Pulmonary arterial hypertension (PAH) is defined as pulmonary vascular disease affecting the pulmonary arterioles resulting in an elevation in pulmonary artery pressure and pulmonary vascular resistance but with normal or only mildly elevated left-sided filling pressures (1). PAH is caused by a constellation of diseases that affect the pulmonary vasculature. PAH can be caused by or associated with collagen vascular disorders such as systemic sclerosis (scleroderma), uncorrected congenital heart disease, liver disease, portal hypertension, HIV infection, Hepatitis C, certain toxins, splenectomy, hereditary hemorrhagic teleangiectasia, and primary genetic abnormalities. In particular, a mutation in the bone morphogenetic protein type 2 receptor (a TGF-b receptor) has been identified as a cause of familial primary pulmonary hypertension (PPH)(2, 3). It is estimated that 6% of cases of PPH are familial, and that the rest are “sporadic.” The incidence of PPH is estimated to be approximately 1 case per 1 million population. Secondary causes of PAH have a much higher incidence. The pathologic signature of PAH is the plexiform lesion of the lung which consists of obliterative endothelial cell proliferation and vascular smooth muscle cell hypertrophy in small precapillary pulmonary arterioles. PAH is a progressive disease associated with a high mortality. Patients with PAH may develop right ventricular (RV) failure. The extent of RH failure predicts outcome (4).
The evaluation and diagnosis of PAH is reviewed by McLaughlin and Rich (1) and McGoon et al (5). A clinical history, such as symptoms of shortness of breath, a family history of PAH, presence of risk factors, and findings on physical examination, chest x-ray and electrocardiogram may lead to the suspicion of PAH. The next step in the evaluation will usually include an echocardiogram. The echocardiogram can be used to estimate the pulmonary artery pressure from the Doppler analysis of the tricuspid regurgitation jet. The echocardiogram can also be used to evaluate function of the right ventricle and left ventricle, and the presence of valvular heart disease such as mitral stenosis and aortic stenosis. The echocardiogram can also be useful in diagnosing congenital heart disease such as an uncorrected atrial septal defect or patent ductus arteriosus. Findings on echocardiogram consistent with a diagnosis of PAH would include: 1) Doppler evidence for elevated pulmonary artery pressure; 2) right atrial enlargement; 3) right ventricular enlargement and/or hypertrophy; 4) absence of mitral stenosis, pulmonic stenosis, and aortic stenosis; 5) normal size or small left ventricle; 6) relative preservation of or normal left ventricular function. To confirm the diagnosis of PAH a cardiac catheterization to directly measure the pressures on the right side of the heart and in the pulmonary vasculature is mandatory. An accurate measurement of the pulmonary capillary wedge pressure (PCWP) which gives an accurate estimate of the left atrial and left ventricular end-diastolic pressure is also required. If an accurate PCWP cannot be obtained then direct measurement of LV end-diastolic pressure by left heart catheterization is advised. By definition, patients with PAH should have a low or normal PCWP. However, in the late stages of PAH the PCWP can become somewhat elevated though usually not greater than 16 mm Hg (1, 5). The upper limit of normal for mean pulmonary artery pressure in an adult human is 19 mm Hg. A commonly used definition of mean pulmonary artery pressure is one-third the value of the systolic pulmonary artery pressure plus two-thirds of the diastolic pulmonary artery pressure. Severe PAH may be defined as a mean pulmonary artery pressure greater than or equal to 25 mm Hg with a PCWP less than or equal to 15-16 mm Hg, and a pulmonary vascular resistance (PVR) greater than or equal to 240 dynes sec/cm5. Pulmonary vascular resistance is defined as the mean pulmonary artery pressure minus the PCWP divided by the cardiac output. This ratio is multiplied by 80 to express the result in dyne-secs/cm5. The PVR may also be expressed in millimeters Hg per liter per minute which is referred to as Wood Units. The PVR in a normal adult is 67±23 dyne sec/cm55 or 1 Wood Unit (1, 5, 6). In clinical trials to test efficacy of drugs for PAH, patients with left sided myocardial disease or valvular heart disease are typically excluded (6).
