WO2008019106A1

WO2008019106A1 - Methods and compositions for the treatment of pulmonary hypertension using a combination of a calcium channel blocker and a phosphodiesterase inhibitor

Info

Publication number: WO2008019106A1
Application number: PCT/US2007/017410
Authority: WO
Inventors: Jeff Bechard; Beth Allison; John K. Gibson
Original assignee: Artesian Therapeutics, Inc.
Priority date: 2006-08-04
Filing date: 2007-08-03
Publication date: 2008-02-14

Abstract

The present invention provides novel methods for the treatment of pulmonary hypertension. In particular, the present invention includes methods of treating pulmonary hypertension using a combination of a calcium channel blocker and a phosphodiesterase inhibitor, as well as pharmaceutical compositions comprising a calcium channel blocker and a phosphodiesterase inhibitor, including, e.g., admixtures suitable for delivery by inhalation.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PULMONARY

HYPERTENSION USING A COMBINATION OF A CALCIUM CHANNEL

BLOCKER AND A PHOSPHODIESTERASE INHIBITOR

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is directed to the treatment of pulmonary hypertension. In particular, the present invention includes methods of treating pulmonary hypertension using a combination of a calcium channel blocker and a phosphodiesterase inhibitor, as well as pharmaceutical compositions comprising a calcium channel blocker and a phosphodiesterase inhibitor, including, e.g., admixtures suitable for inhaled delivery.

Description of the Related Art

Pulmonary hypertension is a complex, life-threatening disease for which there is no cure. Despite limited advances in therapy, pulmonary hypertension associated mortality remains high, with a mean survival of approximately 2.8 years from the onset of disease. Patients with pulmonary hypertension suffer shortness of breath, fatigue, temporary blackouts, chest pain, bluish skin color, a racing heart, and chest pain.

Pulmonary hypertension is characterized by abnormally high blood pressure in the blood vessels of the lungs. It generally begins when the lung's tiny arteries narrow or become blocked, causing increased resistance to blood flow and increased pressure within the arteries. As the pressure builds, the heart's right ventricle works harder to pump blood through the lungs, especially when increased flow is needed, as during exercise. Eventually, the heart weakens, and oftentimes fails completely. Heart failure is the leading cause of death in patients with pulmonary hypertension.

Generally, pulmonary hypertension falls into one of two categories; primary pulmonary hypertension, for which no underlying cause is identified, and secondary pulmonary hypertension, for which an underlying cause is identified.

Secondary pulmonary hypertension is the most common form of the disease, and may be caused by congenital heart defects, connective tissue disease (e.g., scleroderma), medications (e.g., the diet pill fen-phen), HIV infection, blood clots, liver disease, and chronic blood clots in the pulmonary artery, among others causes.

Pulmonary hypertension treatment is complex, controversial and potentially dangerous (Rubin L.J., Primary Pulmonary Hypertension, N. Engl. J.

Med. 336:111-7 (1997)). Patients often require referral to specialized health centers. Most often, pulmonary hypertension is treated with medications to lower the pressure in the lungs, making the heart work more efficiently. Vasodilators, or drugs that dilate blood vessels, are often used in managing secondary pulmonary hypertension.

For example, prostacyclins dilate the blood vessels in the lungs, and are commonly considered the most effective treatment for pulmonary hypertension. A popular synthetic version is given intravenously via continuous infusion that requires a semi-permanent central venous catheter. The delivery system, however, may cause sepsis and thrombosis. Additionally, the drug is unstable, and must therefore be kept on ice during its continuous administration, 24 hours a day, 7 days a week. Interruption can be fatal. Subcutaneously delivered prostacyclins have been developed, but they can be very painful.

Oral pills are also available, but drug stability is still an issue.

Further, all such forms of systemic treatment create a higher risks of side effects.

Delivery via inhalation may provided a more targeted approach. However, there is currently only one inhaled form of this class of drug approved for limited use in the

U.S. market, and it has a short half-life, requiring 6-9 daily administrations involving

10-15 minutes of inhalation at each administration.

Other current treatments include alternate vasodilators, anticoagulants, digoxin, diuretics, supplemental oxygen, and surgical procedures such as thromboendartectomies and lung transplants in select patients. Current therapeutic strategies, however, are rarely satisfactory. There is considerable room for improvement in the art of treating pulmonary hypertension, especially with respect to lowering dosage levels and targeting drug delivery so as to minimize the inconvenience, pain, and adverse side effects associated with current treatments.

BRIEF SUMMARY OF THE INVENTION The present invention provides methods of treating or preventing pulmonary hypertension in a subject, comprising providing a phosphodiesterase inhibitor and a calcium channel blocker to the subject by inhalation or intranasally. In certain embodiment, a composition comprising the phosphodiesterase inhibitor and the calcium channel blocker is provided to the subject. In other embodiments, a single dual pharmacophore molecule comprising the phosphodiesterase and the calcium channel blocker is provided to the subject. In other related embodiments, the phosphodiesterase inhibitor and the calcium channel blocker are provided separately. In one embodiment, the phosphodiesterase inhibitor and the calcium channel blocker are administered simultaneously, while in another embodiment they are administered sequentially in any order.

In another embodiment, the present invention provides a pharmaceutical composition comprising a calcium channel blocker and a phosphodiesterase inhibitor, wherein the composition is formulated for inhalation.

In a further embodiment, the present invention provides a drug delivery device adapted for nasal administration of a pharmaceutical composition, wherein the delivery device comprises a pharmaceutical composition comprising a calcium channel blocker and a phosphodiesterase inhibitor.

In various embodiments of the present invention, the calcium channel blocker is selected from the group consisting of: amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil, nisoldipine, nitrendipine, lacidipine, lercaninidipine, gallopimil, mibefradil, diltiazem, and isradipine.

In certain embodiments of the present invention, the phosphodiesterase inhibitor is selected from the group consisting of sildenafil, tadafil, inoximone, pimobendan, vardenafil, milrinone, and amrinone. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Figure 1 is a graph showing decrease of pulmonary vascular resistance in anesthetized dogs following administration of a dual pharmacophore comprising calcium channel blocker and phosphodiesterase inhibitor moieties.

DETAILED DESCRIPTION OF THE INVENTION

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

Definitions

"Alkyl" refers to a saturated straight or branched chain hydrocarbon radical. Examples include without limitation methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl.

"Alkylene" refers to a divalent alkyl radical.

"Alkylthio" refers to a sulfur substituted alkyl radical.

"Alkenyl" refers to an unsaturated straight or branched chain hydrocarbon radical comprising at least one carbon to carbon double bond. Examples include without limitation ethenyl, propenyl, iso-propenyl, butenyl, iso- butenyl, tert-butenyl, n-pentenyl and n-hexenyl.

