Use of integrin a5b1 inhibitors in treatment of pulmonary hypertension and heart failure

Abstract

The present invention provides methods and compositions for treating lung or heart diseases and / or diseases associated with increased expression or activity of integrin alpha 5 beta 1 comprising administering to a subject an integrin alpha 5 beta 1 inhibitor. Also provided herein are methods of treating or preventing pulmonary hypertension, pulmonary arterial hypertension (PAH), right ventricular failure, heart failure, and fibrosis.

Description

Background Art

[0001] Fibronectin (Fn) is an extracellular matrix protein that coordinates complex cell adhesion and signaling during tissue development, remodeling, and diseases such as hypertension and heart failure through cell surface integrin receptors (fibronectin binds integrin (e.g., a5b1)). Heart failure (HF) is a debilitating disease in which abnormal function of the heart leads to inadequate perfusion of body tissues and organs. Hypertension is responsible for a variety of deleterious effects and has high morbidity and mortality, including heart failure. One form of hypertension is pulmonary arterial hypertension (PAH). PAH is a rare but devastating disease in which the normally low pulmonary artery pressure is elevated due to vasoconstriction and pulmonary vascular remodeling. Vasoconstriction and vascular remodeling increase the workload on the right side of the heart, leading to right heart hypertrophy, fibrosis, and ultimately heart failure.

[0002] Current treatments include vasodilators that target calcium channels or endothelin receptors. New approaches to treat pulmonary hypertension, PAH, heart failure, and related diseases are needed. Summary of the invention

[0003] The present invention is based in part on the discovery that integrin signaling promotes cell proliferation and resistance to apoptosis that contributes to pulmonary arteriolar and arterial (PA) vascular remodeling. In the right ventricle (RV), integrin signaling leads to maladaptive hypertrophy and fibrosis, which can lead to right ventricular failure in pulmonary hypertension. The use of α5β1 inhibitors (e.g., small molecule compounds and antibodies) provided herein reverses PA vascular remodeling and prevents RV dysfunction.

[0004] In one aspect, the present invention provides a method of treating a subject's heart or lung disease comprising administering an integrin α5β1 inhibitor. As described herein, a5b1 inhibition not only maintains cardiac output, but also prevents RV maladaptation. Thus, the present invention provides methods of treating hypertension or cardiac disorders, including but not limited to heart failure, RV failure (e.g., RV failure due to RV volume overload caused by septal defects or valvular regurgitation), RV pressure overload due to other WHO groups of pulmonary hypertension or outflow tract obstruction (such as pulmonary stenosis), and RV cardiomyopathy due to infarction, arrhythmia, or fibrosis.

[0005] The present invention provides a method for treating diseases involving α5β1 function using integrin α5β1 inhibitors (e.g., small molecule compounds and antibodies). In one aspect, the present invention provides a method for treating a disease associated with increased expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor.

[0006] In some embodiments, the disease is characterized by a World Health Organization (WHO) panel.

[0007] In some embodiments, the disease is pulmonary hypertension, WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, and WHO Group 5 pulmonary hypertension.

[0008] In some embodiments, the disease is characterized by the World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by the WHO functional classification based on cardiac function. In some embodiments, the disease is pulmonary hypertension, WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension. In some embodiments, the disease is heart failure or right ventricular failure.

[0009] In some embodiments, the disease is fibrosis. In some embodiments, the disease is cardiac fibrosis.

[0010] In one aspect, the invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject comprising administering an integrin α5β1 inhibitor.

[0011] In some embodiments, the integrin α5β1 inhibitor is a Fab, a single-chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single domain antibody-linked Fab (s-Fab), an antibody, or a combination thereof.

[0012] In some embodiments, the integrin α5β1 inhibitor is an antibody drug conjugate (ADC).

[0013] In some embodiments, the integrin α5β1 inhibitor is an antibody.

[0014] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5.

[0015] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin β1.

[0016] In some embodiments, the integrin α5β1 inhibitor is an anti-CD29 / β1 integrin / ITGB1 monoclonal antibody. In some embodiments, the integrin α5β1 inhibitor antibody is the anti-CD29 / β1 integrin / ITGB1 monoclonal antibody OS2966. In some embodiments, the integrin α5β1 inhibitor antibody competes with OS2966 for integrin binding.

[0017] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to the integrin α5β1 heterodimer.

[0018] In some embodiments, the antibody is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A. In some embodiments, the antibody is volociximab (M200). In some embodiments, the antibody is PF-04605412. In some embodiments, the antibody is MINT1526A.

[0019] In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from the group consisting of volociximab (M200), P1D6, PF-04605412, MINT1526A, BMA5, BMB5, BMC5, HA5, JBS5, LS-C509074, LS-C24758, 1D9, 22B5, 24C7, 2D2, 3C2.2A8, 3C5, 5B11, MOR04055, MOR04624, P8D4, MOR04974, MOR04977, SG / 19, and 18C12.

[0020] In some embodiments, the antibody is an integrin α5β1 antibody that competes with the anti-α5β1 antibody clone 339.1 for integrin binding.

[0021] In some embodiments, the antibody is the integrin α5β1 antibody 3C5 or 5B11. In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from 3C5 and 5B11. 3C5 and 5B11 are integrin α5β1 antibodies described in WO2010072740A2, which is incorporated herein by reference.

[0022] In some embodiments, the α5β1 antibody is an anti-integrin α5 (CD49e) antibody (clone mAb16). In some embodiments, the α5β1 antibody is an antibody that competes with the anti-integrin α5 (CD49e) antibody (clone mAb16) for integrin binding.

