Methods for improving cardiovascular function and treating cardiovascular disease using recombinant ectonucleotide pyrophosphatase / phosphodiesterase (NPP1)

Abstract

To provide methods for improving the cardiovascular function and treating a cardiovascular disease using recombinant ectonucleotide pyrophosphatase / phosphodiesterase (NPP1).SOLUTION: The present invention provides methods for improving the cardiovascular function in a human patient (e.g., reducing hypertension) and methods of treating a cardiovascular disease, by administering a recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1) or an active fragment or fusion protein thereof. In one embodiment, the improvement is a reduction in elevated blood pressure (e.g., a reduction in blood pressure higher than 140 / 90 millimeters of mercury (mmHg) to within a normal blood pressure range, namely, about 120 / 80 mm of mercury (mmHg)).SELECTED DRAWING: None

Description

[Technical field]

[0001] Related information paragraph This application claims the benefit of the priority date of U.S. Provisional Patent Application No. 62 / 563,829, filed September 27, 2017, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy (created on August 22, 2018) is named AXJ-241PC_SL.txt and is 88,772 bytes in size. [Background technology]

[0003] background The cardiovascular system (also known as the circulatory system) includes the heart, arteries, veins, capillaries, and blood. The heart functions as a pump that moves blood throughout the body. The arterial circulation pumps blood from the heart to the body, and the venous circulation returns it to the heart. Capillaries are very small blood vessels at the border of the arterial and venous circulations, where exchange of materials between blood and body tissues occurs. The cardiovascular system has three main functions: (1) transport of nutrients, oxygen, and hormones to cells throughout the body and removal of metabolic waste products (carbon dioxide, nitrogenous wastes), (2) defense of the body by white blood cells, antibodies, and complement proteins that circulate in the blood and protect the body from foreign microorganisms and toxins, as well as the clotting mechanism that protects the body from blood loss after injury, and (3) regulation of body temperature, fluid pH, and water content of cells.

[0004] Cardiovascular disease is the leading cause of death worldwide. Cardiovascular disease includes coronary artery disease (CAD) (e.g., angina and myocardial infarction (commonly known as heart attack)), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, and venous thrombosis (see GBD 2013 Mortality and Causes of Death, Collaborators (17 December 2014), Lancet. 385(9963): 117-71).

[0005] Approximately 85 million people in the United States have hypertension (also known as high blood pressure). Normal blood pressure is 120 / 80 mm of mercury (mmHg). Clinical practice guidelines define hypertension as blood pressure higher than 140 / 90 mmHg. Blood pressure is the force that blood exerts against the walls of blood vessels. A systolic reading of 140 mmHg refers to the pressure when the heart pumps blood around the body. A diastolic reading of 90 mmHg refers to the pressure when the heart relaxes and refills with blood. Hypertension severely impacts quality of life and increases the risk of heart disease, stroke, and death.

[0006] Despite considerable research in the field, there is a continuing need for therapies that effectively treat cardiovascular disease, including hypertension. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The present invention relates to the use of isolated recombinant human soluble NPP1 lacking N-terminal cytosolic domain and transmembrane domain, and its fusion protein, for improving cardiovascular function in human patients, including preventing and / or treating cardiovascular disease (e.g., hypertension). Any cardiovascular disorder is within the scope of the present invention.

[0008] In one aspect, a method is provided for improving cardiovascular function in a human patient, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment thereof, or the fusion protein. In one embodiment, the improvement is a reduction in elevated blood pressure (e.g., reducing blood pressure above 140 / 90 millimeters of mercury (mmHg) to within the normal blood pressure range (about 120 / 80 mmHg). In another embodiment, the improvement is a reduction in elevated blood pressure by at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70)%. In another embodiment, the improvement is a reduction in elevated blood pressure by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70). In another embodiment, the improvement is a normalization of blood pressure (e.g., to about 120 / 80 mm of mercury (mmHg)). In another embodiment, the improvement is a reduction (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% or more) in left ventricular end diastolic pressure (EDP) and end systolic pressure (ESP). % decrease in left ventricular EDP and ESP. In another embodiment, the improvement is about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold decrease in left ventricular EDP and ESP. In another embodiment, the improvement is a reduction (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%) in ventricular stiffness (EDPVR). ... In another embodiment, the improvement is in an increase (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%) in contractility (PRSW). In another embodiment, the improvement is in an increase (e.g., at least 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% in contractility).

[0009] In another aspect, a method is provided for treating a human patient having a cardiovascular disorder, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment thereof, or a combination thereof. Exemplary cardiovascular diseases include, but are not limited to, coronary artery disease (CAD) (e.g., angina and myocardial infarction (commonly known as heart attack)), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolism, and venous thrombosis.

[0010] In another aspect, a method is provided for treating a human patient having hypertension, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment thereof, or a combination thereof. or administering a fusion protein.

[0011] In another aspect, a method is provided for treating a human patient having hypertension comprising: a) identifying the human patient as having hypertension; and b) administering to the identified patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion protein thereof.

[0012] In another aspect, a method is provided for reducing hypertension in a human patient, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment thereof, or a combination thereof. or administering a fusion protein.

[0013] In one embodiment, the methods described herein result in a drop in the patient's blood pressure (e.g., from an elevated blood pressure greater than about 140 / 90 mmHg to within the normal blood pressure range (about 120 / 80 mmHg). In another embodiment, the patient's blood pressure is reduced by at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70)% following treatment. In another embodiment, the patient's blood pressure is reduced by about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold following treatment. In another embodiment, the patient's blood pressure normalizes (e.g., to about 120 / 80 mmHg) following treatment.

[0014] In one embodiment, an NPP1 fusion protein is administered. A preferred fusion protein comprises an NPP1 component, an Fc region of an immunoglobulin, and optionally a targeting moiety. In one embodiment, the targeting moiety is Asp 10 (SEQ ID NO: 18). In another embodiment, the targeting moiety comprises at least 8 consecutive aspartic acid or glutamic acid residues (SEQ ID NOs: 20 and 21, respectively). Certain NPP1 fusion proteins for administration according to the methods disclosed herein have the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0015] Any suitable amount of recombinant hsNPP1 may be administered to the human patient. In one embodiment, the hsNPP1 is administered in one or more doses including about 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10.0 mg / kg, 11.0 mg / kg, 12.0 mg / kg, 13.0 mg / kg, 14.0 mg / kg, 15.0 mg / kg, 16.0 mg / kg, 17.0 mg / kg, 18.0 mg / kg, 19.0 mg / kg, or 20.0 mg / kg. In another embodiment, the hsNPP1 is administered in one or more doses including about 1.0 mg / kg to about 5.0 mg / kg NPP1. In another embodiment, the hsNPP1 is administered in one or more doses including about 1.0 mg / kg to about 10.0 mg / kg NPP1.

[0016] The period between doses of hsNPP1 is at least 2 days and may be longer (e.g., at least 3 days, at least 1 week, 2 weeks, or 1 month). In one embodiment, the administration is weekly, biweekly, or monthly.

[0017] The recombinant hsNPP1 can be administered in any suitable manner, for example, intravenously, subcutaneously, or intraperitoneally.