"Alkenylene" refers to a divalent alkenyl radical.

"Alkyπyl" refers to an unsaturated straight or branched chain hydrocarbon radical comprising at least one carbon to carbon triple bond. Examples include without limitation ethynyl, propynyl, iso-propynyl, butynyl, iso- butynyl, tert-butynyl, pentynyl and hexynyl.

"Alkynylene" refers to a divalent alkynyl radical.

"Cycloalkyl" refers to a cyclic alkyl radical. Examples include without limitation cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

"Cycloalkenyl" refers to a cyclic alkenyl radical. Examples include without limitation cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

"Alkoxy" refers to an alkyl group bonded through an oxygen linkage. "Alkenoxy" refers to an alkenyl group bonded through an oxygen linkage.

"Substituted phenyl" refers to a phenyl that is substituted with one or more substituent(s). Examples of such substituents include without limitation Ci- C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Ci-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, tπfluoromethyl, iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, disilanyl, siloxy, silyl, silylene and carbocyclic and heterocyclic moieties.

"Aryl" refers to a cyclic aromatic hydrocarbon moiety having one or more closed ring(s). Examples include without limitation phenyl, benzyl, naphthyl, anthracenyl, phenanthracenyl and biphenyl.

"Heteroaryl" refers to a cyclic aromatic moiety having one or more closed rings with one or more heteroatom(s) (for example, sulfur, nitrogen or oxygen) in at least one ring. Examples include without limitation pyrryl, furanyl, thienyl, pyridinyl, oxazolyl, thiazolyl, benzofuranyl, benzothienyl, benzofuranyl and benzothienyl.

"Halo" refers to a fluoro, chloro, bromo or iodo radical.

"Isosteres" refer to elements, functional groups, substituents, molecules or ions having different molecular formulae but exhibiting similar or identical physical properties. For example, tetrazole is an isostere of carboxylic acid because it mimics the properties of carboxylic acid even though they have different molecular formulae. Typically, two isosteric molecules have similar or identical volumes and shapes. Ideally, isosteric molecules should be isomorphic and able to co-crystallize. Other physical properties that isosteric molecules usually share include boiling point, density, viscosity and thermal conductivity.

However, certain properties may be different: dipolar moments, polarity, polarization, size and shape since the external orbitals may be hybridized differently. The term "isosteres" encompasses "bioisosteres."

"Bioisosteres" are isosteres that, in addition to their physical similarities, share some common biological properties. Typically, bioisosteres interact with the same recognition site or produce broadly similar biological effects.

"Effective amount" refers to the amount required to produce a desired effect, for example: regulating calcium homeostasis; treating a disease, condition in which disregulation of calcium homeostasis is implicated; treating pulmonary hypertension, a cardiovascular disease, stroke, epilepsy, ophthalmic disorder or migraine; or inhibiting PDE (for example, PDE-3) or L-type calcium channels.

"Metabolite" refers to a substance produced by metabolism or by a metabolic process.

"Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ or portion of the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and suitable for use with the patient. Examples of materials that can serve as a pharmaceutically acceptable carrier include without limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

"Pharmaceutically acceptable equivalent" includes, without limitation, pharmaceutically acceptable salts, hydrates, solvates, metabolites, prodrugs and isosteres. Many pharmaceutically acceptable equivalents are expected to have the same or similar in vitro or in vivo activity as the compounds of the invention.

"Pharmaceutically acceptable salt" refers to an acid or base salt of the inventive compounds, which salt possesses the desired pharmacological activity and is neither biologically nor otherwise undesirable. The salt can be formed with acids that include without limitation acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexaπoate, hydrochloride hydrobromide, hydroiodide, 2- hydroxyethane-sulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate and undecanoate. Examples of a base salt include without limitation ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine. In some embodiments, the basic nitrogen-containing groups can be quartemized with agents including lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as phenethyl bromides.

"Prodrug" refers to a derivative of the inventive compounds that undergoes biotransformation, such as metabolism, before exhibiting its pharmacological effect(s). The prodrug is formulated with the objective(s) of improved chemical stability, improved patient acceptance and compliance, improved bioavailability, prolonged duration of action, improved organ selectivity, improved formulation (e.g., increased hydrosolubility), and/or decreased side effects (e.g., toxicity). The prodrug can be readily prepared from the inventive compounds using conventional methods, such as that described in BURGER'S MEDICINAL CHEMISTRY AND DRUG CHEMISTRY, Fifth Ed., Vol. 1 , pp. 172- 178, 949-982 (1995).

"Isomers" refer to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms.

"Stereoisomers" refer to isomers that differ only in the arrangement of the atoms in space.

"Diastereoisomers" refer to stereoisomers that are not mirror images of each other. Diastereoisomers occur in compounds having two or more asymmetric carbon atoms; thus, such compounds have 2ⁿ optical isomers, where n is the number of asymmetric carbon atoms. "Enantiomers" refers to stereoisomers that are non-superimposable mirror images of one another.

"Enantiomer-enriched" refers to a mixture in which one enantiomer predominates.

"Racemic" refers to a mixture containing equal parts of individual ^' enantiomers.

"Non-racemic" refers to a mixture containing unequal parts of individual enantiomers.

"Animal" refers to a living organism having sensation and the power of voluntary movement, and which requires for its existence oxygen and organic food. Examples include, without limitation, members of the human, equine, porcine, bovine, murine, canine and feline species. In the case of a human, an

"animal" may also be referred to as a "patient."

"Mammal" refers to a warm-blooded vertebrate animal.

"Calcium homeostasis" refers to the internal equilibrium of calcium in a cell. "Cardiovascular disease" refers to a disease of the heart, blood vessels or circulation.

"Heart failure" refers to the pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirements of the metabolizing tissues.

"Congestive heart failure" refers to heart failure that results in the development of congestion and edema in the metabolizing tissues.

"SA/AV node disturbance" refers to an abnormal or irregular conduction and/or rhythm associated with the sinoatrial (SA) node and/or the atrioventricular (AV) node.

"Arrhythmia" refers to abnormal heart rhythm. In arrhythmia, the heartbeats may be too slow, too fast, too irregular or too early. Examples of arrhythmia include, without limitation, bradycardia, fibrillation (atrial or ventricular) and premature contraction. "Hypertrophic subaortic stenosis" refers to enlargement of the heart muscle due to pressure overload in the left ventricle resulting from partial blockage of the aorta.

"Angina" refers to chest pain associated with partial or complete occlusion of one or more coronary arteries in the heart. "Treating" refers to: (i) preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting a disease, disorder or condition, i.e., arresting its development; and/or (iii) relieving a disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.

