized de novo. Antibodies that are useful in the present invention are optionally derived from libraries of recombinant antibodies in phage or similar vectors (see for example Huse et al., Science, 246, pp. 1275-81 (1989); Ward et al., Nature, 341, pp. 544-46

(1989); Vaughan et al., Nature Biotech., 14, pp. 309-14

(1996).

As used herein, "pharmaceutically effective amount" means the amount necessary to reduce or decrease the severity of vasoconstriction and/or improve pulmonary hemodynamics over a period of time. A pharmaceutically effective amount also means the amount necessary to improve the clinical symptoms in a patient.

As used herein, "pharmaceutically acceptable carrier or adjuvant" means a non-toxic carrier or adjuvant which can be administered to a patient, together with a compound of the present invention, and which does not destroy the pharmacological activity thereof.

As used herein, "pulmonary edema" means a condition in which fluid accumulates in the lungs. The clinical signs and symptoms of pulmonary edema can start as a primary manifestation of a certain pathology or as a development of a pre-existing disease. Patients present with a variety of symptoms including dyspnea, tachypnea, orthopnea, tachycardia, hypertension, chest tightness, cold extremities with or without cyanosis, cough with frothy or rose-colored sputum, extensive use of accessory muscles when breathing, moist rales with or without wheezing . Diagnosis of pulmonary edema is within the competence of ordinary professionals.

As used herein, "pulmonary hemodynamics" means the forces or mechanisms involved in circulating blood through the lungs. "Improved pulmonary hemodynamics" or "improvement of pulmonary hemodynamics" includes but is not limited to a decrease in pulmonary vascular resistance, decrease in pulmonary artery pressure, decrease in pulmonary capillary wedge pressure, increase in arterial oxygen saturation, decrease in "rattle", improvement in " shortness of breath", and increase in training capacity when limited by pulmonary function.

As used herein, "pulmonary hypertension" means abnormally increased blood pressure within the pulmonary circuit (pulmonary arteries). Pulmonary hypertension can be secondary to another disease process or occur as a primary disease process known as primary pulmonary hypertension. Diagnosis of pulmonary hypertension is within the skill of the ordinary person

competence.

As used herein, "pulmonary vasoconstriction" means the narrowing of the lumen of blood vessels in the lungs, particularly as a result of vasomotor action. Pulmonary vasoconstriction results in a decrease in blood flow through the lungs or an increase in resistance to blood flow through the pulmonary vasculature. "Reduction of pulmonary vasoconstriction" includes a decrease in vasoconstriction or an increase in pulmonary vasodilation. "Pulmonary vasodilatation" refers to an expansion of the lumen of the blood vessels. This is an increase in the internal diameter of a blood vessel that originates from the relaxation of smooth muscle within the wall of the chamber. This causes an increase in blood flow and/or a decrease in pulmonary artery pressure.

Synthesis of the Adenosine Antagonist Compounds

Compounds that are applicable in the invention can be prepared by conventional methods known in the art. For example, the synthesis of the compounds of formula I is described in International Publication Nos. WO 01/34604 and WO 01/34610.

The synthesis of the compounds 8-(3-oxa-tricyclo[3.2.1.0 2'4] oct-6-yl)-1, 3-dipropyl-3, 7-dihydro-purine-2, 6-dione and 8-bicyclo[ 2.2.1]hept-5-en-2-yl-1,3-dipropyl-3,7-dihydro-purine-2,6-dione is described in U.S. Pat. patent 5,446,046.

In some embodiments, the compounds may be in the form of an achiral compound, an optically active compound, a pure diastereomer, a mixture of diastereomers, a prodrug, or a pharmacologically acceptable salt thereof.

Preparation of Aj adenosine receptor antibody

The human Ax adenosine receptor has been cloned and the DNA sequence encoding the receptor as well as the protein sequence for the receptor have been identified (see, for example, Libert et al. Biochem Biophys Res Commun, 187(2), p. 919 -926

(1992); Townsend-Nicholson et al., Brain Res Mol Brain Res, 16(3-4), pp. 365-370 (1992)).

