Method of treating a vasculopathy in a human subject

Abstract

The present invention is based upon the observation that inhibition of NPR-C Signaling pathway leads to the development of pulmonary arterial hypertension (PAH). Accordingly, the invention provides a mouse model for PAH, and proposes a method of using synthetic analogs of the NPR-C signaling pathway, specifically synthetic C-type atrial natriuretic factor or intermediates for, or modulators of, the NPR-C signaling pathway as anti-pulmonary vasculopathy agents. Activators of the NPR-C signaling pathway are disclosed to treat or prevent vasculopathy, including but not limited to PAH and other types of pulmonary hypertension, peripheral vascular disease, critical limb ischemia, coronary artery disease, and diabetic vasculopathy.

Claims

1. A method of treating or preventing a vasculopathy in a subject, the method comprising: administering to the subject a therapeutically effective amount of an activator of NPR-C signaling, wherein the activator binds the NPR-C and reduces intercardiac and/or pulmonary pressure, wherein the subject is a human subject and wherein the activator of NPR-C signaling is cANF or a functional analog thereof.

2. The method of claim 1, wherein said vasculopathy comprises pulmonary arterial hypertension (PAH).

3. The method of claim 1, wherein said vasculopathy comprises pulmonary hypertension (PH).

4. The method of claim 3, wherein said pulmonary hypertension is a complication of a medical condition selected from the group consisting of: left sided heart disease, heart failure, chronic hypoxia and thromboembolic disease.

5. The method of claim 1, wherein the activator of NPR-C signaling is cANF.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a schematic representation of a cascade of events following an impairment of NPR-C signaling pathway. The impaired NPR-C signaling pathway may be the result of several factors, including but not limited to an abnormal NPR3 gene, an inhibition of NPR3 gene expression, or an inhibition or abnormal NPR-C protein. Impaired activation of this signaling pathway leads to failure of the antiproliferative effect of NPR-C in the pulmonary vasculature, which in turn results in vascular pulmonary injury, including endothelial dysfunction, vascular smooth muscle dysfunction, matrix changes, and platelets, as well as inflammatory cell activation. The proliferation of smooth muscle in pulmonary arterioles, secondary to remodelling, would then ultimately lead to PAH.

(2) FIG. 2 illustrates the loss of homeostasis with NPR-C signaling pathway. The imbalance between impaired activation and physiological activation may cause or prevent the development of PAH. Impaired activation of NPR-C signaling pathway leads to failure of the antiproliferative effect of NPR-C in the pulmonary vasculature, which in turn results in PAH. Activators to NPR-C pathway initiate signaling that results in the inhibition of cell proliferation in pulmonary artery smooth muscle cells and therefore reverses the remodelling that is typical to PAH.

(3) FIGS. 3a and 3b illustrate the results of apical four-chamber echocardiography in NPRC.sup.+/+ and NPRC.sup.−/− mice and illustrates right atrial dilation as evidenced by the RA notation on the respective figures.

(4) FIGS. 4a and 4b show a 2D view of the parasternal short-axis echocardiographic view at the mid papillary level showing the paradoxical ventricular septum motion including flattening and bulging into the left ventricle during systole in the NPRC.sup.−/− mice.

(5) FIGS. 5a and 5b show an M-Mode view of the parasternal short-axis echocardiographic view at the mid papillary level showing the paradoxical ventricular septum motion including flattening and bulging into the left ventricle during systole in the NPRC.sup.−/− mice.

(6) FIG. 6 demonstrates the estimation of RVSP/PASP by using the maximum velocity of the tricuspid regurgitation jet, and reveals a higher RVSP/PASP in the NPRC.sup.−/− mice.

(7) FIGS. 7a and 7b show representative examples of the right atrium pressure tracings in the NPRC.sup.+/+ and NPRC.sup.−/− mice, demonstrating the presence of an increased atrial pressure in the NPRC.sup.−/− mice.