[0018] The recombinant hsNPP1 may be administered in combination with one or more additional therapeutic agents. Exemplary therapeutic agents include, but are not limited to, thiazide diuretics (e.g., hydrochlorothiazide (Microzide) or chlorthalidone), beta-blockers (e.g., acebutolol (Sectral) or atenolol (Tenormin)), angiotensin-converting enzyme (ACE) inhibitors (e.g., lisinopril (Zestril), benazepril (Lotensin), or captopril (Capoten)), angiotensin II receptor blockers (ARBs) (e.g., candesartan (Atacand) or losartan (Cozaar)), calcium channel blockers (e.g., amlodipine (Norvasc)), and the like. or diltiazem (Cardizem)), renin inhibitors (e.g., aliskiren (Tekturna)), alpha blockers (e.g., doxazosin (Cardura) or prazosin (Minipress)), alpha beta blockers (e.g., carvedilol (Coreg) or labetalol (Trandate)), centrally acting drugs (e.g., clonidine (Catapres, Kapvay), guanfacine (Intuniv, Tenex) and methyldopa), vasodilators (e.g., hydralazine and minoxidil), and / or aldosterone antagonists (e.g., spironolactone (Aldactone) or eplerenone (Inspra)). In one embodiment, the recombinant hsNPP1 and the additional therapeutic agent are administered separately, simultaneously or sequentially. In one embodiment, the recombinant hsNPP1 is administered prior to administration of the additional therapeutic agent. In another embodiment, the recombinant hsNPP1 is administered after administration of the additional therapeutic agent, hi another embodiment, the recombinant hsNPP1 and the additional therapeutic agent are administered together.

[0019] In another aspect, the use of isolated recombinant human sNPP1, its fragment or fusion protein is provided. In one embodiment, the use of isolated recombinant human sNPP1, its fragment or fusion protein for the manufacture of a medicament for improving cardiovascular function is provided. In another embodiment, the use of isolated recombinant human sNPP1, its fragment or fusion protein for the manufacture of a medicament for reducing hypertension is provided. In another embodiment, the present invention provides the use of isolated recombinant human sNPP1, its fragment or fusion protein for reducing hypertension. In another embodiment, the present invention provides the use of isolated recombinant human sNPP1, its fragment or fusion protein for treating hypertension. In another embodiment, the present invention provides the use of isolated recombinant human sNPP1, its fragment or fusion protein for improving cardiovascular function in a patient. The present invention provides, for example, the following items: (Item 1) A method for improving cardiovascular function in a human patient, said method comprising administering to said patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion protein thereof. The method comprises administering a (Item 2) A method for treating cardiovascular disease in a human patient, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion protein thereof. The method comprises administering a (Item 3) A method for treating a human patient having hypertension, said method comprising administering to said patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion protein thereof. The method of claim 1, further comprising administering (Item 4) 1. A method for reducing hypertension in a human patient, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion protein thereof. The method of claim 1, further comprising administering (Item 5) The method of any one of the preceding items, wherein the hsNPP1 is a fusion protein. (Item 6) 6. The method of claim 5, wherein the fusion protein comprises an immunoglobulin Fc region. (Item 7) 7. The method of claim 5 or 6, wherein the fusion protein comprises a targeting moiety. (Item 8) 8. The method of claim 7, wherein the targeting moiety comprises at least eight consecutive aspartic acid or glutamic acid residues (SEQ ID NOs: 20 and 21, respectively). (Item 9) 6. The method of claim 5, wherein the fusion protein comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. (Item 10) The method of any one of the preceding items, wherein the one or more doses comprise from about 1.0 mg / kg to about 20.0 mg / kg NPP1. (Item 11) The method of any one of the preceding items, wherein the one or more doses comprise from about 1.0 mg / kg to about 5.0 mg / kg NPP1. (Item 12) 10. The method of any of items 1-9, wherein the one or more doses are about 0.5 mg / kg, about 1 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg. (Item 13) The method of any one of the preceding items, wherein the two or more doses of NPP1 are administered at least 3 days, 1 week, 2 weeks, or 1 month apart. (Item 14) 13. The method of any one of items 1 to 12, wherein said administration is weekly, biweekly, or monthly. (Item 15) The method of any one of the preceding items, wherein the administration is intravenous, subcutaneous, or intraperitoneal. (Item 16) The method of any one of the preceding items, wherein an additional therapeutic agent is administered to the patient. (Item 17) 2. The method according to item 1, wherein said improvement is a reduction in elevated blood pressure, a normalization of blood pressure, a reduction in left ventricular end diastolic pressure (EDP), a reduction in left ventricular end systolic pressure (ESP), a reduction in ventricular stiffness, and / or an increase in contractility. (Item 18) 18. The method of claim 17, wherein the lowering of elevated blood pressure is a lowering of blood pressure from greater than about 140 / 90 millimeters of mercury (mmHg) to about 120 / 80 mmHg. (Item 19) Use of isolated recombinant human sNPP1, a fragment or a fusion protein thereof to improve cardiovascular function in a human patient. (Item 20) Use of an isolated recombinant human sNPP1, a fragment or a fusion protein thereof for treating cardiovascular disease in a human patient. (Item 21) Use of an isolated recombinant human sNPP1, a fragment thereof or a fusion protein thereof for the manufacture of a medicament for treating hypertension in a human patient. (Item 22) Use of an isolated recombinant human sNPP1, a fragment thereof or a fusion protein thereof for the manufacture of a medicament for reducing hypertension in a human patient.

[0020] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0021] [Figure 1] Figures 1A-1E show that in ENPP1-treated 2J mice after 6 weeks of treatment, there was no reduction in calcification (Figures 1A-1C), no increase in plasma pyrophosphate (PPi) levels (Figure 1D), and high plasma anti-drug antibody ("ADA") levels (Figure 1E).

[0022] [Diagram 2] FIG. 2 shows that in ENPP1-treated 2J mice, there was no increase in pyrophosphate (PPi) levels 24 hours after the last dose of 6 weeks of treatment.

[0023] [Diagram 3] 3A-3D show that Enpp1 treatment reduces elevated blood pressure in Asj-2J mice.

[0024] [Figure 4] 4A-4B show that Enpp1 treatment reduces elevated left ventricular end-diastolic pressure (EDP) and end-systolic pressure (ESP) in Asj-2J mice.

[0025] [Diagram 5] 5A-5C are pressure volume loop data showing that Enpp1 treatment reduces ventricular stiffness (EDPVR) and increases contractility (PRSW) in Asj-2J mice.

[0026] [Figure 6-1] 6A-6J are echocardiogram data suggesting that Asj-2J mice do not have left ventricular hypertrophy (no changes in LV anterior / posterior diastolic wall thickness, internal dimensions, end-diastolic area, and estimated LV mass). [Figure 6-2]6A-6J are echocardiogram data suggesting that Asj-2J mice do not have left ventricular hypertrophy (no changes in LV anterior / posterior diastolic wall thickness, internal dimensions, end-diastolic area, and estimated LV mass). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0028] For clarity, "NPP1" and "ENPP1" refer to the same protein and are used interchangeably herein.

[0029] "About," as used herein when referring to a measurable value (e.g., amount, temporal duration, etc.), is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the particular value, where such variations are appropriate for practicing the disclosed methods.

[0030] As used herein, the term "normal", when used to modify the term "individual" or "subject", refers to an individual or group of individuals that do not have a particular disease or condition (e.g., cardiovascular disorder) and are not suspected of being at risk or at risk of developing the disease or condition as well. The term "normal" is also used herein to qualify a biological specimen or sample isolated from a normal or healthy individual or subject (or group of such subjects) (e.g., a "normal control sample" or a "normal control").

[0031] As used herein, the term "fragment" refers to an active subsequence of full-length NPP1 in relation to the NPP1 protein. A "fragment" of a protein or peptide can be at least about 20 amino acids long; for example, at least about 50 amino acids long; at least about 100 amino acids long; at least about 200 amino acids long; at least about 300 amino acids long; or at least about 400 amino acids long (and any integer value therebetween). The fragment can range in size from 4 amino acid residues to the entire amino acid sequence minus 1 amino acid. Thus, a protein "comprising at least a portion of the amino acid sequence of SEQ ID NO:1" encompasses full-length NPP1 and its fragments.