Antibody directed against the Ai adenosine receptor can be produced by immunization of a suitable host organism. Such antibodies can be polyclonal or monoclonal. Preferably they are monoclonal. Production of polyclonal and monoclonal antibodies is within the common knowledge in the field. For an overview of methods that are applicable in carrying out the invention, see, for example, Harlow and Lane

(1988), Antibodies, A Laboratory Manual, Yelton, D.E. et al. (1981); Ann. Fox. of Biochem., 50, pp. 657-80., and Ausbel et al. (1989); Current Protocols in Molecular Bioloqy (New York: John Wiley & Sons), updated annually. Determination of immunoreactivity with an A1 adenosine receptor can be performed by any of several methods well known in the art, including, for example, immunoblot assay and ELISA.

Monoclonal antibodies with affinities of 10<-8> M<-1> or preferably 10<-9> to 10<-10> M<-1> or stronger are typically prepared by standard procedure described as for example in Harlow and Lane, (1988) supra. Briefly, appropriate animals are selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells are fused, typically to immortalized myeloma cells under appropriate selection conditions. Then the cells are clonally separated and the supernatants from each clone are examined for their production of an appropriate antibody specific to the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic A1 adenosine receptor, or alternatively, selection of libraries for antibody in phage or similar vectors. See Huse et al., Science, 246, pp. 1275-81 (1989). Antigens (in this case the Ai adenosine receptor) and antibody can be labeled by joining together, either covalently or non-covalently, a substance that provides a detectable signal. Various markers and conjugation techniques are known in the art. Suitable markers include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent substances, chemiluminescent substances, magnetic particles and the like. Patents describing the use of such markers include U.S. Pat. patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In addition, recombinant immunoglobulins can be prepared (see U.S. Patent 4,816,567).

Application for Aj adenosine receptor antagonists

The compositions prepared by the application according to the present invention can be used to treat pulmonary diseases. The pulmonary disease can be, for example, pulmonary edema or pulmonary hypertension. These diseases can be caused by a number of physical traumas.

In some embodiments, the relevant compositions are used in the treatment of a pulmonary disease characterized by at least one condition selected from the group consisting of global pulmonary hypoxia, regional pulmonary hypoxia, pulmonary edema, increased pulmonary arterial pressure, increased pulmonary vascular resistance, increased central venous pressure, decreased arterial oxygen saturation, shortness of breath, "rattling" and "crawling".

As used herein, "rattles" and "crawling" mean abnormal sounds that are heard and accompany the normal respiratory sounds on auscultation of the chest.

The relevant drugs can be used to treat pulmonary edema caused by a number of conditions. These include an imbalance in Starling forces, altered alveolar-capillary membrane permeability (acute respiratory distress syndrome), lymphatic insufficiency. Furthermore, pulmonary edema can be caused by a number of other conditions, including pulmonary elevation edema, neurogenic pulmonary edema, drug overdose, pulmonary embolism, eclampsia, after cardioversion, after anesthesia, after cardiopulmonary bypass.

The topical drugs can be used to treat pulmonary edema caused by an imbalance in Starling forces. Causes of the imbalance in Starling forces include increased pulmonary capillary pressure, increased oncotic plasma pressure, underlying hypoalbuminemia and increased negativity of interstitial pressure. Increased pulmonary capillary pressure has both cardiac and non-cardiac causes. Cardiac causes include left ventricular failure, mitral stenosis or subacute bacterial endocarditis. Non-cardiac causes include pulmonary venous fibrosis, congenital stenosis of the origin of the pulmonary veins or pulmonary veno-occlusive disease. Increased pulmonary capillary pressure can also be caused by overperfusion of fluids.

The topical drugs can be used to treat pulmonary edema caused by increased negativity of interstitial pressure. Reasons for increased negativity of interstitial pressure include rapid removal of pneumothorax with large applied

negative pressure or asthma.

The relevant drugs can be used to treat pulmonary edema caused by altered alveolar-capillary membrane permeability. The causes of altered alveolar-capillary membrane permeability include infectious pneumonia (viral or bacterial), inhaled toxins, circulating toxins, vasoactive substances (eg histamine, kinins), disseminated intravascular coagulation, immunological reactions, radiation pneumonia, uremia, near drowning, aspiration pneumonia, smoke inhalation, adult respiratory distress syndrome.

The relevant drugs can be used to treat pulmonary edema caused by lymphatic insufficiency. Causes of lymphatic insufficiency include post-lung transplant insufficiency, lymphangitic carcinomatosis, or fibrosing lymphangitic.