[0023] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

[0024] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5.

[0025] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin β1.

[0026] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to the integrin α5β1 heterodimer.

[0027] In some embodiments, the integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0028]

[0029] in:

[0030] R 1 is hydrogen or OMe;

[0031] R 2 is optionally C 1-4 Alkoxy substituted C 1-4 Alkyl; and

[0032] R 3 It is C 1-4 Alkyl or C 3-5 Cycloalkyl.

[0033] In some embodiments, R2 is methyl or ethyl.

[0034] In some embodiments, R3 is methyl, ethyl, isopropyl, or cyclopropyl.

[0035] In some embodiments, R1 is hydrogen.

[0036] In some embodiments, the compound of formula (I) is selected from:

[0037]

[0038] as well as

[0039]

[0040] In some embodiments, the integrin α5β1 inhibitor is a compound of formula (II).

[0041]

[0042] In some embodiments, the integrin α5β1 inhibitor is administered by inhalation, orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intrapulmonary. In some embodiments, the integrin α5β1 inhibitor is administered by inhalation.

[0043] In some embodiments, the methods of treatment described herein further comprise administering an additional therapy to the subject. In some embodiments, the methods of treatment comprise administering an integrin α5β1 inhibitor in combination with one or more additional therapies. In some embodiments, the methods of treatment further comprise administering an integrin α5β1 inhibitor and a second therapy to the subject. In some embodiments, the methods of treatment further comprise administering an integrin α5β1 inhibitor and two additional therapies to the subject.

[0044] In some embodiments, the additional therapy is selected from the group consisting of anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostanoids, prostanoid receptor agonists, soluble guanylate cyclase stimulators, and / or surgery.

[0045] In some embodiments, the additional therapy is oxygen, warfarin, furosemide, bumetanide, bendroflumethiazide, metolazone, spironolactone, amiloride, digoxin, nifedipine, diltiazem, nicardipine, amlodipine, ambrisentan, bosentan, macitentan, sildenafil, tadalafil, epoprostenol, iloprost, treprostinil, riociguat, selexipag, surgery, pulmonary endarterectomy, and / or atrial septostomy.

[0046] In some embodiments, the additional therapy is macitentan and / or tadalafil.

[0047] In some embodiments, the additional therapy is a SGLT2 inhibitor. In some embodiments, the SGLT2 inhibitor is dapagliflozin.

[0048] In some embodiments, administration of an integrin α5β1 inhibitor reduces proliferation and / or survival of pulmonary artery smooth muscle cells (PASMC), pulmonary arteries, right ventricular fibroblasts (RVFb), vascular fibroblasts, adventitial fibroblasts, cardiomyocytes, and / or endothelial cells.

[0049] In some embodiments, the method includes administering an integrin α5β1 inhibitor to modulate the level of a biomarker in a subject. In some embodiments, the biomarker is an N-terminal fragment (NT) of a BNP precursor (NT-proBNP), TNFα, IFNγ, IL-6, IL-8, and IL-10. In some embodiments, the biomarker is a brain natriuretic peptide (BNP) or an N-terminal fragment (NT) of a BNP precursor (NT-proBNP).

[0050] In some embodiments, the methods of treatment described herein comprise administering an integrin α5β1 inhibitor at a dose of 1 mg to 1000 mg.

[0051] In some embodiments, the integrin α5β1 inhibitor is administered daily. In some embodiments, the integrin α5β1 inhibitor is administered twice a day.

[0052] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in treating pulmonary hypertension in a subject in need of such treatment, comprising administering to the subject an integrin α5β1 inhibitor and a pharmaceutical excipient.

[0053] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in treating pulmonary arterial hypertension (PAH) in a subject in need of such treatment, comprising administering to the subject an integrin α5β1 inhibitor and a pharmaceutical excipient.

[0054] In some embodiments, the present invention provides an integrin α5β1 inhibitor for use in treating right ventricular failure in a subject in need of treatment, comprising administering to the subject an integrin α5β1 inhibitor and a pharmaceutical excipient.

[0055] definition

[0056] In order to more readily understand the present invention, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0057] "Active agent" and "therapeutic agent" refer to molecules (e.g., small molecule compounds, peptides, antibodies or antibody fragments, etc.) that produce preventive or therapeutic effects on diseases or disease states. Active agent can refer not only to a single active agent, but also to a combination of two or more different active agents.

[0058] "Relieve" and "improve" refer to a process whereby the severity of signs or symptoms of a disease is reduced. Importantly, signs or symptoms may be alleviated, but not eliminated. A therapeutically effective dose is expected to reduce the severity of disease signs or symptoms, thereby alleviating and improving disease signs or symptoms.

[0059] As used herein, the term "affinity" refers to a characteristic of a binding interaction between a binding moiety (e.g., an integrin α5β1 inhibitor) and a target (e.g., α5β, αvβ1), and indicates the strength of the binding interaction. In some embodiments, the measure of affinity is expressed as a dissociation constant (K D In some embodiments, the binding moiety has a high affinity for the target (e.g., less than about 10 -7 M, less than about 10 -8 M or less than about 10 -9 M of K D In some embodiments, the binding moiety has a low affinity for the target (e.g., greater than about 10 -7 M, above about 10 -6 M, above about 10 -5 M or above about 10 -4 M of K D ).

[0060] As used herein, when applied to one or more values ​​of interest, the term "approximately" or "approximately" refers to a value similar to the reference value. In certain embodiments, the term "approximately" or "approximately" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the reference value, unless otherwise specified or otherwise apparent from context (unless the numeral exceeds 100% of the possible value).