Until recently, the only effective long-term therapy for PAH in conjunction with anticoagulant therapy was continuous intravenous administration of prostacyclin, also known as epoprostenol (PGI2) (7, 8). Recently, the non-selective endothelin receptor antagonist, bosentan, has shown efficacy for the treatment of PAH (9). As the first orally bioavailable agent with efficacy in the treatment of PAH, bosentan represents a significant advance. However, a subset of patients treated with bosentan may continue to deteriorate and require the addition of PGI2. Conversely some patients on PGI2 can be weaned off this medication with the addition of bosentan. PGI2 has both anti-platelet, inotropic, and vasodilatory properties. Recent evidence suggests that PGI2 may have additional beneficial effects on vascular remodeling (10). In most cases, incremental dosing is needed because of apparent tachyphylaxis/resistance. The mechanism for this resistance is not known. Selective endothelin type A receptor antagonists are currently in development for the treatment of PAH (6, 11). Sildenafil, a phosphodiesterase type V (PDE-V) inhibitor has recently been approved for the treatment of PAH (12, 13). PDE-V inhibition results in an increase in cyclic GMP which leads to vasodilation of the pulmonary vasculature. Treprostinil, an analogue of PGI2, can be administered subcutaneously to appropriately selected patients with PAH (14, 15). In addition, Iloprost, another prostacyclin analogue, can be administered in nebulized form by direct inhalation (16). These agents are used to treat PAH of multiple etiologies, including PAH associated with or caused by familial PAH (primary pulmonary hypertension or PPH), idiopathic PAH, scleroderma, mixed connective tissue disease, systemic lupus erythematosus, HIV infection, toxins such as phentermine/fenfluramine, congenital heart disease, Hepatitis C, liver cirrhosis, chronic thrombo-embolic pulmonary artery hypertension (distal or inoperable), hereditary hemorrhagic teleangiectasia, and splenectomy.
The rat monocrotaline model is a standard and well accepted model of PAH. Improvement in pulmonary arterial hypertension from drug treatment in the rat monocrotaline model is predictive of therapeutic response in humans with PAH. For example, both non-selective and selective ETA/B receptor antagonists have been shown to improve nitric oxide mediated pulmonary vasodilation, significantly reduce pulmonary hypertension and improve survival in rats with monocrotaline induced pulmonary arterial hypertension (17-20). In particular, the non-selective endothelin receptor antagonist bosentan, has been shown to reduce pulmonary hypertension and decrease pulmonary artery thickening in the rat monocrotaline model of PAH (21). Currently the non-selective endothelin receptor antagonist, bosentan is approved by the FDA for the treatment of PAH, and several selective ETA receptor antagonists are in phase III clinical trials for the treatment of PAH in humans. Oral sildenafil, which is FDA approved as a treatment of PAH, when given alone or in combination with beraprost, also showed efficacy in the rat monocrotaline model of PAH (22). The long-acting prostacyclin analogue, iloprost, which is also approved for the treatment of PAH in humans, showed efficacy in the treatment of monocrotaline induced PAH in the rat (23). Therefore, it has been established that the rat monocrotaline model of PAH predicts therapeutic response, efficacy, and utility of agents and drugs for the treatment of PAH of multiple and different etiologies in humans.
In the rat monocrotaline model, the Rho-kinase inhibitor, fasudil, prevented the progression of PAH and improved survival (24). In this study, fasudil decreased pulmonary arterial hypertension, pulmonary vascular lesions, endothelial cell dysfunction, and RV hypertrophy. In another study, specific inhibition of p38 MAP kinase with FR167653 was shown to decrease the progression of PAH in the rat monocrotaline model (25). The PDGF inhibitor, imantinib, decreased RV systolic pressure, and improved survival in the rat monocrotaline model of pulmonary arterial hypertension (26).