Unless the context clearly dictates otherwise, the definitions of singular terms may be extrapolated to apply to their plural counterparts as they appear in the application; likewise, the definitions of plural terms may be extrapolated to apply to their singular counterparts as they appear in the application. A. Methods of Treating or Preventing Pulmonary Hypertension and Other Diseases Using a Combination of a Calcium Channel Blocker and a Phosphodiesterase Inhibitor

The present invention is based on the discovery that treatment with a combination of a calcium channel blocker and a phosphodiesterase inhibitor reduces both pulmonary vascular resistance and pulmonary wedge pressure and is, therefore, useful in the treatment or prevention of pulmonary hypertension.

Furthermore, the methods and pharmaceutical compositions of the present invention may be administered topically, e.g., by inhalation, to the lungs, resulting in specificity for the pulmonary vasculature and conferring little or no systemic side effects.

Without wishing to be bound by any theory, it is considered that the combination of the two pharmacological effects (PDE inhibition and calcium channel blockade) creates an additive or synergistic effect on pulmonary vascular resistance that is greater than either effect on its own, allowing the administration of lower dose levels and minimizing adverse systemic effects.

Phosphodiesterase inhibition decreases pulmonary vascular resistance by elevating cytosolic cAMP/cGMP, resulting in vasodilation. Similarly, calcium channel blockade results in vasodilation and lowered pulmonary vascular resistance, albeit through a divergent mechanism involving inhibition of L-type calcium channels on vascular smooth muscle cells, resulting in reduction of intracellular calcium levels and ensuing smooth muscle relaxation.

Approaches to pulmonary hypertension therapy are commonly based on reducing pulmonary vascular resistance. Generally, such treatments are often directed towards vasodilation of the blood vessel smooth muscle cells via the individual and separate use of either phosphodiesterase (PDE) inhibitors or calcium channel blockers, among other agents. Phosphodiesterase inhibitors, e.g., sildenafil (a PDE5 inhibitor), and calcium channel blockers have been used independently via oral administration to treat pulmonary hypertension. However, calcium channel blockers require high dose levels, are only effective in approximately 20-25% of patients with pulmonary hypertension, and have adverse effects on systemic vascular resistance. Similarly, sildenafil requires high dose levels, and, although it demonstrates some selectivity for pulmonary vasculature, it is not devoid of unwanted systemic effects.

According to the present invention, the combination of the pharmacological effects of phosphodiesterase inhibition and calcium channel blockade results in an additive or synergistic effect on pulmonary vascular resistance that is greater than either effect alone. Moreover, topical administration affords specificity for pulmonary vasculature, while reducing untoward systemic effects. In light of this discovery, the present invention provides novel methods of treating or preventing pulmonary hypertension, which methods provide increased efficacy and reduced adverse systemic effects as compared to previous methods. In general, these methods comprise providing to a subject diagnosed with or at risk for developing pulmonary hypertension a combination of a calcium channel blocker and a phosphodiesterase (PDE) inhibitor. In various embodiments, PDE inhibitor and calcium channel blocker compounds are administered simultaneously as separate formulations, sequentially and in any order as separate formulations, simultaneously in a single formulation or as a combined admixture, or simultaneously as a single dual pharmacophore molecule. Of course, it is understood that the methods may further include providing two or more of either or both of a calcium channel blocker and/or PDE inhibitor.

Similarly, the present invention includes a method of reducing or preventing pulmonary vascular resistance, comprising providing to a subject diagnosed with or at risk for pulmonary hypertension or pulmonary vascular resistance, a combination of a PDE inhibitor and a calcium channel blocker. In addition, the present invention includes a method of treating or preventing heart failure, e.g., congestive heart failure, comprising providing to a subject diagnosed with or at risk for heart failure, a combination of a PDE inhibitor and a calcium channel blocker. The methods of the present invention may be used in a variety of applications, including both human and veterinary treatment. Thus, a subject may be a human or other animal, and in particular embodiments, a mammal.

In particular embodiments, the PDE inhibitor and calcium channel blocker are provided topically to the pulmonary vasculature, e.g., by nasal or oral delivery or inhalation or, more generally, via delivery to the lungs.

Examples of different classes of pulmonary hypertension that may be treated or prevented include, but are not limited to, pulmonary arterial hypertension, pulmonary venuous hypertension, pulmonary hypertension associated with disorders of the respiratory system and/or hypoxemia, pulmonary hypertension due to chronic thrombotic and/or embolic disease, and pulmonary hypertension due to disorders directly affecting the pulmonary vasculature.

Pulmonary arterial hypertension includes, e.g., primary pulmonary hypertension and secondary pulmonary hypertension related to another disease or condition, such as e.g., collagen vascular disease, congenital systemic to pulmonary shunts, portal hypertension, HIV infection, pregnancy, persistent pulmonary hypertension of the newborn, or drugs and toxins.

Pulmonary venuous hypertension includes or is related to, e.g., left- sided atrial or ventricular heart disease, left-sided valvular heart disease, extrinsic compression of the central pulmonary veins, and pulmonary veno-occlusive disease.

Pulmonary hypertension associated with disorders of the respiratory system and/or hypoxemia is caused primarily by diseases that effect the structure and function of the lungs, rather than heart or other organs. These include, e.g., chronic obstructive pulmonary disease, interstitial lung disease, sleep disordered breathing, alveolar hyperventilation disorders, chronic exposure to high altitude, neonatal lung disease, and alveolar capillary dysplasia.

Pulmonary hypertension due to chronic thrombotic and/or embolic disease is caused by blockages in the pulmonary blood vessels, such as, e.g., thromboembolic obstruction of proximal pulmonary artiers and obstruction of distal pulmonary arteries. Pulmonary hypertension due to disorders directly affecting the pulmonary vasculature results from diseases and disorders that directly affect the pulmonary blood vessels, including, e.g., inflammatory diseases such as schistosomiasis and sarcoidosis, and pulmonary capillary haemangiomatosis. Pulmonary hypertension is further associated with a number of cardiovascular diseases, lupus, and sclerodoma.

Combinations of the present invention may be administered according to any means known to an ordinarily skilled practitioner, and may be determined according to routine factors known in the arts. For example, embodiments of the present invention may be administered orally, parenterally (including subcutaneous, intramuscular and intravenous), rectally, buccally

(including sublingual), transdermal^, or topically (e.g., intranasally, by inhalation).

In particular embodiments of the invention, an admixture or an intravenous admixture comprising a phosphodiesterase inhibitor and a calcium channel blocker is provided topically, e.g., by inhalation, orally, or intravenously. In other particular embodiments of the invention, administration a single dual pharmacophore molecule comprising a phosphodiesterase inhibitor moiety and a calcium channel blocker moiety, both of which are connected by a variable linker, is provided topically, e.g., by inhalation, orally, or intravenously.