The drugs in question can be used to treat pulmonary hypertension caused by a number of conditions. These include pulmonary arterial hypertension, pulmonary hypertension associated with disorders of the respiratory system and/or hypoxaemia, pulmonary venous hypertension, pulmonary hypertension arising from chronic thrombotic and/or embolic disease, pulmonary hypertension arising from disorders directly affecting the pulmonary vasculature.

The relevant drugs can be used to treat pulmonary hypertension caused by pulmonary arterial hypertension. The causes of this include primary pulmonary hypertension (including sporadic and familial diseases); related conditions such as collagen vascular disease, congenital systemic to pulmonary shunt, portal hypertension, and human immunodeficiency virus infection; drug and toxin induced (that is, anorectic agents (appetite suppressants)); and persistent pulmonary hypertension in newborns.

The relevant drugs can be used to treat pulmonary hypertension caused by pulmonary hypertension associated with diseases of the respiratory system and/or hypoxemia. The causes of pulmonary hypertension associated with diseases of the respiratory system and/or hypoxemia include chronic obstructive pulmonary disease, interstitial lung disease, sleep disordered breathing, alveolar hypoventilation diseases, chronic exposure to altitude, neonatal lung disease, and alveolar-capillary dysplasia.

The relevant drugs can be used to treat pulmonary hypertension caused by pulmonary venous hypertension. Causes of pulmonary venous hypertension include left-sided atreill or ventricular heart disease, left-sided valvular heart disease extrinsic compression of central pulmonary veins (eg, fibrosing mediastinitis, adenopathy and/or tumors) and pulmonary veno-occlusive disease.

The relevant drugs can be used to treat pulmonary hypertension caused by chronic thrombotic and/or embolic disease. The causes of pulmonary hypertension arising from chronic thrombotic and/or embolic disease include thromboembolic obstruction of proximal pulmonary arteries (eg, pulmonary embolism (thrombus, tumor, ova and/or parasites, foreign material), in-situ thrombosis, sick cell disease).

The relevant drugs can be used to treat pulmonary hypertension caused by disorders that directly affect the pulmonary vasculature. Causes of pulmonary hypertension arising from disorders directly affecting the pulmonary vasculature include inflammatory conditions (eg, schistosomiasis, sarcoidosis) and pulmonary capillary hemangiomatosis.

Pharmaceutical compositions

The A1 adenosine receptor antagonists can be formulated into pharmaceutical compositions for administration to animals, including humans. These pharmaceutical compositions preferably include an amount of A1 adenosine receptor antagonist effective to reduce vasoconstriction or increase pulmonary hemodynamics and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carrier materials useful in these pharmaceutical compositions include, for example, ion exchangers, alumina, aluminum stearate, lecintin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, wax types, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool grease .

The compositions according to the present invention can be administered parenterally, orally by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intraethical, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions according to the present invention can be aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any soft fixed oil can be used including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectable preparations, and so are natural pharmaceutically acceptable oils such as olivine oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcoholic diluent or dispersing agent such as carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifiers or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed by a maintenance dose. These compositions can be administered once per day or on an "as needed" basis.

The pharmaceutical compositions may be administered orally in an orally acceptable dosage form including capsules,

tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carrier materials include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavoring or coloring agents can also be added.

Alternatively, the pharmaceutical compositions may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will consequently melt in the rectum to release the drug. Such materials include coconut butter, beeswax and polyethylene glycols.

The pharmaceutical compositions may also be administered topically. Topical application can be carried out in a rectal suppository formulation (see above) or in a suitable rinse formulation. Topical transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carrier materials. Carrier materials for topical administration of the compounds of the present invention include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetaryl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

The pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and may be prepared as solutions in physiological saline, using benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability, fluorocarbons, and/or other conventional solubility increasing or dispersing agents.

The amount of Ax adenosine receptor antagonist that can be combined with the carrier materials to form a single dosage form will vary depending on the host being treated and the particular mode of administration. The compositions may be formulated such that a dose of between 0.01-100 mg/kg body weight of the A1 adenosine receptor antagonist is administered to a patient receiving these compositions. In some embodiments of the invention, the dose is 0.1-10 mg/kg body weight. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion.