(8) FIGS. 8a and 8b show representative examples of right ventricular pressure tracings in the NPRC.sup.+/+ and NPRC.sup.−/− mice, demonstrating the presence of an increased right ventricular systolic pressure (RVSP) in the NPRC.sup.−/− mice.

(9) FIG. 9 demonstrates the effect of cANF administration on the right ventricular systolic pressure and heart rate in a diabetic mouse.

(10) FIG. 10 demonstrates the effect of cANF administration on the right ventricular systolic pressure in NPR-C.sup.+/+ mice. cANF significantly reduces RVSP.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

(11) As used herein, “a” or “an” means “one or more.”

(12) Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

(13) As used herein, the term “subject” (also referred to herein as a “patient”) includes warm-blooded animals, preferably mammals, including mice, rats, rabbits, pigs, sheep, goats, cattle, and other domestic farm animals, zoo animals, as well as higher primates and humans.

(14) As used herein, the term “vasculopathy” includes, but is not limited to, pulmonary vasculopathy, PAH and other types of PH, peripheral vascular disease, critical limb ischemia, coronary artery disease, and diabetic vasculopathy.

(15) As used herein the terms “treating”, “treat” or “treatment” refer to obtaining a desired physiological or pharmacological effect that may be partially or completely effective in preventing a disease, or may be partially or completely effective in preventing, reducing, or improving one or more symptoms of, or other adverse effects caused by, a disease. The desired physiological or pharmacological effect may be achieved by administering a therapeutically effective amount of an NPR-C signalling pathway activator as defined herein, wherein said amount of activator is sufficient to reduce or eliminate at least one symptom of vasculopathy.

(16) As used herein, an “effective amount” is an amount that achieves the stated goal, which may be treatment and/or prevention of vasculopathy, or any symptom associated with vasculopathy. It is contemplated that in the context of treatment an effective amount produces a therapeutic benefit, which includes, but may not be necessarily limited to the following characteristics with respect to pulmonary arterial hypertension: reducing mean pulmonary pressure, increasing cardiac output/cardiac index measured by either thermodilution or Fick, improving timed walk distance (e.g., six-minute walk), improving metabolic equivalents (MET) {e.g., exercise treadmill test), reducing anginal pain frequency, reducing dyspnea, synocope, presyncope, symptoms of right heart failure including edema and ascites, preventing need for lung or heart transplant, reducing length of stay in intensive care, reducing length of stay in hospital, or prolonging life.

(17) As used herein, the terms “comprises,” “comprising,” “containing” and “having” and the like are open-ended as defined by U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

(18) This application is directed to the surprising and unexpected discovery that NPR-C knockout (NPR-C.sup.(−/−)) mice are PAH-prone and, therefore, represent an experimental animal model for PAH. The NPR-C.sup.(−/−) mice show similar pathology when compared to human PAH patients, including right atrial dilation, tricuspid regurgitation as well as echocardiographic signs of right ventricular pressure overload, including paradoxical bulging of the septum into the left ventricle during systole, and hypertrophy of the right ventricular free wall and trabeculae. The left ventricular systolic and diastolic function are within normal limits in NPR-C.sup.(−/−) mice. Doppler echocardiography assessment reveals a higher right ventricular systolic pressure (RVSP) and thus a higher pulmonary artery systolic pressure (PASP) in NPR-C.sup.(−/−) mice compared with wild-type littermates. This significant increase in RVSP and PASP in NPR-C.sup.(−/−) mice was confirmed with right heart catheterization. Accordingly, the invention is directed to the use of the NPR-C.sup.(−/−) mouse as a model system for PAH.

(19) The mouse model of the present invention may be used in a wide variety of assays of screening agents for their potential effect on a patient with PAH. In this embodiment, the agent is administered to the mouse and the effect on the mouse is evaluated. For example, the model can be used to evaluate, i.e., screen, potential therapeutic agents for preventing or treating conditions associated with PAH.