[0032] An "isolated" or "purified" soluble NPP1 protein or a biologically active fragment or fusion protein thereof is substantially free of cellular material or other contaminating proteins derived from the cell or tissue source from which the NPP1 protein, biologically active fragment or NPP1 fusion protein is obtained, or, if chemically synthesized, is substantially free of chemical precursors or other compounds. The phrase "substantially free of cellular material" includes preparations of NPP1 protein, biologically active fragment, or NPP1 fusion protein in which the protein has been separated from cellular components of the cell from which it was isolated or recombinantly produced. In one embodiment, the phrase "substantially free of cellular material" includes preparations of NPP1 protein, biologically active fragments, or NPP1 fusion proteins having less than about 30% (by dry weight) of non-NPP1 proteins / fragments / fusion proteins (also referred to herein as "contaminating proteins"), more preferably less than about 20% of non-NPP1 proteins / fragments / fusion proteins, even more preferably less than about 10% of non-NPP1 proteins / fragments / fusion proteins, and most preferably less than about 5% of non-NPP1 proteins / fragments / fusion proteins. When the NPP1 protein, fusion protein, or biologically active fragment thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0033] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and simplicity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). This is true regardless of the breadth of the range.

[0034] As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0035] As used herein, the term "therapeutically effective amount" refers to a non-toxic but sufficient amount of an agent (e.g., hsNPP1 protein) that results in improved treatment, cure, prevention, or amelioration of a disease, disorder, or side effect, or a slowing of the rate of progression of a disease or disorder (e.g., a cardiac disease or disorder), compared to a comparable subject that does not receive a non-toxic but sufficient amount of the agent (e.g., hsNPP1 protein). The term also includes within its scope an amount effective to enhance normal physiological function.

[0036] As used herein, a cardiovascular disease or disorder involves the heart or blood vessels. Exemplary cardiovascular diseases include, but are not limited to, coronary artery disease (CAD) (e.g., angina and myocardial infarction (commonly known as heart attack)), stroke, heart failure, hypertension, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolism, and venous thrombosis.

[0037] As used herein, "hypertension" (also known as high blood pressure) is defined as blood pressure higher than 140 / 90 millimeters of mercury (mmHg). Blood pressure is the force that blood exerts against the walls of blood vessels. A systolic reading of 140 mmHg refers to the pressure when the heart pumps blood around the body. A diastolic reading of 90 mmHg refers to the pressure when the heart relaxes and refills with blood. Normal blood pressure is 120 / 80 mmHg. Hypertension severely impacts quality of life and increases the risk of heart disease, stroke, and death.

[0038] Hypertension that is not caused by another condition or disease is called "primary" or "essential" hypertension. If the hypertension occurs as a result of another condition, it is called "secondary" hypertension. Primary hypertension can result from a number of factors, including the activity of hormones that regulate plasma volume, blood volume and blood pressure, as well as environmental factors (e.g., stress and lack of exercise). Secondary hypertension has a specific cause and is a complication of another problem (e.g., diabetes, kidney disease due to both kidney problems and nerve damage, pheochromocytoma (a rare cancer of the adrenal gland), Cushing's syndrome (which can be caused by corticosteroid drugs), congenital adrenal hyperplasia (a disorder of the adrenal gland that secretes cortisol), hyperthyroidism (overactive thyroid gland), hyperparathyroidism (which affects calcium and phosphorus levels), pregnancy, sleep apnea, obesity, and / or chronic kidney disease (CKD)).

[0039] "Heart rate" (HR) as used herein refers to the rate at which the heart beats as measured by the number of contractions of the heart per minute (bpm). Heart rate can vary according to the physical needs of the body, including the need to take in oxygen and expel carbon dioxide. It is usually equal to or close to the pulse rate measured at any peripheral point. Activities that can cause changes include physical exercise, sleep, anxiety, stress, illness, and drug intake. Many sources cite the normal resting adult human heart rate as in the range of 60 to 100 bpm (e.g., "Target Heart Rates." American Heart Association, 2007). (See, e.g., "Tachycardia, Fast Heart Rate". American Heart Association. 4 Apr 2014). Tachycardia is a fast heart rate, defined as more than 100 bpm at rest (See, e.g., "Tachycardia, Fast Heart Rate". American Heart Association. 2 May 2013). Bradycardia is a slow heart rate, defined as less than 60 bpm at rest. Several studies, as well as expert consensus, indicate that a normal resting adult heart rate is probably closer to the range between 50 and 90 bpm (e.g., Aladin, et al., The American Journal of Cardiology. 114(11): 1701-06(2014-12-01); Hjalmarson, A., et al., The American Journal of Cardiology. 65(9): 547-53(1990-03-01); Spodick, DH, The American Journal of Cardiology. 72(5): 487-88(1993-08-15); and Mason, Jay W. et al., (See Journal of Electrocardiology. 40(3): 228-34(2007-07-01)).

[0040] As used herein, "fractional shortening" (FS) is the percentage reduction in length of the end-diastolic diameter or any diastolic dimension that occurs by end-diastole or is lost during systole.

[0041] As used herein, "arterial blood pressure" in the great vessels is made up of several separate components: systolic and diastolic blood pressure, pulse pressure, and mean arterial pressure.

[0042] When "systemic arterial blood pressure" (SAP) is measured, it is recorded as the ratio of two numbers, expressed as systolic blood pressure / diastolic blood pressure (e.g., 120 / 80 is normal adult blood pressure). Systolic blood pressure is the higher number (typically around 120 mmHg) and reflects the arterial pressure resulting from the ejection of blood during ventricular contraction, or systole. Diastolic arterial blood pressure (DAP) is the lower number (usually around 80 mmHg) and represents the arterial pressure of blood during ventricular relaxation, or diastole.

[0043] As used herein, "mean arterial pressure" (MAP) refers to the average blood pressure in an individual during one cardiac cycle, i.e., the average force driving blood into the blood vessels supplying the tissues (e.g., Zheng L, et al. (July 2008), Stroke. 39(7): 1932-7). The average is a statistical concept, calculated by dividing the sum of values ​​by the number of values. Although complex to measure directly and complex to calculate, MAP can be approximated by adding the diastolic pressure to the pulse pressure or 1 / 3 of (systolic pressure - diastolic pressure). Normally, MAP falls within the range of 70-110 mmHg. When the value falls below 60 mmHg for an extended period of time, the blood pressure is not high enough to ensure circulation to and through the tissues, which results in ischemia, or insufficient blood flow. A condition called hypoxia (insufficient oxygenation of tissues) commonly accompanies ischemia. The term hypoxia refers to low levels of oxygen in systemic arterial blood. Neurons are particularly sensitive to hypoxia and can die or become damaged if blood flow and oxygen supply are not quickly restored.

[0044] As used herein, "pulse pressure" (PP) refers to the difference between the measured systolic and diastolic blood pressures. The up and down fluctuations in arterial pressure result from the pulsatile nature of cardiac output, i.e., the heart beats. Pulse pressure is determined by the interplay of the stroke volume of the heart, the compliance (ability to expand) of the arterial system, and the resistance to flow in the arterial tree. By expanding under pressure, the aorta absorbs some of the force of the blood surge from the heart during the heartbeat. In this way, the pulse pressure drops from what would be present if there was no compliance of the aorta. The loss of arterial compliance that occurs with aging explains the elevated pulse pressure found in older patients.

[0045] "Ventricular pressure" is a measure of blood pressure within the chambers of the heart. The pressure generated in the ventricles of the heart can be measured and used in a variety of ways to understand changes in cardiac function. "Left ventricular pressure" (LVP), which is related to ventricular volume measurements, has a history of being used to characterize the pressure-volume relationship in the beating heart. In addition, LVP is studied to ensure that drugs developed do not negatively alter cardiac contractility. Left ventricular dP / dt is the first derivative of LVP, which is calculated by a software algorithm using calculus. Its peak value, dP / dt max is a general, powerful and sensitive indicator of changes in cardiac contractility when experimental parameters (eg, preload, afterload and heart rate) are well controlled.