A particular dosage and treatment regimen for any particular patient will depend on a number of factors, including the particular A!-adenosine receptor antagonist, the patient's age, body weight, general health, gender and diet and the time of administration, the degree of excretion, drug combination , and the severity of the particular disease being treated. Assessment of such factors by medical practitioners is within the usual competence in the field. The amount of antagonist will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amounts of antagonists can be determined by pharmacological and pharmacokinetic principles well known in the art.

In order for the invention described herein to be more fully understood, the following examples are described.

Example 1

Animal model

Nineteen male Yorkshire pigs (20-25 kg, Hambone Farms, SC) were instrumented to induce ongoing myocardial heart failure as described in Tomita et al., Circulation, 83, pp. 635-644 (1991). Briefly, under isoflurane anesthesia (3% in 1.5 L/min oxygen) and through a left thoracotomy, a shielded pacing lead was sutured into the left atrium, connected to a modified programmable pacemaker (8329 Medtronic, Inc., Minnepolis, MN) and buried in a subcutaneous pocket. Ten to fourteen days after recovering from the surgical procedure, a baseline echocardiographic study was performed and running was initiated at 240 beats per minute for 3 weeks. A further group of 7 normal control animals were treated in an identical manner with the exception of the running control. At the end of the 3-week run-in period, the pacemakers were deactivated and echocardiographic studies were performed. For these studies, the animals were brought to the laboratory and the pacemakers were deactivated. Two-dimensional and M-method echocardiographic studies (ATL Ulmark VI, 2.25 MHz transducer, Bothell, WA) were used to image the left ventricle from a right parasternal angle. After the echocardiographic study, the animals were prepared for acute instrumentation and initiation of the study protocol.

Acute instrumentation

The pigs were anesthetized with intravenous boluses of sufentanil 2.0 ug/kg, etomidate 0.3 mg/kg, and vecuronium 10 mg, during which a tracheostomy was performed. A tubocurarine 12 mg intravenous bolus was administered after finding arterial pressure. Anesthesia was maintained throughout the procedures by continuous intravenous infusion of morphine sulfate 3 mg/kg/hour and tubocurarine 2 mg/hour. Etomidate 0.1 mg/kg intravenous was also given at 30 minute intervals. A maintenance infusion of 10 mL/kg/hour of lactated Ringer's solution was maintained throughout the procedure. This anesthetic procedure resulted in a deep anesthetic plane and stable hemodynamic profiles for up to 6 hours. A multilumen thermodilution catheter (7.5 Fr, Baxter Healthcare Corp., Irvine, CA) was placed in a pulmonary arterial via the right external carotid artery and a large bore catheter (7 Fr) was placed in the left external carotid artery for fluid administration. The carotid artery was exposed and cannulated, and a catheter (7 Fr) was advanced to the aortic root for main pulse year blood pressure measurements and blood sampling.

Hemodynamic and new functional measurements

An instrumentation and a 15 minute stabilization period, baseline hemodynamics were recorded and digitized. Thermodilution-derived myocardial power and ejection fraction were obtained from the pulmonary arterial catheter in triplicate. All measurements were recorded simultaneously with the ventilator temporarily suspended to prevent respiratory misreadings of the findings. An arterial sample was drawn for electrolyte analyses. Pulmonary and systemic vascular resistances were calculated from thermodilution cardiac output and pressure measurements using standard formulas.

Experimental method

After instrumentation and collection of baseline measurements, animals were randomly selected to receive either vehicle infusion (polyethylene glycol, 3 ml intravenous, n=10) or an A1 adenosine receptor antagonist (1 mg/kg 1, 8-(3-oxa-tricyclo [3.2 .1.02'4]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (BG9719); n=9). After infusion of vehicle or BG9719, the hemodynamic measurements described in the previous section were repeated at 10, 30, 60, 90, 120 minutes after infusion.

Data analysis

Changes in hemodynamics were initially examined between the control and Ai adenosine receptor antagonist (BG9719) groups by ANOVA. Comparisons between these baseline values after randomization were performed with a 2-way ANOVA. Post-infusion comparisons of these parameters were compared using a repeated-measures multiway ANOVA. Pairwise comparisons were performed with a Bonferroni adjusted t-test. All statistical analyzes were performed using statistical software programs (BMDP statistical software Inc. University of California Press, Los Angeles, CA). Results were as mean ± standard deviation of that mean (SEM). Values of p<0.05 were considered to be statistically significant.