The JAK/STAT pathway has recently been implicated in the pathophysiology of PAH. JAKs are kinases which phosphorylate a group of proteins called Signal Transduction and Activators of Transcription or STATs. When phosphorylated, STATs dimerize, translocate to the nucleus and activate expression of genes which lead to proliferation of endothelial cells and smooth muscle cells, and cause hypertrophy of cardiac myocytes. There are three different isoforms of JAK: JAK1, JAK2, and JAK3. Another protein with high homology to JAKs is designated Tyk2. An emerging body of data has shown that the phosphorylation of STAT3, a substrate for JAK2, is increased in animal models of PAH. In the rat monocrotaline model, there was increased phosphorylation of the promitogenic transcription factor STAT3. In this same study pulmonary arterial endothelial cells (PAECs) treated with monocrotaline developed hyperactivation of STAT3 (27). A promitogenic agent or protein is an agent or protein that induces or contributes to the induction of cellular proliferation. Therefore, one effect of JAK2 inhibition would be to decrease proliferation of endothelial cells or other cells, such as smooth muscle cells. A contemplated effect of a JAK2 inhibitor would be to decrease the proliferation of endothelial cells or other cells which obstruct the pulmonary arteriolar lumen. By decreasing the obstructive proliferation of cells, a JAK2 inhibitor could be an effective treatment of PAH.
However, whether or not inhibition of JAK2, and consequent reduction in phosphorylation of STAT3, ameliorates PAH was not previously known. Examples of the use of a JAK2 inhibitor to treat heart failure and systemic hypertension have been reported in PCT/US02/23444 “Method for Reducing Hypertension and Heart Failure.” PAH is a substantially different disease than systemic hypertension. PAH is characterized by high pulmonary artery and right ventricular pressures due to increased pulmonary vascular resistance; systemic hypertension is characterized by elevated pressure in the systemic circulation. Typically patients with PAH do not have systemic hypertension. PCT/US02/23444 does not contemplate the use of a selective JAK2 inhibitor as a treatment for PAH.
Because a drug may be effective as a treatment for systemic hypertension does not mean that it will also be effective for treating PAH. For example a vasodilator drug that is effective for treating systemic hypertension, such as the ACE-inhibitor Captopril, can worsen pulmonary arterial hypertension and RV failure in patients with PAH. Evidence for the potential deleterious effect of drugs used to treat systemic hypertension on PAH are given by Packer M, Vasodilator Therapy for Primary Pulmonary Hypertension, Annals of Internal Medicine 1985; 103: 258-270, which is hereby incorporated by reference (28). The one known exception to this limitation is that approximately 15-20% of patients with idiopathic PAH respond to calcium channel blockers, agents which also may be used to treat systemic hypertension. In order to determine if a patient has so-called “reactive” PAH and may be a responder to therapy with a calcium channel blocker, the diagnostic evaluation of PAH includes a pulmonary artery catheterization and acute challenge with adenosine, prostacyclin, or inhaled nitric oxide. If the patient has a greater than 10 mm Hg reduction in the mean pulmonary artery pressure and the mean pulmonary artery pressure decreases to less than or equal to 40 mm Hg with one of these agents, then testing to determine if the patient will respond to a calcium channel blocker may be performed (29, 30). Some clinicians will consider PAH reactive if there is a 20% or greater decrease in the mean pulmonary artery pressure in response to adenosine, prostacyclin, or inhaled nitric oxide. The reason that testing for acute vasoreactivity with protstacyclin, adenosine, or inhaled nitric oxide is performed prior to testing with a calcium channel blocker is that some patients given a calcium channel blocker who were not previously shown to have acute vasoreactivity have died (30). This complicated evaluation and treatment algorithm emphasizes that drugs used to treat systemic hypertension are not necessarily appropriate for patients with PAH.