[0046] As used herein, cardiac "contractility" is an inherent property of myocardium that affects cardiac performance and can be modified by autonomic systems, circulating hormones, drugs and disease. Evaluation of drug effects on contractility is important in safety evaluation studies, because either increase or decrease can be harmful under certain clinical conditions. Increased contractility dramatically increases cardiac energy consumption, which translates into increased oxygen consumption and increased coronary blood flow. This can have serious consequences in the presence of heart disease and / or coronary insufficiency. Decreased contractility in already diseased hearts can exacerbate the symptoms and consequences of clinical heart failure.

[0047] The treatment methods described herein employ administration of an NPP1 protein, active fragment or fusion protein as described herein to a subject (e.g., a human) to cure, slow, reduce the severity of, or ameliorate one or more symptoms of a cardiovascular disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment.

[0048] The term "treating" includes administering or administering the NPP1 protein, active fragments and fusion proteins of the present invention to a subject, or administering or administering the NPP1 protein, active fragments and fusion proteins of the present invention to a subject with a cardiovascular disorder (e.g., hypertension) for the purpose of curing, curing, mitigating, alleviating, altering, treating, ameliorating, preventing, improving or affecting the cardiovascular disease or disorder. The term "treating" refers to any indication of success in treating or ameliorating an injury, pathology or condition, including any objective or subjective parameter, such as a reduction in symptoms; remission; reduction or making the injury, pathology or condition more tolerable to the subject; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; or improving the physical or mental well-being of the subject. Treatment can be therapeutic or prophylactic. Treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination.

[0049] 1. Treatment method The present invention relates to the use of isolated recombinant human soluble NPP1 ("sNPP1") lacking the N-terminal portion (i.e., lacking the cytosolic and transmembrane domains) and fusion proteins thereof for the treatment of cardiovascular disorders (e.g., hypertension).

[0050] In one embodiment, a method for improving cardiovascular function in a human patient is provided, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), its active fragment, The method includes administering a fusion protein or a fusion protein to a mammal.

[0051] In one embodiment, the improvement is a reduction in elevated blood pressure (e.g., lowering blood pressure above 140 / 90 mmHg to within the normal blood pressure range (about 120 / 80 mmHg)). In another embodiment, the improvement is at least a 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70)% reduction in elevated blood pressure. In another embodiment, the improvement is about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold reduction in elevated blood pressure. In another embodiment, the improvement is normalization of blood pressure (e.g., to about 120 / 80 mmHg). In another embodiment, the improvement is a drop (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%) in left ventricular end-diastolic pressure (EDP) and end-systolic pressure (ESP). In another embodiment, the improvement is about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold drop in left ventricular EDP and ESP. In another embodiment, the improvement is in a reduction (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70%) in ventricular stiffness (EDPVR). In another embodiment, the improvement is about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold reduction in ventricular stiffness. In another embodiment, the improvement is in an increase in contractility (PRSW) (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%). In another embodiment, the improvement is in about a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold increase in contractility.

[0052] In another embodiment, a method is provided for treating a human patient having a cardiovascular disorder, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active flagellar enzyme (AFGHA), or a combination thereof. The present invention includes administering a fusion protein or a fusion protein to a patient having a vascular endothelial cell. Exemplary cardiovascular diseases include, but are not limited to, coronary artery disease (CAD) (e.g., angina and myocardial infarction (commonly known as heart attack)), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolism, and venous thrombosis.

[0053] In another embodiment, a method is provided for treating a human patient having hypertension, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), its active fragment, The method includes administering a fusion protein or a fusion protein to a mammal.

[0054] In another embodiment, a method for treating a human patient having hypertension is provided, the method comprising the steps of: a) identifying a human patient as having hypertension; and b) administering to the identified patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), an active fragment or a fusion thereof. The method includes administering the fusion protein.

[0055] In another embodiment, a method for reducing hypertension in a human patient is provided, the method comprising administering to the patient one or more doses of recombinant human soluble ectonucleotide pyrophosphatase / phosphodiesterase (hsNPP1), its active fragment, The method includes administering a fusion protein or a fusion protein to a mammal.

[0056] In another embodiment, the methods described herein result in a drop in the patient's blood pressure (e.g., from an elevated blood pressure of greater than about 140 / 90 mmHg to within the normal blood pressure range (about 120 / 80 mmHg)). In another embodiment, the patient's blood pressure is reduced by at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70)% after treatment. In another embodiment, the patient's blood pressure is reduced by about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold after treatment. In another embodiment, the patient's blood pressure is normalized (e.g., to about 120 / 80 mmHg) after treatment.

[0057] Generally, the dosage of the fusion protein administered to a subject will vary depending on known factors (e.g., the age, health and weight of the recipient, the type of concurrent treatment, the frequency of treatment, etc.). Typically, the dosage of the active ingredient (i.e., the fusion protein) may be between about 0.0001 and about 50 milligrams per kilogram of body weight. The exact dosage, frequency of administration and duration of treatment can be determined by a physician skilled in the art of administering therapeutic proteins.

[0058] As defined herein, a therapeutically effective amount (i.e., an effective dosage) of a protein ranges from about 0.001 to 50 mg / kg body weight. One of skill in the art will recognize that certain factors may affect the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease, previous treatments, the general health and / or age of the subject, and other existing diseases. Furthermore, treatment of a subject with a therapeutically effective amount of a protein may include a single treatment or, preferably, may include a series of treatments. It will also be recognized that the effective dosage of a protein used for treatment may increase or decrease over the course of a particular treatment.

[0059] As defined herein, a therapeutically effective amount (i.e., an effective dosage) of a protein or polypeptide ranges from about 0.001-50 mg / kg body weight, preferably from about 0.01-25 mg / kg body weight, more preferably from about 0.1-20 mg / kg body weight, and even more preferably from about 1-10 mg / kg, 2-9 mg / kg, 3-8 mg / kg, 4-7 mg / kg, or 5-6 mg / kg body weight. In one embodiment, the hsNPP1 is about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10.0 mg / kg, 11.0 mg / kg, 12.0 mg / kg, 13.0 mg / kg, 14.0 mg / kg, 15.0 mg / kg, 16.0 mg / kg, 17.0 mg / kg, 18.0 mg / kg, 19.0 mg / kg, 20.0 mg / kg, 21.0 mg / kg, 22.0 mg / kg, 23.0 mg / kg, 24.0 mg / kg, 25.0 mg / kg, 26.0 mg / kg, 27.0 mg / kg, 28.0 mg / kg, 29.0 mg / kg, 30.0 mg / kg, 31.0 mg / kg, 32.0 mg / kg, 33.0 mg / kg, 34.0 mg / kg, 35.0 mg / kg, 36.0 mg / kg, 37.0 mg / kg, 38.0 mg / kg, 39.0 mg / kg, 40.0 mg / kg, 41.0 mg / kg, 42.0 mg / kg, 43.0 mg / kg, 44.0 mg / kg, 45.0 mg / kg, 46.0 mg / kg, 47.0 mg / kg, 48.0 mg / kg, 49.0 mg / kg, 50.0 mg / kg, 51.0 mg / kg, 52.0 mg / kg, 0 mg / kg, 23.0 mg / kg, 24.0 mg / kg, 25.0 mg / kg, 26.0 mg / kg, 27.0 mg / kg, 28.0 mg / kg, 29.0 mg / kg, 30.0 mg / kg, 31.0 mg / kg, 32.0 mg / kg, 33.0 mg / kg, 34.0 mg / kg, 35.0 mg / kg, 36.0 mg / kg, 37.0 mg / kg, 38.0 mg / kg, 39.0 mg / kg, 40.0 mg / kg, 41.0 mg / kg, 42.0 mg / kg, 43.0 mg / kg, 44.0 mg / kg, or 45.0 mg / kg. In another embodiment, about 0.5 to about 30 mg, about 0.5 to about 20 mg, about 0.5 to about 10 mg, or about 0.5 to about 5 mg is administered to the patient. In another embodiment, the hsNPP1 is administered in one or more doses including about 1.0 mg / kg to about 5.0 mg / kg hsNPP1. In another embodiment, the hsNPP1 is administered in one or more doses including about 1.0 mg / kg to about 10.0 mg / kg hsNPP1.One of skill in the art will recognize that certain factors can affect the dosage required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present.