Systemic and pulmonary haemodynamics in heart failure

In the ongoing heart failure group, left ventricular and diastolic dimensions increased (5.68 + 0.15 vs. 4.09 0.12 cm; p<0.05) and fractional shortening decreased (24 2 vs. 42 2%; p, 0, 05) compared to normal control values. In the heart failure group, heart rate, pulmonary arterial pressure and pulmonary capillary wedge pressure were increased and cardiac output and mean aortic pressure were reduced compared to normal control values. There was no difference in any of the baseline parameters in the animals randomized to A1 adenosine receptor antagonist or vehicle infusions. No changes from baseline in hemodynamic measurements were noted in the normal control group throughout the study.

Systemic and pulmonary haemodynamics - Effects of Aj adenosine receptor antagonists in heart failure

No changes from baseline in heart rate (Figure 1), mean arterial pressure (Figure 1), cardiac output (Figure 2), or systemic vascular resistance were noted after treatment with the A1 adenosine receptor antagonist BG9719. Mean pulmonary arterial pressure decreased from baseline at 30 minutes after treatment with BG9719 and remained low (30 + 1 vs. 23 + 3 mmHg; p<0.05) (Figure 2). Pulmonary capillary wedge pressure (PCWP) decreased at 90 minutes after treatment with BG9719 (9 + 2 mg Hg; p<0.05) (Figure 2). Pulmonary vascular resistance fell by 38% from baseline at 10 min after treatment with BG9719 and expected to return to baseline levels (Figure 3). In the vehicle group, no changes in hemodynamics were noted. Selective A1 adenosine receptor antagonism with BG9719 was associated with an acute decrease in pulmonary resistive properties without reducing systemic vascular tone or blood pressure.

Example 2

Animal model

Four male Yorkshire pigs (25-30 kg, Hambone Farms, SC) were implanted with a pacemaker (8329, Medtronic, Inc., Minneapolis, MN) to induce ongoing CHF as described above. Ten to 14 days after recovering from the surgical procedure, a baseline echocardiographic study was performed

(ATL Ultramark VI, 2.25 MHz transducer, Bothell, WA) performed and running initiated at 240 beats per minute for 3 weeks. A further group of 6 normal control animals were treated identically except for the running procedure. At the end of the 3-week run-in period, the pacemakers were deactivated and echocardiographic studies were used to image the LV from a right parasternal angle. After the echocardiographic study, the animals were prepared for acute instrumentation and initiation of the study protocol.

Acute instrumentation

The pigs were anesthetized (i.v. sufentanyl 2.0 g/kg, etomidate 0.3 mg/kg) and paralyzed (vecuronium 10 mg, tubocurarine 12 mg). A maintenance infusion of 10 mL/kg/hour of lactated Ringer's solution was maintained throughout the procedure. A thermodilution catheter (7.5 Fr, Baxter Healthcare Corp., Irvine, CA) was placed in the pulmonary arterial via the right external carotid artery and a large-bore catheter (7 Fr) was placed in the left external carotid artery for fluid administration. The carotid artery was exposed and cannulated, and the catheter (7 Fr) was advanced to the main artery root for aortic blood pressure measurements and blood sampling.

Hemodynamic function measurements

After instrumentation and a 10-minute stabilization period, baseline hemodynamics were recorded and digitized. Thermodilution derived myocardial output and ejection fraction were obtained from the pulmonary arterial catheter in triplicate. Pulmonary and systemic vascular resistances were calculated from the pressure measurements and myocardial output values using standard formulas.

Experimental protocol

After instrumentation and collection of baseline measurements, the A1 receptor antagonist (3-[4-(2,4-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-bicyclo [2.2.2]oct-l-yl]-propionic acid (BG9928), 1 mg/kg) infused intravenously. Hemodynamic measurements described in the previous section were repeated at 10, 20, 30, 60, 90 and 120 minutes after infusion.

Data analysis

Comparisons of basal hemodynamics between control and

The HF groups were performed using Student's t-test. Time-dependent changes in hemodynamics after Ai block infusion were investigated with ANOVA. Pairwise comparisons were performed with a Bonferroni adjusted t-test. All statistical analyzes were performed using statistical software programs (BMDP Statistical Software Inc. University of California Press, Los Angeles, CA). Results are presented as mean + standard deviation of the mean (SEM). Values of p<0.05 were considered to be statistically significant.