(20) In addition, the invention pertains to the use of NPR3 gene polymorphism for diagnosis of vasculopathy. The invention is based on the discovery that loss of function or mutations of NPR3 gene, particularly loss of function or mutations of the gene encoding NPR-C play a role in the development of PAH. Likewise, mutants of the NPR-C protein, as well as related derivatives, fragments and homologs thereof, and NPR-C nucleic acids encoding them, may also have a role in the development of PAH.

(21) The invention provides a method for the use of one or more activators of the NPR-C signaling pathway for the treatment and prevention of PH and disorders related to vasculopathy, comprising administering to the subject a therapeutically effective amount of said activator. These compounds and compositions may be administered to humans in a manner similar to other therapeutic agents. Therapeutics of the invention may be administered either alone or in combination with other therapies, e.g., therapeutics effective against PAH and PH. Other therapeutic agents that have been used to treat PAH include, but are not limited to, the following: anticoagulants (such as Coumadin or Warfarin), calcium channel blockers (such as amlodipine, diltiazem, nifedipine, felodipine, isradipine, nicardipine, or verapamil), prostacyclins (such as epoprostenol, treprostinil, iloprost), nitric oxide (only used in acute settings), soluble GC stimulators and activators (riociguat), diuretics, cardiac glycosides (digoxin), endothelin antagonists (including nonselective inhibition with bosentan), phosphodiesterase inhibitors (such as sildenafil), endopeptidase inhibitors, lipid lowering agents, thromboxane inhibitors (such as terbogrel), or oxygen.

(22) The invention further provides a method for acute administration of a therapeutically effective amount of cANF to the subject suffering from PH or other disorders related to vasculopathy, in order to significantly reduce RVSP and PASP without alteration of the systemic arterial pressure. The magnitude of this reduction may be greater in subjects with concomitant diabetes or coronary artery disease, including heart failure, who may have endothelial dysfunction. This observation is supported by the finding that, in the coronary vasculature, the vasorelaxant effect of the NPR-C pathway may be increased in the presence of nitric oxide (NO) synthase inhibition (Hobbs et al. (2004)). This observation supports synergistic and complementary functions for NPR-C pathway and NO-mediated signaling. The inhibition of one pathway may thus be compensated for by the upregulation of the other. This may be of particular clinical significance in patients with PAH who are known to have endothelial dysfunction and thus reduced or loss of NO pathway (Fagan et al. 1999; Champion et al. 2002).

(23) The precise nature of the role of NPR-C signaling pathway in pulmonary vascuolopathy is not yet fully elucidated. Evidence suggests that chronic hypoxia causes a significant down regulation of NPR-C expression in several tissues, including pulmonary vascular smooth muscle, independently of changes in NPs levels and expression of other NPs' receptors (Sun et al. 2000). This down regulation of NPR-C expression and associated impaired NPR-C pathways may lead to failure of the antiproliferative effect in the pulmonary vasculature, which would then ultimately lead to PAH (FIG. 1). Therefore, an impaired NPR-C pathway is a common underlying cause of all hypoxia-associated vasculopathy, including, but not limited to, pulmonary vasculopathy and chronic thromboembolic pulmonary hypertension (CTEPH). The most common causes of PH are chronic lung and left sided heart disease. The development of PH in these conditions occurs, at least partially, as the result of chronic hypoxia. This observation suggests that NPR-C pathway represents, therefore, a therapeutic target to inhibit pulmonary vascular remodeling and maladaptive increases in pulmonary arterial pressures in patients with heart failure or CTEPH.

(24) It is hypothesized that the NPR-C pathway may prevent cellular proliferation in some types of cells, with the result that abnormal NPR-C pathway activity may permit excess cell growth and proliferation in response to a variety of injuries. The proliferation of smooth muscle in pulmonary arterioles would then ultimately lead to PAH. These affirmations are supported by the proposed observation that transgenic mice with genetic deletion of NPR-C exhibit PAH. Therefore, the fundamental mechanism of NPR-C-related PAH may be an imbalance of growth signaling caused, at least partially, by an impaired or reduction in the braking function of NPR-C. (FIGS. 1 and 2). In summary, an abnormal NPR-C pathway plays an important role in the pathogenesis of PAH and in particular IPAH, and is likely causally linked to some cases in familial PAH (FPAH) and a substantial percentage of IPAH patients.