In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration. As such, the compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

Dosages and routes of administration are readily determined by a physician, based upon the particular subject being treated and knowledge available in the art regarding appropriate dosages of each of the PDEi and calcium channel blockers. B. Compositions Comprising a Calcium Channel Blocker and a Phosphodiesterase Inhibitor

The methods and compositions of the present invention may be practiced using any calcium channel blocker and any phosphodiesterase inhibitor. Accordingly, in certain embodiments, the present invention includes compositions comprising one or more calcium channel blockers and one or more phosphodiesterase inhibitors. In particular embodiments, only one phosphodiesterase inhibitor and one calcium channel blocker is utilized, although the present invention contemplates that use of combinations comprising more than one of either or both of a PDE inhibitor and/or a calcium channel blocker. A variety of each of these types of compounds are known and available in the art, and any may be used according to the present invention.

1. Calcium Channel Blockers

Calcium channel blockers typically affect L-type voltage-sensitive calcium channels in the cellular plasma membrane, blocking the movement of calcium into cells of the heart and the blood vessels. As a result, intracellular calcium levels remain low upon stimulation, thereby relaxing vascular smooth muscle cells, dilating the blood vessels, and decreasing total peripheral resistance. The heart remains responsive to sympathetic nervous system stimulation, allowing more effective maintenance of lowered blood pressure. Calcium channel blocking agents have been used in subjects with hypertension, including subjects with pulmonary hypertension.

In various embodiments, calcium channel blockers are represented by the five structurally divergent families, including the benzothiazepines, bihydropyridines, phenylalkylamines, diarylaminopropylamine ethers, benzimidazole-substituted tetralines. Individual representative examples of calcium channel blockers include, but are not limited to, amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil, nisoldipine, nitrendipine, lacidipine, lercaninidipine, gallopimil, mibefradil, diltiazem, isradipine, or combinations thereof. 2. Phosphodiesterase Inhibitors

Phosphodiesterase (PDE) inhibitors inhibit one or more of the PDE enzyme subtypes, including PDEi-PDEn. Members of the PDE family target a variety of biochemical pathways, and vary with respect to their tissue, cellular and subcellular distribution. In particular, some PDE enzymes degrade the cyclic nucleotides cAMP and cGMP, which are common regulators of ion channel conductance and play a role in relaxing smooth muscles. PDE inhibitors prevent cGMP/cAMP degradation, encouraging relaxation of vascular smooth muscles in blood vessels, and leading to vasodilation and increased blood flow. PDE inhibitor-mediated vasodilation decreases pulmonary vascular resistance in subjects with hypertension-related problems, including subjects with pulmonary hypertension in particular.

PDE inhibitors are commonly categorized into specific subtypes, or families (see, W. J. Thompson, Pharmac. Ther. (1991) 51: 13-33; and J. P. Hall, Br J. CHn. Pharmac. (1993) 35: 1-7). For example, embodiments of the present invention include, but are not limited to, the families of PDEi inhibitors (e.g., Ca^/Calmodulin-activatable), PDE₂ inhibitors (e.g., cGMP activatable), PDE₃ inhibitors (e.g., cGMP inhibitable), PDE₄ inhibitors (e.g., cAMP-specific), and PDE₅ inhibitors (e.g., cGMP-specific). In particular embodiments of methods of the present invention, the PDE inhibitor component is either specific for a particular PDE, or possesses combined inhibition (e.g., PDE₃/PDE5 or PDE₃/PDE4 inhibition).

Examples of PDEi inhibitors include, but are not limited to, calmodulin antagonists (e.g., phenothiazines, W-7, CGS 9343B), vinpocetine (TCV-3B), HA-558, 8-methoxymethyl-3-isobutyl-1-methylxanthine, KW-6 (isoquinoline derivative, 8-methylamino-3-isobutyl-1-methylxantine (MIMAX), and dibenzoquinazoline diones (dihydroisoquinoline derivative), or combinations thereof.

Examples of PDE₂ inhibitors include, but are not limited to, trequinsin, oxindole (2), and erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), and ND7001(Neuro3D), or combinations thereof. Examples of PDE₃ inhibitors include, but are not limited to, milrinone, amrinone, motapizone, cilostazol, indolidan, cilostamide, lixazinone, Y-590, imazodan, SKF 94120, quazinone, IC 153, 110, cilostazol, bemorandan, siguazodan, adibendan, olprinone, enoximone, pimobendan, saterinone, and sulmazole, or combinations thereof.

PDE₄ inhibitor embodiments include, but are not limited to, those that fall generally into one of six chemical structural classes: rolipram-like, xanthines, nitraquazones, benzofurans, naphthalenes, and quinolines. Representative examples of rolipram-like analogs include imidazolidinones and pyrrolizidinone mimetics of rolipram and Ro 20-1724, as well as benzamide derivatives of rolipram such as RP 73401 (Rhone-Poulenc Rorer). Representative examples of xanthine analogs include denbufylline and arofylline; nitraquazone analogs include CP- 77,059 (Pfizer) and a series of pyrid[2,3d]pyridazin-5-ones; benzofuran analogs include BAY 19-8004 and EP-685479 (Bayer); napthalene analogs include T-440 (Tanabe Seiyaku); and quinoline analogs include SDZ-ISQ-844 (Novartis). Examples of PDE₄ inhibitors may also include cilomilast, roflumilast, and pumafentrine, cipamfylline, atizoram, darbufelone, and arofylline, or combinations thereof.

Representative examples of PDE₅ inhibitors include, but are not limited to, sildenafil, tadafil, zaprinast, vardenafil, dipyridamole, pyrazolopyrimidinones, griseolic acid derivatives, 4-aryl-1-isoquinolinone derivatives, 1 ,7- and 2,7-naphthyridine derivatives, 2-phenylpurinones, phenylpyridone derivatives, pyrimidiπes, pyrimidopyrimidines, purines, quinazolines, phenylpyrimidinones, imidazoquinoxalinones or aza analogues thereof, phenylpyridones, 4-bromo-5-(pyridylmethylamino)-6-[3-(4- chlorophenyl)propoxy]-3(2H)pyridazinone, 1-[4-[(1 ,3-benzodioxol-5- ylmethyl)amiono]-6-chloro-2-quinazolinyl]-4-piper idine-carboxylic acid, monosodium salt, (+)-cis-5,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)- phenylmethyl-S-meth yl-cydopenM.δlimidazo^.i-blpurin^SHJone. furazlocillin, cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]imidazo[2,1- b]purin-4-one, 3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate, 4-bromo- 5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl)propoxy)-3-(2H)pyridazinone, 1- methyl-5-(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1 ,6-dihydro-7 H- pyrazolo(4,3-d)pyrimidin-7-one, and 1-[4[(1 ,3-benzodioxol-5-ylmethyl)amino]-6- chloro-2-quinazolinyl]4-piperidi necarboxylic acid, MBCQ, MY-5445, or combinations thereof.