[0061] As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable region, for example, an amino acid sequence of an immunoglobulin variable domain or an immunoglobulin variable domain sequence is provided. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. An antibody generally includes three complementary determining regions (abbreviated as CDR) in an immunoglobulin light chain and three complementary determining regions (CDR) in an immunoglobulin heavy chain. The three CDRs in an immunoglobulin light chain are respectively referred to as CDR1, CDR2, and CDR3 from the N-terminal side. The three CDRs in an immunoglobulin heavy chain are also respectively referred to as CDR1, CDR2, and CDR3 from the N-terminal side. "CDR" can be identified according to the accumulation, AbM, contact, and / or conformational definitions of Kabat, Chothia, Kabat, and Chothia, or any CDR determination method known in the art. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv and dAb fragments) as well as intact antibodies, such as intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and subtypes thereof). The light chain of an immunoglobulin may be of the kappa or lambda type.

[0062] "Combination therapy" and "synergistic therapy" refer to the administration of a first active agent and at least one different second active agent as part of a specific treatment regimen, intended to provide a beneficial effect through the joint action of at least two active agents. The beneficial effects of the combination may include, but are not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The administration of the combined therapeutic agents may be performed over a determined period of time (e.g., minutes, hours, days or weeks, depending on the selected combination). In some embodiments, the integrin α5β1 inhibitor is administered by inhalation. Combination therapy does not encompass the administration of two or more different therapeutic agents as part of a separate monotherapy regimen, which accidentally and arbitrarily leads to the combination therapy of the present invention. Combination therapy includes the administration of at least two different therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at a different time, and the administration of at least two different therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration may be by, for example, administering to the subject a single capsule of each therapeutic agent having a fixed ratio, or in a separate capsule for each therapeutic agent. The sequential or substantially simultaneous administration of each therapeutic agent may be affected by any suitable route, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption through mucosal tissues. The two different therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while the second therapeutic agent of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered by inhalation, orally, or all therapeutic agents can be administered by intravenous injection. Unless otherwise indicated, the order in which the therapeutic agents are administered is not important.

[0063] Combination therapy also includes further administration of different therapeutic agents as described above in combination with other biologically active ingredients and non-drug therapies (e.g., surgery or physical therapy). In the case where combination therapy includes non-drug therapy, the non-drug therapy can be performed at any appropriate time, as long as a beneficial effect is obtained from the combined effect of the therapeutic agent combination and the non-drug therapy. For example, in appropriate cases, when the non-drug therapy is temporarily separated from the administration of the therapeutic agent, it may be a few days or even a few weeks, and a beneficial effect can still be obtained.

[0064] "Compound" refers to a molecule and encompasses not only a specific molecular entity, but also, if the compound is an active agent or drug, pharmaceutically acceptable pharmacologically active analogs thereof, including but not limited to active metabolites, amides, conjugates, esters, hydrates, polymorphs, prodrugs, salts, solvates and other such derivatives, analogs, including deuterated analogs and analogs containing radioactive atoms or other labeled moieties, and related compounds. In some embodiments, the compound is a small molecule compound.

[0065] "Dosage form" refers to any form of a pharmaceutical composition for administration to a subject, typically a human or animal suffering from a disease or condition to be treated. "Dose" refers to the amount of an active agent. "Unit dosage form" refers to a dosage form containing a fixed amount of an active agent. A single tablet or capsule is a unit dosage form. Multiple unit dosage forms may be administered to provide a therapeutically effective dose. Dosage forms may include combinations of dosage forms.

[0066] "Effective amount" and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an active agent to achieve the desired therapeutic effect.

[0067] "Integrin inhibitor" or "integrin α5β1 inhibitor" or "VLA5 inhibitor" refers to a molecule that can bind to integrin α5 (ITGA5) and has α5β1 integrin and / or ITGA5 inhibitory activity. In some embodiments, the inhibitory activity includes inhibiting the binding of α5β1 integrin and / or ITGA5 to smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes and / or other cells of mesenchymal origin. In some embodiments, the inhibitory activity includes inhibiting the migration of smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes and / or other cells of mesenchymal origin. In some embodiments, the inhibitory activity includes inhibiting the differentiation of smooth muscle cells, fibroblasts, stellate cells, myofibroblasts, pericytes and / or other cells of mesenchymal origin. In some embodiments, the inhibitory activity includes inhibiting the synthesis and / or deposition of extracellular matrix.

[0068] As used herein, “K D ” refers to the dissociation constant, which is composed of K d With K a The ratio (K d / K a ) and expressed as molar concentration (M). K D The value can be determined using methods established in the art, for example by using surface plasmon resonance, or using a biosensor system such as system).

[0069] As used herein, "pulmonary arterial hypertension (PAH)" refers to a rare disease in which the normally low pulmonary artery pressure is elevated due to vasoconstriction and pulmonary vascular remodeling. This in turn increases the workload of the right side of the heart, leading to right heart hypertrophy, fibrosis, and ultimately heart failure.

[0070] As used herein, "peptide" refers to a peptide or polypeptide comprising a plurality of amino acids. The terms "peptide" and "polypeptide" can be used interchangeably. The amino acid sequence or variant thereof can be part of a larger peptide, i.e., part of a peptide extended with one or more additional amino acids at the N-terminus and / or the C-terminus. The amino acid sequence of the peptide of the present invention or a variant thereof can also be N-terminally and / or C-terminally modified, preferably by comprising an N-terminal extension group and / or a C-terminal extension group. Alternatively, the amino acid sequence or variant thereof is N-terminally and / or C-terminally extended.