[0060] Furthermore, treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody may include a single treatment or, preferably, a series of treatments. In one embodiment, in the range of between about 0.1 and 20 mg / kg body weight, once a week, twice a week, once about 10 days, once about 12 days, once about 14 days, once about 17 days, once about 20 days, once about 25 days, or once about 30 days. In one embodiment, the period between doses of the hsNPP1 is at least 2 days and may be longer (e.g., at least 3 days, at least 1 week, 2 weeks, or 1 month). In another embodiment, a therapeutically effective dose of sNPP1, a biologically active fragment thereof, or a fusion protein thereof is administered to the patient for a period determined by one skilled in the medical arts, between once every 5 days and once every 30 days. In another embodiment, the period is the remainder of the patient's life. In another embodiment, the administration frequency is between once every 5 days and once every 25 days. In another embodiment, the administration frequency is between once every 5 days and once every 21 days. In another embodiment, the administration frequency is between once every 7 days and once every 14 days. hsNPP1, a biologically active fragment thereof, or a fusion protein may be administered once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, or once every 14 days. In some embodiments, hsNPP1, a biologically active fragment thereof, or a fusion protein is administered approximately every week. In other embodiments, sNPP1, a biologically active fragment thereof, or a fusion protein is administered approximately every other week. In one embodiment, the administration frequency is about once every 30 days. It will also be recognized that the effective dosage of the soluble sNPP1 protein, biologically active fragment thereof, or fusion protein used for treatment may increase or decrease over the course of a particular treatment.

[0061] In one embodiment, about 1 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 2 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 3 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 4 mg / kg of sNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 5 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 6 mg / kgm of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 7 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 8 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 9 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week. In one embodiment, about 10 mg / kg of hsNPP1, biologically active fragment or fusion protein is administered to the patient once a week.

[0062] The hsNPP1, biologically active fragment or fusion protein can be administered, for example, by subcutaneous injection, intramuscular injection, and intravenous (IV) infusion or injection.

[0063] In one embodiment, hsNPP1, biologically active fragments or fusion proteins are administered intravenously by IV infusion by any useful method.In one example, hsNPP1, biologically active fragments or fusion proteins can be administered intravenously through a peripheral line.In another example, hsNPP1, biologically active fragments or fusion proteins can be administered intravenously through a peripherally inserted central catheter.

[0064] In another embodiment, hsNPP1, biologically active fragments or fusion proteins are administered intravenously by IV injection. In another embodiment, hsNPP1, biologically active fragments or fusion proteins are administered via intraperitoneal injection. In another embodiment, hsNPP1, biologically active fragments or fusion proteins are administered by subcutaneous injection. In another embodiment, hsNPP1, biologically active fragments or fusion proteins are administered by intramuscular injection.

[0065] In yet another embodiment, the hsNPP1, biologically active fragment or fusion protein is administered via a pharma- ceutically acceptable capsule of the therapeutic protein, e.g., the capsule can be an enteric coated gelatin capsule.

[0066] In one embodiment, the method includes administering a soluble NPP1 protein or NPP1 fusion protein of the invention, alone or in combination with other agents. Exemplary therapeutic agents include thiazide diuretics (e.g., hydrochlorothiazide (Microzide) or chlorthalidone), beta-blockers (e.g., acebutolol (Sectral) or atenolol (Tenormin)), angiotensin-converting enzyme (ACE) inhibitors (e.g., lisinopril (Zestril), benazepril (Lotensin), or captopril (Capoten)), angiotensin II receptor blockers (ARBs) (e.g., candesartan (Atacand) or losartan (Cozaar)), calcium channel blockers (e.g., amlodipine (Norvasc) or diltiazem (Cardizem)). , renin inhibitors (e.g., aliskiren (Tekturna)), alpha blockers (e.g., doxazosin (Cardura) or prazosin (Minipress)), alpha beta blockers (e.g., carvedilol (Coreg) or labetalol (Trandate)), centrally acting medications (e.g., clonidine (Catapres, Kapvay), guanfacine (Intuniv, Tenex) and methyldopa), vasodilators (e.g., hydralazine and minoxidil), and / or aldosterone antagonists (e.g., spironolactone (Aldactone) or eplerenone (Inspra)).

[0067] In one embodiment, the isolated sNPP1 protein, fragments and fusion proteins can be administered before, after or simultaneously with the agent, or can be administered simultaneously with other known treatments.The simultaneous administration of the isolated sNPP1 protein, fragments and fusion proteins of the present invention with other therapeutic agents can provide two agents that function through different mechanisms to produce enhanced therapeutic effects.Such simultaneous administration can solve the problem caused by the development of resistance to drugs.

[0068] 2. sNPP1 The present invention employs soluble NPP1 (e.g., hsNPP1) having a biologically active NPP1 domain of NPP1 (i.e., an NPP1 component that includes at least one extracellular catalytic domain for the pyrophosphatase and / or phosphodiesterase activity of naturally occurring NPP1). The soluble NPP1 protein of the present invention includes at least the NPP1 domains essential for carrying out its pyrophosphatase and / or phosphodiesterase activity.

[0069] In one embodiment, soluble NPP1, its fragments and fusion proteins can form functional homodimers or monomers. In another embodiment, soluble NPP1 protein or its NPP1 fusion proteins can be assayed for pyrophosphatase activity and the ability to increase pyrophosphate levels in vivo.

[0070] Various amino acid sequences of soluble NPP1 compounds, fusion partners, and fusion proteins suitable for use according to the methods provided herein are described herein. SEQ ID NO: 5 shows the amino acid sequence of soluble NPP1 including amino acids 107-925 of SEQ ID NO: 1. SEQ ID NO: 6 shows the amino acid sequence of soluble NPP1 including amino acids 187-925 of SEQ ID NO: 1. SEQ ID NO: 7 shows the amino acid sequence of the Fc region of human IgG1 including the hinge region. SEQ ID NO: 8 shows the amino acid sequence of the Fc of human IgG1 including a partial hinge region. SEQ ID NO: 9 shows the amino acid sequence of an NPP1-Fc fusion protein, the NPP1 component of which includes SEQ ID NO: 5 and the Fc sequence includes the hinge region. SEQ ID NO: 10 shows the amino acid sequence of an NPP1-Fc fusion protein, the soluble NPP1 of which includes SEQ ID NO: 5 and the Fc sequence includes a partial hinge region. SEQ ID NO: 1 shows the amino acid sequence of an NPP1-Fc fusion protein. The soluble NPP1 comprises SEQ ID NO: 6, and the Fc sequence comprises a hinge region. SEQ ID NO: 12 shows the amino acid sequence of an NPP1-Fc fusion protein. The soluble NPP1 comprises SEQ ID NO: 6, and the Fc sequence comprises a partial hinge region.

[0071] Preferred soluble NPP1 proteins and NPP1 fusion proteins of the invention are enzymatically active in vivo (e.g., in humans). In one embodiment, the soluble protein comprises an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the following sequence:

[0072] [ka]

[0073] SEQ ID NO:2 is the amino acid sequence of sNPP1 including the cysteine-rich region, the catalytic region and the C-terminal region.