Examples of combined PDE3/PDE4 inhibitors include, but are not limited to, theophylline, isobutylmethylxanthine, AH-21-132, Org-30029 (Organon), Org-20241 (Organon), Org-9731 (Organon), zardaverine, vinpocetine, EHNA (MEP-1), milrinone, siguazodan, zaprinast, SK+F 96231 , tolafentrine, filaminast, roflumilast, pumafentrine, cipamfylline, atizoram, darbufelone, and arofylline or combinations thereof.

3. Dual Pharmacophores

Compounds comprising both a phosphodiesterase inhibitor moiety and a calcium channel blocker moiety may be used according to the methods and compositions of the present invention, including, e.g., compounds possessing inhibitory activity against PDE-3 and L-type calcium channels, of the general formula

Y-L-X wherein: Y is a dihydropyridine L-type calcium channel blocker moiety;

L is a linking group; and X is a PDE-3 inhibitory moiety.

Examples of dihydropyridine L-type calcium channel blockers include without limitation:

Nifedipine

Nitrendipine

Nimodipine

Amlodipine

One embodiment of the present invention encompasses a compound of formula

or a pharmaceutically acceptable equivalent, an isomer or a mixture of isomers thereof, wherein:

R¹ and R⁴ are independently hydrogen, halo, nitro, cyano, trifluoromethyl, amino, -NR⁵R⁶, Ci-C₄ alkoxy, C₁-C₄ alkylthio, Ci-C₈ alkyl, C₂-C₈ alkenyl or C₂-C₈ alkynyl, wherein one or more — CH₂— group(s) of the alkyl, alkenyl or alkynyl is/are optionally replaced with -O— , -S-, — SO2— and/or -NR⁵-, and the alkyl, alkenyl or alkynyl is optionally substituted with one or more carbonyl oxygen(s) and/or hydroxyl(s); R⁵ and R⁶ are independently hydrogen, Ci-C₈ alkyl, C₂-C₈ alkenyl or C₂-Ce alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with phenyl or substituted phenyl;

R² and R³ are independently -COOR⁷, nitro, cyano ortrifluoromethyl;

R⁷ is Ci-Ce alkyl, C₂-C₈ alkenyl or C₂-C₈ alkynyl, wherein the alkyl, alkenyl or alkynyl is optionally substituted with Ci-C₄ alkoxy Or -NR⁵R⁶;

L is a direct bond, C1-C1₂ alkylene, C₂-Ci₂ alkenylene or C₂-Ci₂ alkynylene, wherein one or more -CH₂- group(s) of the alkylene, alkenylene or alkynylene is/are optionally replaced with -O- -S-, -SO₂- and/or -NR⁵-, and the alkylene, alkenylene or alkynylene is optionally substituted with one or more carbonyl oxygen(s) and/or hydroxyl(s); and

X is a moiety of formula A, B, C, D, E, F₁ G₁ H, I₁ J, K, L, M. N₁ O₁ P or Q

with X connected to L through any one R; and each R is independently a direct bond, hydrogen, halo, nitro, cyano, trifluoromethyl, amino, -NR⁵R⁶, Ci-C₄ alkoxy, C₁-C₄ alkylthio, -COOR⁷, Ci-Ci₂ alkyl C₂-C₁₂ alkenyl or C₂-C12 alkynyl, wherein one or more — CH₂— group(s) of the alkyl, alkenyl or alkynyl is/are optionally replaced with — O— , -S-, — SO2— and/or — NR⁵-, and the alkyl, alkenyl or alkynyl is optionally substituted with one or more carbonyl oxygen(s) and/or hydroxyl(s).

In one embodiment of formula I, when X is a moiety of formula A and L is a direct bond, then L is connected to the phenyl ring of A.

In another embodiment of formula, I, R¹ and R⁴ are each Ci-C₄ alkyl, R² and R³ are each -COOR⁷, L is a direct bond, and X is a moiety of formula A or P.

Examples of compounds of formula I include without limitation:

ethyl 4-(5-cyano-2-methyl-6-oxo(3-hydropyridyl))-5-(ethoxycarbonyl)-2,6- dimethyl-1 ,4-dihydropyridine-3-carboxylate (Compound 1),

ethyl 4-(5-amino-6-oxo(3-hydropyridyl))-5-(ethoxycarbonyl)-2,6- dimethyl-1 ,4-dihydropyridine-3-carboxylate (Compound 2),

ethyl 5-(ethoxyoaφonyl)-2,6-dimethyl-4-(2-oxo(6-hydroquinolyl))-1 ,4- dihydropyridine-3-carboxylate (Compound 3), and

ethyl 4(3-cyano-2-o>xo(6-hydroquinolyl))-5-(ethoxycarbonyl)-2,6- dimethyl-1 ,4-dihydrg>pyridine-3-carboxylate (Compound 4).

Another embodiment of the present invention encompasses a compound of formula Il

R², R³, R⁴, L and X are as defined above; and Ar is an aryl or heteroaryl that is optionally substituted in 1 to 3 position(s) with hailo, nitro, cyano, trifluoromethyl, amino, -NR⁵R⁶, Ci-C₄ alkoxy, Ci-C₄ alkylthio, -COQR⁷, Ci-C₈ alkyl, C₂-C₈ alkenyl or C₂-C₈ alkynyl, wherein one or more -CH₂- grouφ(s) of the alkyl, alkenyl or alkynyl is/are optionally replaced with — O-, -S-, — SO₂- and/or -NR⁵-, and the alkyl, alkenyl or alkynyl is optionally substituted with one or more carbonyl oxygen(s) and/or hydroxyl(s). In one embodiment of formula II, when R² is — COOCH2CH₃, R³ is cyano, R⁴ is methyl, L is methylene, X is a moiety of formula A₁ each R is hydrogen, and Ar is trifluoromethylphenyl, then L is not connected to the nitrogen atom of A; when R² and R³ are each cyano, R⁴ is amino, L is -SCH₂-, X is a moiety of formula P, and each R is hydrogen, then Ar is not fluorophenyl; and when R² is -COOCH₂CH₃, R³ is -COOCH₃, R⁴ is methyl, X is a moiety of formula P, each R is hydrogen, and Ar is chlorophenyl, then L is not -CH₂OCH₂CH₂-, -CH₂OCH₂CH₂NHCO- or -CH₂OCH₂CH₂NCH₃CO-.

In another embodiment of formula II, R² and R³ are each -COOR⁷, R⁴ is C₁-C4 alkyl, X is a moiety of formula A, and Ar is phenyl that is optionally substituted in 1 to 3 position(s).