[0071] "Pharmaceutically acceptable" means that it is not biologically undesirable, i.e., the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effect or interacting in a deleterious manner with any other component of the composition containing it. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it means that the carrier or excipient has met the required standards of toxicology and manufacturing testing, or it is included in the Inactive Ingredient Guide established by the US Food and Drug Administration.

[0072] "Pharmaceutically acceptable salt" refers to a derivative of an active agent produced by preparing an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (such as amines), alkali metal or organic salts of acidic residues (such as carboxylic acids), and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed by non-toxic inorganic or organic acids. Pharmaceutically acceptable salts include salts formed when acidic protons present in the parent compound are replaced by metal ions (e.g., alkali metal ions, alkaline earth metal ions, or aluminum ions); or coordinated with organic bases (such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.). Pharmaceutically acceptable salts include solvent addition forms (solvates) or crystalline forms (polymorphs) of the same salts as defined herein.

[0073] "Pharmacologically active" derivatives or analogs (or "activity") are those derivatives or analogs that possess the same type of pharmacological activity as the parent compound, to a substantially comparable degree.

[0074] "Preventing" or "prevent" refers to completely avoiding the onset of clinically apparent disease progression or slowing the onset of a preclinically apparent stage of disease in an at-risk individual. Prevention includes prophylactic or preventive treatment of a person at risk of disease.

[0075] As used herein, "selective binding," "selectively binds," "specifically binds," or "specifically binds," with respect to a binding moiety and a target, means that the binding moiety preferentially associates with the target rather than with non-target entities. Some degree of nonspecific binding may occur between a binding moiety and a non-target. In some embodiments, a binding moiety selectively binds to a target if the binding between the binding moiety and the target is greater than 2-fold, greater than 5-fold, greater than 10-fold, or greater than 100-fold as compared to the binding of the binding moiety to the non-target. In some embodiments, a binding moiety selectively binds to a target if the binding affinity is less than about 10 -5 M, less than about 10 -6 M, less than about 10 -7 M, less than about 10 -8 M or less than about 10 -9 M, the binding portion selectively binds to the target.

[0076] "Signs" are indicators of illness and include things that a doctor, nurse, or other health care professional can observe.

[0077] As used herein, "small molecule" refers to a molecule with a relatively low molecular weight, whether naturally occurring or artificially produced (e.g., by chemical synthesis). Preferred small molecules are biologically active because they produce local or systemic effects in animals, preferably mammals, and more preferably humans. In certain preferred embodiments, the small molecule is a drug, and the small molecule is referred to as a "drug molecule" or "drug" or "therapeutic agent". The small molecule can have a MW of less than or equal to about 5 kDa. In other embodiments, the drug molecule has a MW of less than or equal to about 1.5 kDa.

[0078] As used herein, the term "subject" refers to any subject for which diagnosis, prognosis or treatment is desired. For example, the subject can be a mammal, such as a human or non-human primate (such as an ape, monkey, gorilla or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle or cow.

[0079] A "subject in need thereof" refers to a human or other mammal suitable for treatment with an active agent. A subject in need thereof may be suffering from a disease, or may be at increased risk of suffering from a disease relative to the general population.

[0080] “Symptoms” are physical signs or other indicators of disease, illness, or injury. They may be felt or noticed by the person experiencing the symptoms or by other people, including non-health care professionals.

[0081] As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic molecule (e.g., an integrin a5b1 inhibitor described herein) that imparts a therapeutic effect to the treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication of the effect or feels the effect). Specifically, a "therapeutically effective amount" refers to the amount of a therapeutic molecule or composition that effectively treats, ameliorates, or prevents a specific disease or condition, or exhibits a detectable therapeutic or preventive effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or alleviating the severity or frequency of disease symptoms. A therapeutically effective amount may be administered in a dosing regimen that may include multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, according to the route of administration, in combination with other agents. In addition, the specific therapeutically effective amount (and / or unit dose) for any particular subject may depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific agent employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or excretion or metabolic rate of the specific therapeutic molecule employed; the duration of the treatment; and similar factors well known in the medical arts.

[0082] "Treating" or "treat" describes the management and care of a patient to combat a disease, condition, or disorder, and includes the administration of an active agent to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0083] "Pulmonary hypertension (PH)" includes diseases that share the defining element of mean pulmonary artery pressure ≧ 25 mm Hg. PH is classified and divided into 5 groups and 5 categories characterized by the World Health Organization (WHO). In some embodiments, pulmonary hypertension is WHO functional class I pulmonary hypertension, WHO functional class II pulmonary hypertension, WHO functional class III pulmonary hypertension, WHO functional class IV pulmonary hypertension, or pulmonary arterial hypertension (PAH). In some embodiments, the disease is pulmonary hypertension, WHO group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO group 2 pulmonary hypertension, WHO group 3 pulmonary hypertension, WHO group 4 pulmonary hypertension, and WHO group 5 pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The drawings are for illustrative purposes only and are not limiting.

[0085] Figure 1 Shown is the RNA expression of human integrins expressed in PAH patient-derived pulmonary artery smooth muscle cells.

[0086] Figure 2A-2B The results show that the anti-integrin a5b1 antibody M200 ( Figure 2A ) and P1D6( Figure 2B ) treated hPASMCs.

[0087] Figure 3 Exemplary proliferation of hPASMCs on plastic and fibronectin-coated plates is shown.

[0088] Figures 4A-4C Figure 2 shows the p-FAK ( Figure 4B ) and PCNA( Figure 4B ) is an exemplary western blot analysis of the expression levels of .