[0074] Any desired enzymatically active form of soluble NPP1 can be used in the methods described herein.The enzymatically active sNPP1 can increase pyrophosphate (PPi) levels in appropriate enzyme assays and can be assayed for pyrophosphatase activity, phosphodiesterase activity, or pyrophosphatase and phosphodiesterase activity.Typically, the sNPP1 comprises at least the N-terminal cytosolic domain and NPP1 component that lacks the transmembrane domain of naturally occurring transmembrane NPP1.

[0075] SEQ ID NO: 1 is the amino acid sequence of the wild-type NPP1 protein. The cytosolic and transmembrane regions are underlined. Potential N-glycosylation sites are bolded. The bolded amino acid motif "PSCAKE" (SEQ ID NO: 17) is the start of soluble NPP1, including the cysteine-rich region.

[0076] In a preferred aspect, the NPP1 component comprises the cysteine-rich region (amino acids 99-204 of SEQ ID NO:1) and the catalytic region (amino acids 205-591 of SEQ ID NO:1) of naturally occurring human NPP1. Typically, the NPP1 component also comprises the C-terminal region (amino acids 592-925 of SEQ ID NO:1) and has the amino acid sequence of SEQ ID NO:2. However, the C-terminal region can be shortened if desired. Thus, a preferred NPP1 component comprises the cysteine-rich region and catalytic region of human NPP1 (amino acids 99-591 of SEQ ID NO:1), or the cysteine-rich region, catalytic region and C-terminal region of human NPP1 (SEQ ID NO:2). Other preferred NPP1 components comprise only a portion of the cysteine-rich domain and have the sequence of amino acids 107-925 of SEQ ID NO:1 or amino acids 187-925 of SEQ ID NO:1. The cysteine-rich region of NPP1 (i.e., amino acids 99-204 of SEQ ID NO:1) can promote dimerization of the sNPP1. The sNPP1 can be in the form of a monomer of a functional homodimer, including a fusion protein.

[0077] The amino acid sequence of the NPP1 component can be a variant of a naturally occurring NPP1 sequence, provided that the NPP1 component is enzymatically active. NPP1 variants are enzymatically active and have at least 80%, at least 85%, at least 90%, at least 95%, and more preferably at least 96% amino acid sequence identity with the corresponding portion of human NPP1 (e.g., over the length of the cysteine-rich region, the catalytic region, the C-terminal region, the cysteine-rich region + catalytic region, the cysteine-rich region + catalytic region + C-terminal region). Preferred NPP1 variants have at least 90%, preferably at least 95%, and more preferably at least 97% amino acid sequence identity to (i) the amino acid sequence of residues 205-591 of SEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ ID NO: 1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO: 1, (iv) the amino acid sequence of residues 107-925 of SEQ ID NO: 1, or (v) the amino acid sequence of residues 187-925 of SEQ ID NO: 1. Suitable locations for amino acid variations are well known from NPP1 structural studies and analysis of disease-associated mutations in NPP1. For example, the following amino acid substitutions occur in certain disease-associated mutations that reduce NPP1 enzymatic activity, and amino acid variations at these positions should be avoided: Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471, His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586; Tyr659, Glu668, Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901 (see, e.g., Jansen, S. et al., Structure 20:1948-1959(2012)).

[0078] In one embodiment, the soluble NPP1 protein can be a fusion protein that is recombinantly fused or chemically linked (e.g., covalent, ionic, hydrophobic, and van der Waals forces) to a fusion partner. In another embodiment, the fusion protein has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:4. SEQ ID NO:4 is the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO:4). The Fc sequence is underlined.

[0079] To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., sNPP1 amino acid sequence of SEQ ID NO:2; amino acids 107-925 of SEQ ID NO:1 or amino acids 187-925 of SEQ ID NO:1). The amino acid residues or nucleotides at corresponding amino acid positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0080] The comparison of sequences and the determination of percent identity between two sequences can be achieved using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol Biol 1970, 48, 444-453) algorithm incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either Blosum 62 matrix or PAM250 matrix, and gap weighting of 16, 14, 12, 10, 8, 6, or 4 and length weighting of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acids is determined using the E. Meyers and W. Miller (CABIOS, 1989, 4, 11-17) algorithm incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, gap length penalty of 12, and gap penalty of 4.

[0081] The sNPP1 may consist of or essentially consist of the NPP1 component as described herein. Alternatively, the sNPP1 may be in the form of a fusion protein comprising the NPP1 component and, optionally, one or more other polypeptides (referred to as fusion partners) through a suitable linker in each case, or in the form of a conjugate between the NPP1 component and another molecule (e.g., PEG). When the sNPP1 is in the form of a fusion protein, each fusion partner is preferably located C-terminally to the NPP1 component. Without wishing to be bound by any particular theory, it is believed that fusion proteins comprising the NPP1 component comprising the cysteine-rich region and the catalytic region, and one or more fusion proteins located C-terminally to the NPP1 component, are preferred over other configurations of NPP1 fusion proteins, because they may be expressed at sufficient levels and be sufficiently stable to be used as therapeutic proteins.

[0082] Any suitable fusion partner can be included in the fusion protein. Advantageously, many fusion partners are well known in the art, which can provide certain advantages (e.g., reduced aggregation and immunogenicity, increased solubility, improved expression and / or stability, and improved pharmacokinetic and / or pharmacodynamic performance). See, for example, Strohl, WR BioDrugs 29:215-239 (2015). For example, it is well known that albumin, albumin fragments or albumin variants (e.g., human serum albumin and its fragments or variants) can be incorporated into fusion proteins, and such fusion proteins can be easily purified, stable, and have improved plasma half-life. Suitable albumins, albumin fragments and albumin variants that can be used in sNPP1 fusion proteins are disclosed, for example, in WO 2005 / 077042A2 and WO 03 / 076567A2, each of which is incorporated herein by reference in its entirety. Fusions to human transferrin are also known to improve half-life. See, for example, Kim BJ et al., J Pharmacol Expr Ther 334(3):682-692(2010); and WO 2000 / 020746. Peptides that bind to albumin or transferrin (e.g., antibodies or antibody fragments) can also be used. See, for example, EP 0486525 B1, US 6,267,964 B1, WO 04 / 001064A2, WO 02 / 076489A1, WO 01 / 45746, WO 2006 / 004603, and WO 2008 / 028977.Similarly, immunoglobulin Fc fusion proteins are well known.See, for example, Czajkowsky DM et al., EMBO Mol Med 4(10):1015-1028(2012), U.S. Patent No. 7,902,151; and U.S. Patent No. 7,858,297 (the entire teachings of which are incorporated herein by reference in their entirety).The fusion protein may also include a CTP sequence (see also Fares et al., Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992, 89, 4304-4308; and Furuhashi et al., Mol Endocrinol 1995, 9, 54-63). Preferably, the fusion partner is an immunoglobulin Fc (e.g., Fc or human IgG1). The Fc may include the CH1, CH2 and CH3 of human IgG1, and optionally, if desired, the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO: 13)) or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ ID NO: 14) or PKSCDKTHTCPPCP (SEQ ID NO: 15)). In some fusion proteins, the Fc may comprise the CH2 and CH3 of human IgG1, or, if desired, the Fc of human IgG2 or human IgG4.

[0083] Preferably, the sNPP1 fusion protein comprises an NPP1 component and a peptide that increases the half-life of the fusion protein, most preferably the Fc of an immunoglobulin (e.g., Fc or human IgG1). As used herein, a "protein that increases the half-life of a fusion protein" refers to a protein that, when fused to a soluble NPP1 or biologically active fragment, increases the half-life of the soluble NPP1 polypeptide or biologically active fragment compared to the half-life of the soluble NPP1 polypeptide alone or the biologically active fragment of NPP1 alone.