Examples of compounds of formula Il include without limitation:

methyl 4-(2-chlorophenyl)-5-(ethoxycarbonyl)-2-methyl-6-({2-[4-(2-oxo(6- hydroquinolyloxy))butanoylamino]ethoxy}methyl)-1 ,4-dihydropyridine-3-carboxylate (Compound 5),

methyl 5-(methoxycarbonyl)-2-methyl-4-(2-nitrophenyl)-6-({2-[4-(2-oxo(6- hydroquinolyloxy^butaπoylaminoJethoxy^ethyO-IΛ-clihydropyridine-S-carboxylate (Compound 6),

methyl 4-(2-chlorophenyl)-5-(methoxycarbonyl)-2-methyl-6-{[3-(2-oxo(6- hydroquinolyloxy))propoxy]methyl}-1,4-dihydropyridine-3-carboxylate (Compound 7).

methyl 6-[(2-{2-[2-chloro-4-(6-oxo(1 ,4,5-trihydropyridazin-3- yl))phenoxy]acety!|amino}ethoxy)methyl]-4-(2-chlorophenyl)-5-(methoxycarbonyl)- 2-methyl-1 ,4-dihydropyridine-3-carboxylate (Compound 8),

πriethyl 6-[(2-{2-[2-chloro-4-(6-oxo(1 AS-trihydropyridazin-S- yl))phenoxy]acetylamino}ethoxy)methyl]-4-(2-chlorophenyl)-5-(ethoxycarbonyl)-2- methyl-1 ,4-dihydropyridine-3-carboxylate (Compound 9),

methyl 4-(2-chlorophenyl)-5-(methoxycarbonyl)-2-methyl-6-({2-[4-(2-oxo(6- hydroquinolyloxy))butanoylamiπo]ethoxy}methyl)-1 ,4-dihydropyridine-3-carboxylate (Compound 1O)₁

methyl 4-(2-chlorophenyl)-5-(ethoxycarbonyl)-2-methyl-6-({2-[2-(2-oxo(6- hydroquinolyloxy))acetylamino]ethoxy}methyl)-1 ,4-dihydropyridine-3-carboxylate (Compound 11),

methyl 4-(2-chlorophenyl)-5-(methoxycarbonyl)-2-methyl-6-({2-[2-(2-oxo(6- hydroquinolyloxyJJa.cetylaminolethoxy^ethyO-i ^-dihydropyridine-S-carboxylate (Compound 12), and

methyl 4-(2-chIorophenyl)-6-[(2-{2-[4-(5-cyano-2-methyl-6-oxo(3- hydropyridyl))phenoxy]acetylamino}ethoxy)methyl]-5-(ethoxycarbonyl)-2-methyl- 1 ,4-dihydropyridine-3-carboxylate (Compound 13).

Another embodiment of the present invention encompasses a compound of formula III

R¹, R³, R⁴, L, X and Ar are as defined above. In one embodiment of formula III, when R¹ and R⁴ are each methyl,

R³ is -COOCH₃, aad X is a moiety of formula A or O, then L is not alkyl substituted with —COO- connected directly to the pyridine ring.

In another embodiment of formula III, R¹ and R⁴ are each Ci-C₄ alkyl, R³ is -COOR⁷, X is a moiety of formula E, and Ar is phenyl that is optionally substituted in 1 to 3 position(s).

Examples of compounds of formula III include without limitation:

2-(2-oxo-4,3a-dihydFθimidazolidino[2,1-b]quinazolin-6-yloxy)ethyl 5- (methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1 ,4-dihydropyridine-3- carboxylate (Compound 14), and

methyl 4-(2-chlorophenyl)-2,6-dimethyl-5-[N-(2-{2-[4-(6-oxo(1 , 4, 5-trihydropyridazin- S-yOJphenoxylacetylaminolethyOcarbamoylJ-i ^-dihydropyridine-S-carboxylate (Compound 15).

Every variable substituent is defined independently at each occurrence. Thus, the definition of a variable substituent in one part of a formula is independent of its definition(s) elsewhere in that formula and of its defιnition(s) in other formulas.

Since the inventive compounds may possess one or more asymmetric carbon center(s), they may be capable of existing in the form of optical isomers as well as in the form of racemic or non-racemic mixtures of optical isomers. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes. One such process entails formation of diastereoisomeric salts by treatment with an optically active acid or base, then separation of the mixture of diastereoisomers by crystallization, followed by liberation of the optically active bases from the salts. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid.

A different process for separating optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers. Still another available process involves synthesis of covalent diastereoisomeric molecules, for example, esters, amides, acetals and ketals, by reacting the inventive compounds with an optically active acid in an activated form, an optically active diol or an optically active isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure compound. In some cases hydrolysis to the "parent" optically active drug is not necessary prior to dosing the patient, since the compound can behave as a prodrug. The optically active compounds of this invention likewise can be obtained by utilizing optically active starting materials. The compounds of this invention encompass individual optical isomers as well as racemic and non-racemic mixtures. In some non-racemic mixtures, the R configuration may be enriched while in other non-racemic mixtures, the S configuration may be enriched.

Methods of producing these compounds are described in U.S. Patent Application Nos. 11/499.377 and 10/679,303 (U.S. Patent No. 7,098,211), the entire contents of which are herein incorporated by reference.

4. Pharmaceutical Compositions and Formulations

Particular embodiments of the present invention include formulations, or admixtures, comprising one or more PDE inhibitors (PDEi) and one or more calcium channel blockers (CCB), and possibly a pharmaceutically acceptable carrier. Formulations, or admixtures, of the present invention typically comprise an amount of each component that is sufficient to achieve a therapeutic or prophylactic effect upon administration to a subject at a prescribed dosage. Formulations, or admixtures, may be in any form, e.g., solid, liquid, gaseous, aerosolized, etc. The PDEi and CCB components in an admixture may comprise any

PDE inhibitor or CCB compound or molecule known by the skilled artisan, including those disclosed herein, and may be used to treat or prevent any form of cardiovascular disease, including heart failure and the various forms of hypertension generally, including pulmonary hypertension, particularly. The effective amount of each PDEi or CCB component in an admixture can vary with respect to the other component, depending on the effective amount of any given component, and/or the additive or synergistic effects resulting from the combination of the two components. Thus, each separate PDEi and CCB component can be present in the admixture at an effective ratio with respect to the other component. A skilled artisan would understand that the effective amount of each component can be calculated by such variables as mass, mass per volume, molarity, weight percentage, or the like, and the corresponding effective ratio can be determined therefrom.