[0089] Figures 5A-5C Shown are exemplary proliferation (Ki67) and apoptosis (Annexin V) of PAH patient-derived pulmonary artery smooth muscle cells (PAH-PASMCs) treated with integrin α5β1 inhibitors (MRT, SMi, P1D6, or M200) on fibronectin (Fb)-coated plates.

[0090] Figure 6A-6B Shown are exemplary Western blot analyses measuring the expression levels of ITGα5, ITGαV, ITGβ1, p-FAK, survivin, p-ERK / ERK, MCM2, PLK1, and PCNA in hPASMCs following exposure to SMi at 0.25 μM, 1 μM, and 4 μM on Fb-coated plates.

[0091] Figures 7A-7C Shown are exemplary proliferation (Ki67) and apoptosis (Annexin V) of hPASMCs treated with an integrin α5β1 inhibitor (SMi) on Fb-coated plates.

[0092] Figure 8 Shown are exemplary histological staining of EVG, αSMA, PCNA, and C3C, which demonstrate vascular remodeling in sugar / hypoxia (SuHx)-induced PAH rats.

[0093] Figures 9A-9F Exemplary results demonstrating the efficacy of SMi in SuHx rats following treatment with SMi and / or endothelin receptor antagonists and PDE5 are shown. Cardiac output ( Fig.9A ), stroke volume ( Fig. 9B ), the thickness of the cell wall of the culture medium ( Fig. 9C ), vascular remodeling ( Fig.9D ),proliferation( Fig.9E) and apoptosis ( Fig.9F ). Dashed lines indicate disease windows.

[0094] Figures 10A-10C Shown are exemplary results demonstrating reduced cardiac hypertrophy and cardiac fibrosis in SuHx rats following treatment with SMi and / or SoC (Maci and Tadalafil (Tada)).

[0095] Fig.11 Shown are exemplary histological staining of EVG, αSMA, PCNA, and C3C demonstrating vascular remodeling in the monocrotaline (MCT) rat pulmonary hypertension model.

[0096] Figures 12A-12D Exemplary results of treatment with SMi in MCT rats are shown. Occlusion scores ( Fig. 12A ), vascular remodeling ( Fig. 12B ), apoptosis ( Fig. 12C ) and proliferation ( Fig.12D ).

[0097] Fig.13 Exemplary pharmacokinetics of SMi and MRT treatment in MCT rats are shown.

[0098] Figures 14A-14E The results show that after SMi and MRT treatment in MCT rats, the EVG ( Fig.14A ), the thickness of the cell wall of the culture medium ( Fig. 14B ), mean pulmonary artery pressure ( Fig. 14C ), cardiac output ( Fig.14D ) and right ventricular area fraction change ( Fig.14E ) demonstrated exemplary improved vascular and cardiac function as demonstrated by histological staining of .

[0099] Figures 15A-15C Exemplary histological results are shown, which demonstrate improvement in cardiomyocyte hypertrophy and right ventricular (RV) fibrosis in MCT rats treated with SMi and / or SOCs (eg, macitentan (Maci) and tadalafil (Tada)).

[0100] Figures 16A-16D Shown are the study design and exemplary histological results demonstrating improvements in hypertrophy and right ventricular (RV) fibrosis in the pulmonary artery ligation (PAB) rat model treated with SMi.

[0101] Fig.17 Representative echocardiographic images of PAB rats treated with SMi are shown.

[0102] Figures 18A-18HWe demonstrated that integrin α5β1 inhibition with SMi showed direct improvements in cardiac function over time compared to vehicle controls in the RV PAB model. Fig.18A ), stroke volume ( Fig.18B )、TAPSE( Fig. 18C )、S wave( Fig.18D )、RVFAC( Fig.18E )、RVEDD( Fig.18F )、CI( Figure 18G )、PG( Fig.18H )

[0103] Figures 19A-19E Exemplary cardiac parameters following right heart catheterization of PAB rats treated with SMi are shown.

[0104] Figures 20A-20D Exemplary expression of integrin-binding fibronectin in PAH patient samples is shown.

[0105] Figures 21A-21C showed a dose-dependent reduction in PE-induced hypertrophy of adult rat cardiomyocytes after exposure to SMi treatment.

[0106] Figures 22A-22B Exemplary results showing inhibition of proliferation and activation of human PAH-RVFb by a5bl.

[0107] Figures 23A-23C Exemplary exemplary results are shown demonstrating improvement in hemodynamics and vascular remodeling in MCT rats with established PAH following treatment with SMi alone or in combination with SoCs (macitenttan (Maci) and tadalafil (Tada)) in the MCT rat pulmonary hypertension model.

[0108] Figures 24A-24G An exemplary study design is shown ( Fig.24A ) and results, which confirm the efficacy of integrin α5β1 inhibitors (SMi and anti-α5β1 antibody 339.1) in SuHx mice. Measurement of cardiac output ( Fig. 24B ), stroke volume ( Fig.24C ), right ventricular systolic pressure ( Fig.24D ), mean pulmonary artery pressure ( Fig.24E )、Fulton index( Fig.24F ) and vascular remodeling ( Figure 24G ). Dashed lines indicate disease windows.

[0109] Figures 25A-25B Shows people ( Fig.25A ) and mouse tissues ( Fig.25B ) Exemplary protein levels of a5bl integrin in ). DETAILED DESCRIPTION

[0110] The present invention provides methods and compositions for treating diseases associated with increased expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor. In one aspect, the present invention provides a method of treating a cardiac or pulmonary disease in a subject, comprising administering an integrin α5β1 inhibitor. In some embodiments, the disease is characterized by a World Health Organization (WHO) panel.