(25) Although the foregoing description refers to particular embodiments, it will be understood by one of skill in the art that the present invention is not limited to the disclosed embodiments. Those of ordinary skill in the art will recognize that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.

EXAMPLES

(26) Statistical Analysis

(27) All data are presented as means±SEM. Data were analyzed using Student's t-test. P<0.05 was considered significant.

Example 1: Echocardiography

(28) A study was conducted to examine the differences in cardiac structure and function in NPR-C.sup.+/+ (wild type) and age matched, littermate NPR-C.sup.−/− mice by echocardiography. Two-dimensional, Doppler echocardiography measurements and quantification were performed according to recommendations of the European Society of Echocardiography.

(29) Wild type (n=10) and NPRC.sup.−/− (n=10) mice were scanned at 12 months. Mice were placed in an induction chamber with constant inflow of 5% isoflurane mixed with 100% oxygen. Once each mouse was asleep, it was removed from the induction chamber, weighed and placed on a heating platform with electrocardiogram contact pads. The mouse's nose was placed into a nose cone providing a flow of 1-2% isoflurane in 100% oxygen. Excess gases were evacuated passively using an activated charcoal absorption filter. The eyes were covered with a petroleum based ophthalmic ointment. Electrode gel was placed on the paws and the paws were taped over the electrocardiogram contact pads on the heating platform. A rectal probe was lubricated with gel, placed in the rectum and taped to the platform. The temperature was maintained at 36.5 to 37.5° C. Depilatory cream was applied to the chest of the mouse and removed after two minutes. Ultrasound gel was placed on the chest of the anesthetized mouse. The ultrasound probe was placed in contact with the ultrasound gel and scanning was performed over 20 minutes. B-mode, M-mode and spectral Doppler images were obtained. The temperature and heart rate (HR) were constantly monitored during the scanning. Once completed, all probes and monitors were removed from the mouse. The mouse was cleaned with water and allowed to recover on the heated platform. Once awake, the mouse was returned to its cage.

(30) Estimation of RVSP by Doppler Echocardiography assessment of tricuspid valve regurgitation (TR) jet peak velocity accurately predicts the pulmonary artery systolic pressure (PASP) observed by invasive measurement. TR was graded as none, trace, mild, moderate, or severe by assessment of the colour-flow jet in relation to the right atrium (RA) area in apical 4 chamber view. With pulse-wave Doppler, the maximum peak TR velocity (V) recorded from any view was used to determine the RVSP with the simplified Bernoulli equation (RVSP=4V2+RAP), with RA pressure (RAP) obtained via right heart catheterization. PASP equates to RVSP in the absence of pulmonic stenosis and RV outflow tract obstruction, as was the case in this study. Other calculations were performed using echocardiographic derived values. Percent shortening fraction was calculated from M-mode measurements using the leading edge to leading edge method via the formula % Shortening Fraction (% SF)=left ventricular internal diameter (diastole) [LVID(d)]−left ventricular internal diameter (systole) [LVID(s)]/LVID(d).