For example, each separate active component can be present in the admixture at an effective molar ratio with respect to the other. The effective molar ratio may be based on the actual effective amount of each component, e.g., (XmM PDEi) or (XmM CCB), wherein XmM reflects for example the molar concentration of the given component, and may be represented by the formulas (XmM PDEi/XmM CCB) or (XmM CCB/XmM PDEi). In particular embodiments, the effective molar ratio with respect to the above-recited formulas is recited in the following non-limiting examples: (0.001 :1), (0.005:1), (0.01 :1), (0.02:1), (0.04:1), (0.06:1), (0.08:1), (0.1 :1), (0.2:1), (0.3/1), (0.4:1), (0.5:1), (0.6:1), (0.7:1), (0.8:1), (0.9:1), (1:1), (1.1 :1), (1.2:1), (1.3:1), (1.4:1), (1.5:1), (1.6:1), (1.7:1), (1.8:1), (1.9:1), (2:1 ), (3:1), (4:1), (5:1), (6:1), (8:1), (10:1), (15:1), (20:1), (30:1), (40:1), (50:1). (60:1), (70:1), (80:1), (90:1), (100:1), (500:1), (1000:1), and the like. There are no limitations with respect to the effective ratio or combination of components in an admixture, except regarding therapeutic efficacy.

Pharmaceutical or physiologically acceptable embodiments for use in accordance with the present invention may be formulated in any manner known to the ordinarily skilled artisan, and may comprise one or more PDE inhibitor (PDEi) components, one or more calcium channel blocker (CCB) components, and a carrier. For example, the PDEi and CCB components may be formulated as separate compounds, as a combined admixture, or as a single dual pharmacophore molecule. In particular embodiments, the PDE inhibitor may be varied, e.g., using representative examples described herein, or it may be specific to a particular PDE {e.g., PDE₁, PDE₂, PDE₃, PDE₄, or PDE₅), or it may have mixed inhibition (e.g., PDE₃/PDE₅, PDE3/PDE4), or any combination thereof. The calcium channel blocker component can also be varied, e.g., using representative examples described herein, e.g., amlopidine, verapimil, nifedipine, diltiazem, etc., or it may be a combination thereof. With respect to the dual pharmacophore embodiments, the linker between the PDE inhibitor and calcium channel blocker components can also be varied to alter physiochemical properties, e.g., stability, solubility, size, etc. In particular embodiments, pharmaceutical compositions of the present invention are formulated so as to provide extended release or enhanced stability of one or more compounds. For example, compositions may be formulation in lipid emulsions, liposomes, or polymeric matrixes.

Typically, embodiments of the present invention are formulated so as to comprise an amount of each component that is sufficient to achieve a therapeutic or prophylactic effect upon administration to a subject at a prescribed dosage.

The actual effective amount of each component will depend upon the particular PDEi or CCB component chosen, as each individual drug within the family of PDEi and CCB components has its own prescribed effective dosage range known to those skilled in the art. The actual effective amount will also depend on the condition being treated, the route of administration, the drug treatment used to treat the condition, the medical history of the patient, and the additive or synergistic effects resulting from the combination of the two components. Determination of the effective amount is well within the capabilities of those skilled in the art. As a starting point, the effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating concentrations that have been found to be effective in animals.

In particular embodiments, an effective amount of an active component, e.g. , PDEi or CCB or dual pharmacophore, is from about 0.0001 mg to about 500 mg active agent per kilogram body weight of a patient, from about 0.001 to about 250 mg active agent per kilogram body weight of the patient, from about 0.01 mg to about 100 mg active agent per kilogram body weight of the patient, from about 0.5 mg to about 50 mg active agent per kilogram body weight of the patient, or from about 1 mg to about 15 mg active agent per kilogram body weight of the patient. In terms of weight percentage, in particular embodiment, a pharmaceutical formulation of an active agent comprises an amount from about 0.0001 wt. % to about 10 wt. %, from about 0.001 wt. % to about 1 wt. %, or from about 0.01 wt. % to about 0.5 wt. %. Embodiments of the present invention may be administered by any route known to an ordinarily skilled artisan. The selection of the most appropriate delivery route will vary, and may be influenced by the pharmacological properties of the selected calcium channel blocker and phosphodiesterase inhibitor, the nature and severity of the condition being treated, and the physical condition of the recipient. In certain embodiments, the pharmaceutical compositions of the present invention comprise one or more excipients and may be in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, e.g., Mathiowitz et ai. Nature 1997 Mar 27;386(6623):410-4; Hwang et ai, Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. In certain circumstances it is desirable to deliver the pharmaceutical compositions or admixtures disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneal^. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U. S. Patent 5,543,158, U. S. Patent 5,641 ,515 and U. S. Patent 5,399,363. In certain embodiments, solutions of the active compounds as free-base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirπerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

In another embodiment of the invention, the compositions disclosed herein are formulated in a neutral or salt form. Illustrative pharmaceutically- acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

The carriers may further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

In certain embodiments, the formulation may be delivered topically, e.g., by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering formulations topically to the lungs via nasal aerosol sprays have been described, e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212, the entire contents of which are herein incorporated by reference. Methods for delivering both dry and liquid aerosolized formulations topically to the lungs via oral and other inhalation devices have also been described, e.g., in U.S. Patent 5,775,320, U.S. Patent 7,059,321 , U.S. Patent 5,404,871, the entire contents of which are herein incorporated by reference. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga ef a/., J Controlled Release 1998 Mar 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds, e.g., in U. S. Patent 5,725,871 , are also well-known in the pharmaceutical arts.

For topically administered embodiments directed towards transmucosal delivery, appropriate penetrants (e.g. , surfactant acids) may also be included to enhance absorption across the mucous membrane. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045, the entire contents of which are herein incorporated by reference. Other materials, such as preservatives, salts to achieve isotonicity, buffers, and the like, may be added to intranasal and inhalation formulations. The present invention further includes medical devices useful for practicing the methods of the present invention, including, e.g., inhalation delivery devices, that comprises both a PDE inhibitor and a calcium channel blocker (or a dual pharmacophore). These may be adapted for either nasal or oral inhalation and include, e.g., nebulizers.

EXAMPLES

EXAMPLE 1 PULMONARY EFFECTS IN ANESTHETIZED DOGS

Studies were performed to demonstrate the pulmonary effects of dual pharmacophores comprising calcium channel blocker and phosphodiesterase inhibitor moieties in anesthetized dogs. The compounds used were Compound 8 and the S enatiomer of Compound 8.