[0111] In some embodiments, the disease is pulmonary hypertension, WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, and WHO Group 5 pulmonary hypertension.

[0112] In some embodiments, the disease is characterized by the World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by the WHO functional classification based on cardiac function. In some embodiments, the disease is pulmonary hypertension, WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension. In some embodiments, the disease associated with increased expression or activity of integrin α5β1 is heart failure or right ventricular failure.

[0113] Due to the limitations of current treatments for pulmonary hypertension, there is still great interest and demand for additional or alternative therapies for treating, stabilizing, preventing and / or delaying pulmonary hypertension. Various approaches have been developed to obtain more effective and / or less toxic drugs for treating pulmonary hypertension. However, these approaches still have serious side effects, and the resulting drugs generally exhibit short half-lives and low bioavailability.

[0114] The present invention provides, in particular, methods and compositions for treating diseases associated with increased expression or activity of integrin α5β1. Many normal physiological and disease processes require cells to contact other cells and / or the extracellular matrix. Cell-matrix and cell-cell adhesion are mediated by several protein families including integrins, selectins, cadherins, and immunoglobulins, and promote a variety of normal cell functions, such as proliferation, migration, differentiation, or survival. Cell adhesion is also key to a range of pathologies, so pharmacological disruption of cell adhesion interactions can provide a mechanism for therapeutic intervention. Members of the integrin superfamily of adhesion molecules play an important role in acute and chronic disease states (such as cancer, inflammatory diseases, stroke, and neurodegenerative disorders). Therefore, integrins represent a complex biological field.

[0115] The integrin superfamily of cell surface receptors is formed by a number of structurally and functionally related surface glycoproteins, each receptor existing as a 20 heterodimer of non-covalently linked α and β subunits. At least 18 different α and 8 β subunits have been identified in mammals, which are known to form more than 24 different receptors. Each integrin specifically interacts with defined extracellular ligands through a linear 25 adhesion motif, including extracellular matrix proteins such as fibronectin, vitronectin, collagen, and cell surface molecules such as VCAM, ICAM, and PECAM.

[0116] Integrin α5β1 (a5b1 or α5β1) is composed of α5 (a5 or α5) and βl (b1 or β1) subunits. The a5 subunit forms a specific dimer with the β1 subunit and is widely expressed in most tissues. Integrin a5b1 mediates cell adhesion almost exclusively through interaction with fibronectin, binding via a short arginine-glycine-30 aspartate (RGD) adhesion motif. However, endothelial cells and platelets can bind to fibrin via a5bl. The interaction of a5b1 with fibronectin plays an important role in physiological and pathological angiogenesis and vascular integrity. Although endothelial cells express multiple integrins, a5b1 is important for endothelial cell survival on temporary matrices in vitro, thereby inhibiting apoptosis and promoting proliferation. a5b1 expression is upregulated in tumor vasculature and in patients with pulmonary hypertension. Consistent with the critical functional role of receptor-ligand pairing, the a5b1 ligand fibronectin is also upregulated in tumor tissue and during wound healing.

[0117] As demonstrated herein, inhibiting integrin α5β1 by blocking the activity of α5β1 or inhibiting α5β1 fibronectin binding can effectively prevent and treat pulmonary hypertension, PAH, heart failure and right ventricular failure. The present invention provides a variety of compounds (e.g., small molecule compounds and antibodies) that inhibit this interaction. These compounds are collectively referred to herein as "integrin a5b1 inhibitors".

[0118] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may apply to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0119] Pulmonary hypertension

[0120] Pulmonary hypertension (PH) is a syndrome characterized by increased pulmonary artery pressure. PH is hemodynamically defined as a pulmonary artery systolic pressure greater than 30 mm Hg or a pulmonary artery mean pressure greater than 25 mm Hg. See Zaiman et al., Am. J. Respir. Cell Mol. Biol. 33: 425-31 (2005). In addition, due to increased pressure, PH damages large and small pulmonary arteries. The smallest blood vessel walls thicken and are no longer able to transfer oxygen and carbon dioxide normally between the blood and the lungs. Over time, pulmonary hypertension causes thickening of the pulmonary arteries and narrowing of the blood flow channels. Once pulmonary hypertension occurs, the right side of the heart works harder to compensate; however, the increased effort causes it to become larger and thicker. The proliferation of smooth muscle and endothelial cells, which usually exist in a quiescent state, leads to remodeling of blood vessels and occlusion of the lumen of the pulmonary vasculature. As blood is pumped through the reduced lumen area, this causes a gradual increase in pulmonary pressure. An enlarged right ventricle puts people at risk of pulmonary embolism because blood tends to pool in the ventricle and legs. If clots form in the pooled blood, they may eventually travel and lodge in the lungs. The gradual increase in pressure also places extra workload on the right ventricle, which eventually fails and leads to premature death in these patients.

[0121] Due to PH, various pathological changes occur in the pulmonary artery. Sustained vasoconstriction and structural remodeling of pulmonary vessels are the main characteristics of PH. Pulmonary vascular smooth muscle cells undergo a phenotypic switch from a contractile normal phenotype to a synthetic phenotype, resulting in cell growth and matrix deposition. Histological examination of tissue samples from patients with PH showed intimal thickening as well as smooth muscle cell hypertrophy, especially for those vessels with a diameter of <100 μm. In addition, abnormal smooth muscle cells often overexpress endothelin and serotonin transporters, which may play a role in the development of PH.