Results

In the ongoing heart failure group, the left ventricular end-diastolic dimension increased (5.8 ± 0.1 vs. 4.1 ± 0.3 cm: p<0.05) and the fractional shortening decreased (20 + 1 vs. 41 2%; p<0 .05) compared to normal control values. Baseline left ventricular function and hemodynamics are summarized in table 1. In the heart failure group, heart rate, pulmonary arterial pressure and pulmonary capillary wedge pressure (PCWP) were increased, and stroke volume was reduced compared to normal control values. Pulmonary vascular resistance was also increased in the HF group compared to the normal group. No changes from baseline in hemodynamics were noted in the normal control group throughout the study.

Systemic and pulmonary haemodynamics: Effects of Aj adenosine receptor antagonist in heart failure

No change from baseline in heart rate, mean arterial pressure, or cardiac output was noted after treatment with the Ai adenosine antagonist BG9928.

Pulmonary vascular resistance decreased by 18% from baseline at 10 minutes after treatment with BG9928 (p<0.05) while there was no change in systemic vascular resistance (Figure

4). Selective A1 adenosine receptor antagonism with BG9228 was associated with an acute decrease in pulmonary resistive properties without a reduction in systemic vascular tone or blood pressure.

Example 3

Assessment of Aj selective antagonists - inhibition of adenosine-mediated vasoconstriction of pulmonary vessels

In order to evaluate a large number of compounds, a model was designed where pulmonary vessels from rodents (rats) were prepared and transgoing rings of the vessel were used in an in vitro tissue bath apparatus. This model enables the evaluation of compounds to determine whether the test compounds reduce pulmonary vasoconstriction.

Male Sprague Dawley rats are anesthetized IP with 90 mg/kg sodium brevital. After achieving a surgical anesthetic plane, the neck area is shaved and the heart and neck area are exposed with a median sternotomy. The pulmonary artery is removed by removing the esophagus, resetting the trachea, and exposing the main blood vessels entering the dorsal surface of the heart. The pulmonary artery is carefully dissected and removed. The isolated chambers are kept in an open container with cold Krebs-Henseleit buffer, pH 7.4 containing D-glucose (2 g/l), MgSO 4 (0.14 g/l), monobasic potassium sulfate (0.16 g/l ), KC1 (0.35 g/l), NaCl (6.9 g/l), CaCl (0.373 g/l), and Na bicarbonate (2.1 g/l) until ready for use. Using a petri dish and a stereomicroscope, the chamber is cleaned of adventitia and cut into 3 more ring segments. The pulmonary rings are then carefully mounted in wire triangles, and placed in pre-warmed 37 °C organ baths containing 10 ml of Krebs-Henseleit buffer bubbled with 95% O2/5% CO2. Two lengths of 3-0 silk wire with triangular wire supports at each end are used to support pulmonary rings; one end of the assembly is suspended in an L-shaped glass rod and another end to an isometric force transducer to measure force in gram tension. Manual preload tension is set at 1 g and rings are allowed to equilibrate for 1 hour, with washing and preload adjustment every 15 minutes or as needed. After equilibration, pulmonary rings are added to 60 mM potassium chloride (KCl) and allowed to plateau for up to 5 minutes and washed.

The reactivity of the chambers is examined by application of PGF2A, phenylephrine, or potassium. After the reactivity is confirmed, the tissue is washed three times and allowed to stabilize under a 1 g load. A concentration response curve is then generated with the Ax selective agonist N-6 cyclopentyladenosine (CPA) while under oxygenation. The tissue is then washed three times and allowed to equilibrate under 1 gram of tension without oxygenation and with incubation with different concentrations of the experimental antagonist. The CPA concentration-response curve is then repeated to confirm the vasoconstrictive response under hypoxia and to determine whether the antagonist causes a rightward, parallel shift in the agonist concentration-response curve (indicating full, competitive antagonism).

Throughout the present specification and claims, the word "comprising", or variations such as "comprising" or "comprising" will be understood to imply the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or group of integers .