[0084] In one embodiment, the half-life of the NPP1 fusion protein is increased by 50% compared to the half-life of the NPP1 polypeptide or biologically active fragment alone. In another embodiment, the half-life of the NPP1 fusion protein is increased by 60% compared to the half-life of the NPP1 polypeptide or biologically active fragment alone. In another embodiment, the half-life of the NPP1 fusion protein is increased by 70% compared to the half-life of the NPP1 polypeptide or biologically active fragment alone. In another embodiment, the half-life of the NPP1 fusion protein is increased by 80% compared to the half-life of the NPP1 polypeptide or biologically active fragment alone. In another embodiment, the half-life of the NPP1 fusion protein is increased by 90% compared to the half-life of the NPP1 polypeptide or biologically active fragment alone.

[0085] In another embodiment, the half-life of the NPP1 fusion protein is increased by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the half-life of the NPP1 polypeptide or biologically active fragment alone.Methods for determining the half-life of proteins or fusion proteins are well known in the art.For example, Zhou et al., Determining Protein Half-Lives, Methods in Molecular Biology 2004, 284, 67-77 discloses many methods for testing the half-life of proteins.If desired, the fusion protein can be conjugated to a polymer or other suitable compound that extends half-life (e.g., polyethylene glycol (PEG)) and conjugated to the NPP1 fusion protein.

[0086] In one embodiment, the peptide that increases the half-life of the fusion protein is a CTP sequence (see also Fares et al., 2010, Endocrinol., 151(9):4410-4417; Fares et al., 1992, Proc. Natl. Acad. Sci, 89(10):4304-4308; and Furuhashi et al., 1995, Molec. Endocrinol., 9(1):54-63).

[0087] In another embodiment, the peptide that increases the half-life of the fusion protein is the Fc domain of an Ig.

[0088] Fusion partners can also be selected to target the fusion protein to a desired site of clinical or biological importance (e.g., a site of calcification). For example, peptides with high affinity for bone are described in U.S. Pat. No. 7,323,542, the entire teachings of which are incorporated herein by reference. Peptides that can increase protein targeting to sites of calcification can include a consecutive stretch of at least about four acidic amino acids (e.g., glutamic acid or aspartic acid). Typically, peptides that target the fusion protein to sites of calcification include between 4 and 20 consecutive acidic amino acids (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids selected from glutamic acid and aspartic acid). The peptide may consist of only glutamic acid residues, only aspartic acid residues, or a mixture of glutamic acid and aspartic acid residues. Particularly preferred moieties for targeting to sites of calcification are Asp 10 (Sequence number 18).

[0089] In one embodiment, an NPP1 fusion protein of the invention comprises an NPP1 polypeptide and a moiety that increases targeting of the protein to sites of calcification (eg, a contiguous stretch of acidic amino acids (eg, glutamic acid or aspartic acid)).

[0090] Suitable peptide linkers for use in fusion proteins are well known and typically adopt a flexible, extended conformation and do not interfere with the function of the NPP1 component or its fusion partner. Peptide linker sequences may include Gly, His, Asn and Ser residues in any combination. Useful peptide linkers include, but are not limited to, poly-Gly, poly-His, poly-Asn, or poly-Ser. Other near neutral amino acids (e.g., Thr and Ala) may also be used in the linker sequence. Amino acid sequences that may be usefully used as linkers include those disclosed in Maratea et al., Gene 1985, 40, 39-46; Murphy et al., Proc Natl Acad Sci USA 1986, 83, 8258-8262; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Other suitable linkers can be derived from naturally occurring proteins (e.g., the hinge region of an immunoglobulin). A preferred synthetic linker is (Gly4Ser) n where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 19). Preferably, n is 3 or 4. For example, in some embodiments, the linker is (Gly4Ser)3 (SEQ ID NO: 16) and the fusion protein comprises a linker having the amino acid sequence GlyGlyGlyGlySerGlyGlyGlyGlyGlyGlyGlySerGlyGlyGlySer (SEQ ID NO: 16). Typically, the linker is 1 to about 50 amino acid residues in length, or 1 to about 25 amino acids in length. Frequently, the linker is between about 8 and about 20 amino acids in length.

[0091] A preferred NPP1 fusion protein comprises, from N-terminus to C-terminus, an NPP1 component, optionally a linker, an Fc region of an immunoglobulin (e.g., human IgG1 Fc, optionally including a hinge or a portion thereof), optionally a second linker, and optionally a targeting moiety. Thus, the Fc region and the optional targeting moiety, if present, are each located C-terminally to the NPP1 component. The NPP1 component preferably comprises the cysteine-rich region and catalytic domain of NPP1, lacks the N-terminal cytosolic and transmembrane domains, and optionally comprises a C-terminal region.

[0092] A preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, catalytic domain and C-terminal region of human NPP1; and an Fc region of a human immunoglobulin (including hinge). Preferably, the Fc region is derived from human IgG1. In certain embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:3. SEQ ID NO:3 is the amino acid sequence of sNPP1-Fc fusion protein.

[0093] A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO:3.

[0094] Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, catalytic domain and C-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO: 16)); and a human immunoglobulin Fc region (including hinge). Preferably, the Fc region is derived from human IgG1.

[0095] Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component including the cysteine-rich domain, catalytic domain and C-terminal region of human NPP1; an Fc region of a human immunoglobulin (including a hinge or a portion thereof); and a moiety that targets the fusion protein to sites of calcification. Preferably, the Fc region is derived from human IgG1. Preferably, the moiety that targets the fusion protein to sites of calcification is Asp. 10 (SEQ ID NO: 18). More preferably, the Fc region is derived from human IgG1 and the moiety that targets the fusion protein to sites of calcification is Asp 10 (SEQ ID NO: 18). In certain embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO: 4.

[0096] Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising the cysteine-rich domain, catalytic domain and C-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO: 16)); an Fc region of a human immunoglobulin (including a hinge or a portion thereof); and a moiety that targets the fusion protein to sites of calcification. Preferably, the Fc region is derived from human IgG1. Preferably, the moiety that targets the fusion protein to sites of calcification is Asp 10 (SEQ ID NO: 18). More preferably, the Fc region is derived from human IgG1 and the moiety that targets the fusion protein to sites of calcification is Asp 10 (Sequence number 18).

[0097] Another preferred fusion protein comprises, from N-terminus to C-terminus, an NPP1 component comprising a portion of the cysteine-rich domain, the catalytic domain and the C-terminal region of human NPP1; optionally a linker (e.g., (Gly4Ser)3 (SEQ ID NO:16)); and a human immunoglobulin Fc region (including a hinge or a portion thereof). Preferably, the Fc region is derived from human IgG1. In certain embodiments, the fusion protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Preferred fusion proteins of this type have the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0098] In a particularly preferred aspect, a fusion protein of SEQ ID NO: 3 is administered according to the methods described herein. In another particularly preferred aspect, a fusion protein of SEQ ID NO: 4 is administered according to the methods described herein. In another particularly preferred aspect, a fusion protein of SEQ ID NO: 9 is administered according to the methods described herein. In another particularly preferred aspect, a fusion protein of SEQ ID NO: 10 is administered according to the methods described herein. In another particularly preferred aspect, a fusion protein of SEQ ID NO: 11 is administered according to the methods described herein. In another particularly preferred aspect, a fusion protein of SEQ ID NO: 12 is administered according to the methods described herein.

[0099] Fusion proteins of the present invention can be prepared using standard methods, including recombinant techniques or chemical conjugation well known in the art. Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those skilled in the art, and standard molecular biology and biochemistry manuals can be consulted to select the appropriate protocol to use without undue experimentation. See, for example, Sambrook et al., 1989, "Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor, the contents of which are incorporated herein by reference in their entirety.