[0122] The most common symptom of pulmonary hypertension is initially shortness of breath on exertion. Some people feel dizzy or tired, and angina-like chest pain is common. General weakness is another problem because the body tissues are not getting enough oxygen. Other symptoms, such as coughing and wheezing, may be caused by the underlying lung disease. Edema, especially in the legs, may occur because fluid may leak from the veins into the tissues, which is a sign of cor pulmonale. Some people with pulmonary hypertension have connective tissue disorders, especially scleroderma. When people have both conditions (pulmonary hypertension and a connective tissue disorder), Raynaud's phenomenon often appears before the symptoms of pulmonary hypertension appear, sometimes years earlier.

[0123] Treatments for certain types of pulmonary hypertension are usually directed at underlying lung disease. Currently, treatment options available for those patients with PH target cellular dysfunction that causes vasculature constriction. Therapies such as prostaglandins, phosphodiesterase-5 inhibitors, and endothelin receptor antagonists work primarily by causing pulmonary vasodilation. Vasodilators (such as calcium channel blockers, nitric oxide, and prostacyclin) are usually helpful for pulmonary hypertension associated with scleroderma, chronic liver disease, and HIV infection. In contrast, these drugs have not been proven to be effective for people with pulmonary hypertension due to underlying lung disease. For most people with unexplained pulmonary hypertension, vasodilators (such as prostacyclin) significantly reduce blood pressure in the pulmonary artery. Intravenous administration of prostacyclin through a catheter surgically implanted in the skin can improve quality of life, increase survival rates, and reduce the urgency of lung transplantation. Unfortunately, over time, many patients respond poorly or stop responding to these therapies. The only remaining option is single or double lung transplantation to treat PH. Although there is evidence that available therapies have secondary effects on vascular remodeling, there is currently no therapy for abnormal cell proliferation in PAH.

[0124] In some embodiments, pulmonary hypertension is pulmonary venous hypertension (PVH). In some embodiments, PVH is caused by left heart failure. In some embodiments, pulmonary hypertension is pulmonary hypertension associated with respiratory disorders and / or hypoxia. In some embodiments, pulmonary hypertension is pulmonary hypertension due to chronic thrombotic and / or embolic diseases. In some embodiments, pulmonary hypertension is mixed pulmonary hypertension. In some embodiments, mixed pulmonary hypertension is related to sarcoidosis, eosinophilic granuloma, X histiocytosis, lymphangioleiomyomatosis or pulmonary vascular compression (e.g., adenosis, tumor or fibrotic enteritis). In some embodiments, pulmonary hypertension is related to chronic obstructive pulmonary disease (COPD). In some embodiments, pulmonary hypertension is related to pulmonary fibrosis. In some embodiments, pulmonary hypertension is related to cardiac fibrosis. In some embodiments, pulmonary hypertension is early pulmonary hypertension or late pulmonary hypertension.

[0125] In some embodiments, the subject suffers from pulmonary venous hypertension (PVH). In some embodiments, PVH is caused by left heart failure. In some embodiments, the subject suffers from respiratory disorders and / or hypoxia. In some embodiments, the subject suffers from chronic thrombotic and / or embolic diseases. In some embodiments, the subject suffers from sarcoidosis, eosinophilic granuloma, X-histiocytosis, lymphangioleiomyomatosis or pulmonary vascular compression (e.g., due to adenopathy, tumor or fibrotic enteritis). In some embodiments, the subject suffers from chronic obstructive pulmonary disease (COPD). In some embodiments, the subject suffers from pulmonary fibrosis. In some embodiments, the subject suffers from cardiac fibrosis. In some embodiments, the subject suffers from early pulmonary hypertension or late pulmonary hypertension.

[0126] In some embodiments, one or more symptoms of pulmonary hypertension are improved. In some embodiments, pulmonary hypertension is delayed. In some embodiments, pulmonary hypertension is prevented. In some embodiments, the methods of treatment provided herein reduce pulmonary pressure. In some embodiments, the methods of treatment provided herein inhibit and / or reduce abnormal cell proliferation in the pulmonary artery.

[0127] In some embodiments, pulmonary hypertension is characterized by the World Health Organization (WHO) panel.

[0128] In some embodiments, the pulmonary hypertension is WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, and WHO Group 5 pulmonary hypertension.

[0129] In some embodiments, pulmonary hypertension is characterized by the World Health Organization (WHO) classification system. In some embodiments, pulmonary hypertension is characterized by the WHO functional classification based on cardiac function. In some embodiments, pulmonary hypertension is characterized by WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension.

[0130] In some embodiments, the subject has pulmonary hypertension as characterized by the World Health Organization (WHO) panel.

[0131] In some embodiments, the subject has WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, or WHO Group 5 pulmonary hypertension.

[0132] In some embodiments, the subject suffers from a disease characterized by the World Health Organization (WHO) classification system. In some embodiments, the subject suffers from a disease characterized by the WHO functional classification based on cardiac function. In some embodiments, the subject suffers from pulmonary hypertension classified by WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension.

[0133] In some embodiments, the pulmonary hypertension is associated with pulmonary capillary hemangiomatosis.

[0134] Pulmonary arterial hypertension (PAH)

[0135] In one aspect, the present invention provides a method for treating pulmonary arterial hypertension (PAH) comprising administering an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies disclosed herein). PAH is characterized by a progressive increase in pulmonary vascular resistance, leading to right ventricular overload and ultimately heart failure. PAH leads to progressive obstruction and decreased compliance of the pulmonary artery (PA), leading to right ventricular (RV) failure and premature death. Like cancer cells, PA smooth muscle cells (PASMC) and endothelial cells (PAEC) exhibit hyperproliferation and resistance to apoptosis in response to increased PA stiffness caused by extracellular matrix (ECM) remodeling. Integrin signaling can promote PAH-PASMC and PAH-PAEC proliferation and resistance to apoptosis, thereby promoting PA vascular remodeling, while in the RV, maladaptive hypertrophy and fibrosis lead to RV failure in PAH. The present invention is based in part on the discovery that a5b1 integrin inhibition can reverse PA vascular remodeling in PAH and prevent RV dysfunction.