A JAK2 inhibitor is any compound that selectively inhibits the activity of JAK2. One activity of JAK2 is to phosphorylate a STAT protein. Therefore an example of an effect of a JAK2 inhibitor is to decrease the phosphorylation of one or more STAT proteins. The inhibitor may inhibit the phosphorylated form of JAK2 or the non-phosphorylated form of JAK2. The JAK2 inhibitor may be any type of compound. For example, the compound may be a small organic molecule or a biological compound, such as an antibody or an enzyme.
Examples of JAK2 inhibitors include some members of small organic molecules called tyrphostins. Tyrphostins inhibit the activity of protein tyrosine kinases and have the basic structure shown in structure 1 below:

A preferred class of tyrphostins for use are those compounds represented by Structure 1 wherein:
R1═C6H5—CH2—NH
R2 and R3═H, OH, lower alkyl, F, NO2, CF3, C6H5—SO2, 0-R4, O—CO—R4, or R4
R4=phenyl or lower alkyl; and
Lower alkyl=C1-C4 branched or unbranched alkyl (for example, methyl or ethyl).
R2 and R3 may be the same or different except R2 and R3 cannot both be H. Preferably, R2 and R3 are OH.
The tyrphostin may be any tyrphostin that selectively inhibits JAK2. Some examples of tyrphostins include structures described by Meydan et al. (1996) Nature, 379:645-648; and Levitzki et al (1995) Science, 267:1782-1788, and referred to in application PCT/US02/23444 (International Publication Number WO 03/020202 A2). These structures are incorporated herein by reference.
The preferred compound is known as Tyrphostin AG490, which is a selective, specific, and potent JAK2 protein tyrosine kinase inhibitor. The structure of AG490 is shown as structure 2 below:

A compound is considered a selective inhibitor of JAK2 when the compound inhibits JAK2 activity to an extent significantly greater than it inhibits the activity of other members of the JAK family, i.e., JAK1, JAK3, and Tyk2. Preferably the selective JAK2 inhibitor inhibits JAK2 at least 2-fold more than it inhibits other members of the JAK family, more preferably at least about 5-fold more, and most preferably at least about 10-fold more.
A Symposium sponsored by the World Health Organization (WHO) was held in 1998 which developed a clinical Classification of PAH. McLaughlin V V and Rich S (Current Problems in Cardiology 2004; 29:575-634) summarized the clinical classification of PAH developed at this symposium (1). The present invention contemplates the use of a JAK2 inhibitor to treat the following specific forms of PAH described in the WHO classification of PAH: familial or idiopathic PAH; PAH associated with connective tissue disease; PAH associated with congenital heart disease; PAH associated with portal hypertension; PAH associated with HIV infection; PAH associated with Drugs/toxins; PAH associated with thromboembolic obstruction of the distal pulmonary arteries.
Evidence of the association of PAH with the connective tissue disease scleroderma is given by Badesch et al (31). In this application connective tissue disease and collagen vascular disorder are synonyms. Evidence of the association of PAH with the collagen vascular disorder called “mixed connective tissue disease”, Sjogren's syndrome, and/or systemic lupus erythematosus is given by Humbert et al (32). Robbins et al (33) previously reported on the use of epoprostenol to treat PAH associated with systemic lupus erythematosus. Evidence for the association of PAH with HIV infection is given by Aguilar and Farber (34). Evidence for the association of PAH with congenital heart defects is given by Rosenzweig et al (35). Evidence for the association of PAH with fenfluramine, an anorexigen, is given by Archer et al (36). Evidence for the association of PAH with phentermine is given by Reeve et al (37). Evidence for the association of PAH with thromboembolic disease is given by Nagaya et al (38). Evidence for the association of PAH with hereditary hemorrhagic teleangiectasia is given by Mc Goon et al (5). Review of diseases associated with PAH are given by McGoon et al (5) and Badesch et al (30).
The present invention also contemplates the use of a JAK2 inhibitor to treat PAH associated with splenectomy. Evidence for the association of PAH with splenectomy is given by Hoeper et al. (39). Evidence for the association of PAH with portal hypertension is given by Hoeper et al. (40).