Claims (16)

1. Use of an Ax adenosine receptor antagonist and possibly a pharmaceutically acceptable carrier for this for the production of a drug which is suitable for reducing pulmonary vasoconstriction or improving pulmonary hemodynamics in an individual or which is suitable for treating a pulmonary disease, where the adenosine Ax adenosine receptor antagonist is selected from the group consisting of: a. a compound comprising formula I: where Ri and R2 are independently selected from the group consisting of: alkyl, having from 1-6 C atoms, R3 is selected from a polycyclic group selected from 3-oxa-tricyclo[3.2.1.0<2-4>]oct -6-yl and bicyclo [2 . 2. 2 ] oct-1-yl-4-propionic acid, X 1 and X 2 are 0, and R 6 is hydrogen, Z is a single bond. 2. Use according to claim 1, where the A1-adenosine receptor antagonist is selected from the group consisting of: 3-[4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purine- 8-yl)-bicyclo[2.2.2]oct-1-yl]-propionic acid, 8-(3-oxa-tricyclo[3.2.1.0 2'4]oct-6-yl)-1,3-dipropyl -3,7-dihydro-purine-2,6-dione; 3. Application according to claim 1, where the individual for which drug is suitable is a human. 4. Use according to claim 1, where the Ax adenosine receptor antagonist is formulated together with a pharmaceutically suitable carrier material into a pharmaceutically acceptable composition. 5. Use according to claim 4, where the pharmaceutically acceptable composition is suitable for administration to a human. 6. Use according to claim 4, where the drug is suitable for the treatment of a disease which exhibits signs or symptoms of a pulmonary disease. 7. Use according to claim 6, where the pulmonary disease is selected from pulmonary oedema, pulmonary hypertension and a combination thereof. 8. Use according to claim 7, where the pulmonary edema is followed by a condition selected from the group consisting of an imbalance of Starling forces, altered alveolar-capillary membrane permeability, lymphatic insufficiency. 9. Use according to claim 7, where the pulmonary hypertension is followed by a condition selected from the group consisting of pulmonary arterial hypertension, pulmonary hypertension associated with disorders in the respiratory system or hypoxemia, pulmonary venous hypertension, pulmonary hypertension originating from chronic thrombotic or embolic disease, pulmonary hypertension arising from disorders that directly affect the pulmonary vasculature. 10. Use according to claim 4, where the drug is suitable for the treatment of a disease in an individual who exhibits signs or symptoms of a pulmonary disease that is characterized by at least one condition selected from the group consisting of global pulmonary hypoxia, regional pulmonary hypoxia, pulmonary edema , increased pulmonary arterial pressure, increased pulmonary vascular resistance, increased central venous pressure, decreased arterial oxygen saturation, shortness of breath, "rattling" and "cackling". 11. Use according to claim 1, where the drug is suitable for treating an individual who exhibits signs or symptoms of a pulmonary disease. 12. Use according to claim 11, where the pulmonary disease is selected from edema and pulmonary hypertension. 13. Use according to claim 12, wherein the pulmonary edema is followed by a condition selected from the group consisting of an imbalance of Starling forces, altered alveolar-capillary membrane permeability, lymphatic insufficiency. 14. Use according to claim 12, where the pulmonary hypertension is followed by a condition selected from the group consisting of pulmonary arterial hypertension, pulmonary hypertension associated with disorders of the respiratory system or hypoxemia, pulmonary venous hypertension, pulmonary hypertension originating from chronic thrombotic or embolic disease, pulmonary hypertension arising from disorders that directly affect the pulmonary vasculature. 15. Use according to claim 1, where the drug is suitable for the treatment of an individual who exhibits signs or symptoms of a pulmonary disease characterized by at least one condition selected from the group consisting of global pulmonary hypoxia, regional pulmonary hypoxia, pulmonary oedema, increased pulmonary arterial pressure , increased pulmonary vascular resistance, increased central venous pressure, decreased arterial oxygen saturation, shortness of breath "rattles" and "cackles". 16. Use according to claim 7, where the pulmonary hypertension is followed by a condition selected from the group consisting of pulmonary arterial hypertension, pulmonary hypertension associated with disorders of the respiratory system or hypoxemia, pulmonary venous hypertension, pulmonary hypertension originating from chronic thrombotic or embolic disease, pulmonary hypertension arising from disorders that directly affect the pulmonary vasculature.