[0100] The isolated recombinant human sNPP1, fragments thereof, and fusion proteins can be produced in any useful protein expression system, including, but not limited to, cell culture (e.g., CHO cells, COS cells, HEK203), bacteria (e.g., Escherichia coli (E. coli)), and transgenic animals (including, but not limited to, mammals and birds (e.g., chicken, quail, duck, and turkey)). For expression, a construct will encode the sNPP1 and contain a suitable signal sequence (e.g., such as from human Ig heavy chain, NPP2, NPP4, NPP7, or human serum albumin) in frame with the sequence of sNPP1 and operably linked to suitable expression control elements.

[0101] sNPP1 (including its fusion protein and physiologically acceptable salt forms) is typically formulated into a pharmaceutical composition for administration according to the methods described herein. The pharmaceutical composition typically includes a pharma- ceutical acceptable carrier or excipient. Compositions including such carriers (including conjugate molecules) are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 14th ed., Mack Publishing Co., Easton, PA), the entire teachings of which are incorporated herein by reference). The carrier may include a diluent. In one embodiment, the pharmaceutical carrier may be liquid and the fusion protein may be in the form of a solution. The pharmaceutical carrier may be a wax, a fat, or an alcohol. In another embodiment, the pharma- ceutical acceptable carrier may be a solid in the form of a powder, a lyophilized powder, or a tablet. In one embodiment, the carrier may include a liposome or a microcapsule. The pharmaceutical composition may be in the form of a sterile lyophilized powder for injection upon reconstitution with a diluent. The diluent may be water for injection, bacteriostatic water for injection, or sterile saline. The lyophilized powder may be produced by lyophilizing a solution of the fusion protein to produce the protein in a dry form. As is known in the art, the lyophilized protein generally has increased stability and a longer shelf life than a liquid solution of the protein.

[0102] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the scope of the following claims. Any combination of the embodiments disclosed in any plurality of dependent claims or examples is contemplated to be within the scope of this disclosure.

[0103] References The disclosures of each and every U.S. and foreign patents, and pending patent applications and publications mentioned herein, as well as the contents of the sequence listing and any figures, are specifically incorporated herein by reference in their entirety. EXAMPLES

[0104] The present invention is further illustrated by the following examples, which are for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner.

[0105] Example 1: Evaluation of hEnpp1 treatment on cardiovascular hemodynamics and function in Asj-2J mice Experiments were performed to determine whether Enpp1 treatment improves elevated blood pressure and left ventricular hypertrophy in Asj-2J mice. asj-2J Mice were given 2100U / kg hEnpp1-Fc (TSAC 2.7) subcutaneously every other day (EOT) over the course of 6 weeks. Vehicle-treated wild-type and Asj-2j mice served as controls. At 7 weeks of age, the mice were shipped to a third-party analyst for evaluation of primary and secondary endpoints. Administration was continued by the analyst throughout the study (i.e., until week 11). Primary endpoints included: echocardiograms (cardiac dimensions, heart rate ("HR") and fractional shortening ("FS")), end-stage hemodynamics (systolic arterial pressure ("SAP"), diastolic arterial pressure ("DAP"), mean arterial pressure ("MAP"), pulse pressure ("PP"), heart rate ("HR"), left ventricular pressure ("LVP")), pressure-volume loops (cardiac compliance and contractility), and aortic and vibrissae calcification. Secondary endpoints included ENPP1 protein, pyrophosphate (PPi), and Including ADA.

[0106] As shown in Figures 1A-1C, there was no reduction in calcification, no increase in plasma pyrophosphate (PPi) levels (Figure 1D), and no increase in high plasma ADA levels (Figure 1E) in ENPP1-treated Asj-2J mice after 6 weeks of treatment. As shown in Figure 2, there was no increase in PPI levels in ENPP1-treated Asj-2J mice 24 hours after the last dose of that 6-week treatment. However, as shown in Figures 3A-3D, ENPP1 treatment lowered the elevated blood pressure in Asj-2J mice. Furthermore, as shown in Figures 4A-4B, ENPP1 treatment lowered the elevated left ventricular end-diastolic and end-systolic pressures in ASJ-2J mice.

[0107] Pressure-volume loops were used to assess intact cardiac function, as well as cardiac compliance (stiffness) and contractility. Pressure-volume loops provide simultaneous real-time measurements of both LV pressure and LV volume during a complete cardiac cycle. "ESPVR" describes the maximum pressure that can be generated in the ventricle at any given LV volume (e.g., it is a measure of myocardial contractility). "EDPVR" describes the passive filling curve for the ventricle and is a measure of passive chamber stiffness (e.g., it is a measure of myocardial contractility). "PRSW" describes the relationship between stroke work and EDV (e.g., it is a measure of ventricular stiffness). As shown in Figures 5A-5C, pressure-volume loops show that ENPP1 treatment reduces ventricular stiffness (EDPVR) and increases contractility (PRSW) in ASJ-2J mice.

[0108] An echocardiogram ("ECHO") is a method of examining the heart's chambers, valves, walls, and associated Echocardiograms are tests that use high-frequency sound waves (ultrasound) to create images of the blood vessels (aorta, arteries, and veins) involved in the left ventricular hypertrophy. As shown in Figures 6A-6J, echocardiogram results suggest that Asj-2J mice do not have left ventricular hypertrophy, as there were no changes in LV anterior / posterior diastolic wall thickness, internal dimensions, end-diastolic area, and estimated LV mass.

[0109] In summary, Enpp1 treatment improved the elevated blood pressure (SBP, DBP, MBP) and left ventricular pressure observed in Asj-2J mice. HR was not affected. Pressure-volume loop data suggest that Asj-2J have increased ventricular stiffness (EDPVR) and reduced contractility (PRSW), both of which were rescued by Enpp1 treatment. ECHO analysis of Asj-2J mice (normalized to BW) showed no changes in cardiac dimensions and area, suggesting no signs of left ventricular hypertrophy. Overall, a significant improvement in cardiovascular function was observed with Enpp1 treatment, although there was no reduction in calcification.

[0110] This suggests that the improvements in cardiovascular function may be due to effects unrelated to calcification (eg, adenosine signaling).

[0111] Example 2: Evaluation of the feasibility of hemodynamic procedures in young Asj-2J mice It was hypothesized that if Enpp1 could improve blood pressure in 4-week-old Asj-2J mice (before elevated calcification was detected), Enpp1-induced improvement in blood pressure would be independent of improvement in calcification, thus suggesting a possible role of Enpp1 in adenosine signaling and myointimal proliferation. Thus, preliminary studies were performed to evaluate the feasibility of hemodynamic procedures in young WT and Asj-2J mice.

[0112] The primary objectives were to determine the feasibility of catheter insertion for hemodynamic procedures in 4-week-old WT and Asj-2J mice, to determine whether blood pressure is elevated in 4-week-old Asj-2J mice similar to 8-week-old mice, and to determine whether isoproterenol (a beta-adrenergic agonist that increases heart rate and myocardial contractility) or phenylephrine (a adrenergic agonist that increases heart rate and myocardial contractility) are associated with increased hemodynamics. The aim of this study was to determine whether challenge with a vasopressor (a vasopressor that increases blood pressure but does not affect myocardial contractility and stroke volume) would reveal any further differences in the responsiveness of WT and Asj-2J mice. Primary endpoints included: terminal hemodynamics (e.g., SAP, DAP, MAP, PP, HR, LVP readouts) at baseline and after isoproterenol / phenylephrine challenge. Secondary endpoints included ECHO as a back-up if hemodynamics were not feasible (heart dimensions, HR, FS). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Claims

[Claim 1] The invention described in the specification.

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