Six male beagle dogs were anesthetized, intravenously (iv), with morphine sulfate (1.5 mg/kg) and 100 mg/kg alpha chloralose + 60 mg/kg/hour, and instrumented to measure a variety of cardiac parameters, including pulmonary vascular resistance. Measurements were made during a baseline period, after a 10 min loading dose, and after a 20 min maintenance dose for vehicle and four escalating iv doses of test article (total of 150 min). Measurements were made over the last min of each loading and maintenance dose with cardiac outputs measured after the acquisition system was marked. The test articles were Compound 8 and the S enantiomer of Compound 8, given in doses 0.2, 0.6, 2.0, and 6.0 mg/kg over 10 minutes (loading dose), and then 0.01% of that dose (maintenance dose) over the next 20 minutes. Pulmonary vascular resistance (PVR) was measured as: PVR = (PAP - RAPyCO, where

PAP is pulmonary trunk mean pressure (mm.Hg); RAP is right atrium mean pressure (mmHg), and CO is cardiac output (l/min).

PVR did not change after the 0.2 mg/kg dose of Compound 8 when compared to vehicle. PVR appeared to decrease with each additional escalating dose of Compound 8. PVR decreased slightly after dosing with 0.2 mg/kg of the S-enantiomer of Compound 8 when compared to vehicle. PVR remained decreased through the 2.0 mg/kg dose of the S-enantiomer of Compound 8 before increasing after the 6.0 mg/kg dose. In addition, Compound 8 reduced vascular resistance and pulmonary wedge pressure. These results are of particular relevance to pulmonary hypertension and demonstrate that the compounds of the present invention are useful in the treatment of pulmonary hypertension.

EXAMPLE 2 EFFECTS IN ACUTE RABBIT MODEL OF PULMONARY ARTERIAL HYPERTENSION

The ability of the methods and compositions of the present invention to reduce pulmonary arterial hypertension is confirmed using an acute rabbit model of the disease. These studies confirm the vasodilatory properties of combination treatment with a calcium channel blocker and a phosphodiesterase inhibitor in the pulmonary circulation following both intravenous and inhalation delivery.

Anesthesia is induced in rabbits with ketamine (35 mg/kg) and xylazine (5 mg/kg), via an intramuscular injection into Biceps Femoris. Anesthesia is monitored by the absence of a pedal reflex. In addition, a pulse oximeter is used to monitor oxygen level in the blood. The animals are shaved at the neck and leg area (groin), and ECG leads (Lead II) are attached to assure sinus rhythm and normal heart rate. Tracheotomy is performed on the anesthetized animal. The animals are ventilated with 95% O₂/5% CO₂ (tidal volume 5-7 ml/kg; frequency 45 /min) + isoflurane (maintained at 1.0 to 1.25% at 1.5 L/min). Alternatively, rabbits are initially anesthetized with a mixture of ketamine (7 mg/kg) and xylazine (2.1 mg/kg), followed by a constant intravenous infusion of ketamine (80 mg/kg/h) and xylazine (25 mg/kg/h).

A 4 F catheter is inserted into the pulmonary artery via the right external jugular vein. A direct cut down to the left femoral artery allows for blood pressure monitoring, and a 3.0 Fr Millar catheter is inserted into the left carotid artery for left ventricular pressure monitoring. A 3.0 Fr catheter is inserted into the left external jugular vein for i.v. drug administration (10 μg/kg/min - 1000 μg/kg/min for 10 min). The cannulae is flushed with heparin sodium (1000 U/ml). In some cases, the test compounds are inhaled from a nebulizer via the inspiratory limb of the ventilation system (0.25-1.0 mg/ml given at a rate of 0.25-0.3 ml/min for 10 min). The animals are covered with a sheet and monitored for 30 minutes before test compound infusion or inhalation. Body temperature is maintained at 37⁰C with a heating pad and monitored using a rectal thermometer.

In some animals, U46619, a thromboxane mimetic, is continually infused (0.5-2 μg/kg) to increase the pulmonary artery pressure from ~13 to ~26 mm Hg within 20 minutes. Hemodynamic parameters are monitored during the infusion or inhalation of ascending doses of reference and test compounds. Blood samples are drawn from the left femoral artery during control conditions and at the end of each dose (6X1 ml_). Dose response curves are developed over 2-3 hours, after which the animals are euthanized by an intravenous injection of sodium pentobarbital (150 mg/kg i.v.).

Delivery of the combinations and compositions of the present invention will reduce pulmonary artery pressure. In addition, topical (e.g., inhalation) delivery of the combinations and compositions will lead to reduced pulmonary artery pressure without systemic effects. All of the above U.S. patents, U.S. patent application publications,

U.S. patent applications, foreign patents, foreign patent applications and non- patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A method of treating or preventing pulmonary hypertension in a subject, comprising, providing a phosphodiesterase inhibitor and a calcium channel blocker to the subject by inhalation.

2. The method according to claim 1 , wherein a composition comprising the phosphodiesterase inhibitor and the calcium channel blocker is provided to the subject.

3. The method according to claim 1 , wherein a single dual pharmacophore molecule comprising the phosphodiesterase and the calcium channel blocker is provided to the subject.

4. The method according to claim 1, wherein the phosphodiesterase inhibitor and the calcium channel blocker are provided separately.

5. The method according to claim 4, wherein the phosphodiesterase inhibitor and the calcium channel blocker are administered simultaneously or sequentially in any order.

6. The method of claim 5, wherein the phosphodiesterase inhibitor and the calcium channel blocker are administered as a dual pharmacophore.

7. The method according to claim 1 , wherein the calcium channel blocker is selected from the group consisting of: amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil, nisoldipine, nitrendipine, lacidipine, lercaninidipine, gallopimil, mibefradil, diltiazem, and isradipine.

8. The method according to claim 1, wherein the phosphodiesterase inhibitor is selected from the group consisting of sildenafil, tadafil, inoximone, pimobendan, vardenafil, milrinone, and amrinone.

9. A pharmaceutical composition comprising a calcium channel blocker and a phosphodiesterase inhibitor, wherein the composition is formulated for inhalation.

10. The composition according to claim 9, wherein the calcium channel blocker is selected from the group consisting of: amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil, nisotdipine, nitrendipine, lacidipine, lercaninidipine, gallopimil, mibefradil, diltiazem, and isradipine.

11. The composition according to claim 9, wherein the phosphodiesterase inhibitor is selected from the group consisting of sildenafil, tadafil, inoximone, pimobendan, vardenafil, milrinone, and amrinone.

12. A drug delivery device adapted for nasal administration of a pharmaceutical composition, wherein the delivery device comprises a pharmaceutical composition comprising a calcium channel blocker and a phosphodiesterase inhibitor.

13. The device according to claim 12, wherein the calcium channel blocker is selected from the group consisting of: amlodipine, bepridil, diltiazem, felodipine, fiunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil, nisoldipine, nitrendipine, lacidipine, lercaninidipine, gallopimil, mibefradil, diltiazem, and isradipine.

14. The device according to claim 12, wherein the phosphodiesterase inhibitor is selected from the group consisting of sildenafil, tadafil, inoximone, pimobendan, vardenafil, milrinone, and amrinone.