(31) As illustrated in FIGS. 3a and 3b, 4a and 4b, and 5a and 5b, the NPR-C.sup.(−/−) mice had right atrial dilation and echocardiographic signs of right ventricular pressure overload, including flattening and paradoxical bulging of the septum into the left ventricle during systole, and hypertrophy of the right ventricular free wall and trabeculae. Similar testing (figures not shown) indicated a severe tricuspid regurgitation jet in the NPRC.sup.−/− mice. All these findings are also typically seen in human patients with PAH. The ejection fraction (EF) (69±2.4 in NPR-C.sup.+/+ vs. 74 W 2 in NPR-C.sup.(−/−), p=0.11) and fractional shortening (FS) (34±4.3 in NPR-C.sup.+/+ vs. 38.3±3.8, P=0.13) tended to be greater in NPR-C.sup.−/− mice, although these did not reach statistical significance as would be expected in the human patient with PAH. Severe TR was detected in all NPR-C(−/−) mice while most NPR-C(+/+) mice had none or trace TR. Consistently, Doppler echocardiography assessment revealed a higher RVSP and thus a higher PASP compared with wild-type littermates (25±1 mmHg vs 7±1 mmHg, P<0.001).

Example 2: Right Heart Catheterization and Administration of cANF

(32) To confirm the presence of increased pulmonary artery systolic pressure among NPR-C.sup.−/− mice, right heart catheterization was performed in both NPR-C.sup.+/+ (wild type) and age matched, littermate NPR-C.sup.−/− mice.

(33) Mice were placed in an induction chamber with constant inflow of 5% isoflurane mixed with 100% oxygen. Once each mouse was asleep, it was removed from the induction chamber, weighed and placed on a heated surgical table and secured with surgical tape. The mouse's nose was placed into a nose cone with a flow of 3% isoflurane in 100% oxygen. The animals were then shaved to expose the surgical area. An incision of ˜1 inch length was made, extending from the animal's chin down to the right armpit. The thyroid gland was then blunt-dissected upward to expose the underlying tissue and the right jugular vein. The jugular vein was then separated from surrounding tissue using dissecting forceps until the body of the vessel was completely free from adherent tissues. The cranial end of the jugular was tied off completely, and a loose tie was then made at the caudal end of the exposed jugular using 4-0 braided silk suture. Four-inch microdissecting scissors were then used to make a small incision in the medial aspect of the right jugular vein. A Millar 1.4 French pressure-volume microtip catheter transducer connected to a PowerLab/8s (AD Instruments) was then inserted through the incision and gently threaded down into the right ventricle. Proper placement within the ventricle was determined through observation of the pressure-volume loop obtained from the catheter. The loose caudal suture was then tightened to secure the catheter in place. Once the catheter was properly placed, data including HR and the right ventricular systolic pressure (RVSP) were recorded and analyzed using a data acquisition system (Chart, AD Instruments).

(34) As illustrated in FIGS. 6a and 6b, right atrial pressure was significantly elevated in the NPR-C.sup.−/− mice compared to their age matched, littermate NPR-C.sup.+/+ mice, at baseline (1.99±0.08 mm Hg vs 0.38±0.02 mm Hg, respectively (P=0.01)). As illustrated in FIGS. 7a and 7b, right ventricular systolic pressure (RVSP) was significantly elevated in NPR-C.sup.−/− mice compared to their age matched, littermate NPR-C.sup.+/+ mice, at baseline (21.95±0.56 mmHg vs 5.3±0.6 mmHg, respectively (P<0.001)).

(35) Each NPR-C.sup.+/+ mouse was administered a dose of the NPRC receptor agonist, cANF, by means of an intraperitoneal (IP) bolus of 1 μL of cANF (50 nM) in 15 mL of distilled water. Consistently, the administration of the cANF in NPR-C.sup.+/+ mice, decreased the RVSP and therefore PASP by 50%, and HR by 11%, but had no effect on blood pressure. PASP equates to RVSP in the absence of pulmonic stenosis and RV outflow tract obstruction, as was the case in this study.

(36) Interestingly, the effect on RVSP and therefore PASP was more striking in the age matched diabetic mice (more than 75% reduction in RVSP) as illustrated in FIG. 9. The increased magnitude of this reduction may be due to the concomitant endothelial dysfunction in diabetic mice, and a similar response is anticipated in subjects with concomitant coronary artery disease including heart failure, which may also have endothelial dysfunction.