[0136] PAH is a chronic condition that involves all layers of the pulmonary vasculature. Vasoconstriction, structural changes in the pulmonary vascular wall (vascular remodeling), and thrombosis lead to increased pulmonary vascular resistance in PAH. Structural and functional changes in the endothelium lead to endothelial dysfunction. Increased vasoconstrictor factors (e.g., endothelin) and reduced vasodilator capacity (e.g., reduced prostacyclin) lead to vasoconstriction and increased pulmonary vascular resistance. Current treatments that seek to address vasoconstriction may slow the progression of PAH or improve clinical symptoms for a limited time, but they have not yet been shown to significantly reduce overall PAH morbidity and mortality. The underlying structural changes in the pulmonary vasculature (vascular remodeling) are not affected by these treatments.

[0137] Vascular remodeling that occurs in PAH is characterized by proliferative and obstructive changes involving many cell types, including endothelial cells, smooth muscle cells, and fibroblasts. Vascular remodeling itself can manifest itself, for example, as thickening of the tunica media of the pulmonary vessels due to smooth muscle cell hyperplasia and hypertrophy, the formation of a neointima composed of smooth muscle cells and / or myofibroblasts, and / or the formation of plexiform lesions, which consist of localized proliferation of endothelial cells, smooth muscle cells, lymphocytes, and mast cells. Vascular remodeling leads to obstruction of the vascular lumen, resulting in pulmonary hypertension. Therapies directed at addressing the proliferative aspects of PAH are needed.

[0138] In some embodiments, pulmonary hypertension is pulmonary arterial hypertension (PAH). In some variations, PAH is idiopathic PAH. In some variations, PAH is familial PAH. In some variations, PAH is associated with persistent pulmonary hypertension of the newborn. In some variations, PAH is associated with pulmonary veno-occlusive disease.

[0139] In some embodiments, pulmonary hypertension is associated with pulmonary disease. In some embodiments, pulmonary disease is idiopathic pulmonary fibrosis (IPF) or interstitial pneumonia (IIP). IPF is an idiopathic interstitial pneumonia (IIP), which is also an interstitial lung disease (also referred to as diffuse parenchymal lung disease (DPLD)). Interstitial lung disease involves alveolar epithelium, pulmonary capillary endothelium, basement membrane, perivascular and perilymphoid tissue. Other forms of idiopathic interstitial pneumonia include nonspecific interstitial pneumonia (NSIP), desquamative interstitial pneumonia (DIP) and acute interstitial pneumonia (AIP). Examples of known causes of interstitial lung disease include sarcoidosis, hypersensitivity pneumonitis, pulmonary Langerhans cell histiocytosis, asbestosis and collagen vascular disease (such as scleroderma and rheumatoid arthritis).

[0140] Thus, in some embodiments, the subject suffers from a lung disease, such as idiopathic pulmonary fibrosis (IPF) or interstitial pneumonia (IIP). In some embodiments, the subject suffers from idiopathic interstitial pneumonia (IIP), diffuse parenchymal lung disease (DPLD), nonspecific interstitial pneumonia (NSIP), desquamative interstitial pneumonia (DIP) or acute interstitial pneumonia (AIP). In some embodiments, the subject suffers from sarcoidosis, hypersensitivity pneumonitis, pulmonary Langerhans cell histiocytosis, asbestosis or collagen vascular disease (such as scleroderma or rheumatoid arthritis).

[0141] Pulmonary fibrosis is the formation or development of excess fibrous connective tissue in the lungs.

[0142] Heart failure (HF)

[0143] In one aspect, the present invention provides a method for treating heart failure (HF) comprising administering an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies disclosed herein). Heart failure refers to any disease characterized by the inability of the heart to pump enough blood to the body. A physiological state in which cardiac output is insufficient to meet the body's needs or can only meet the body's needs at higher filling pressures. HF has many potential causes, including myocardial infarction, coronary artery disease, valvular disease, hypertension, and myocarditis. Chronic heart failure is associated with neurohormonal activation and altered autonomic control. Although these compensatory neurohormonal mechanisms provide valuable support to the heart under normal physiological circumstances, they also play an important role in the development and subsequent progression of HF.

[0144] For example, one of the body's main compensatory mechanisms for reduced blood flow in HF is to increase the amount of salt and water retained by the kidneys. Retaining salt and water (rather than excreting it through urine) increases the volume of blood in the bloodstream and helps maintain blood pressure. However, the large volume of blood also causes the heart muscle, especially the ventricles, to become larger. As the ventricles become larger, the wall thickness decreases and the heart contracts less, resulting in decreased heart function. Another compensatory mechanism is vasoconstriction of the arterial system, which raises blood pressure to help maintain adequate perfusion, thereby increasing the workload that the heart must pump.

[0145] In low ejection fraction (EF) heart failure, the high pressure in the heart is due to the high pressure required by the body to try to maintain adequate peripheral perfusion. However, this derangement becomes more severe because the heart becomes weak due to such high pressures. The pressure in the left atrium may exceed 25 mmHg, at which stage fluid from the blood flowing through the pulmonary circulatory system leaks or flows out of the pulmonary capillaries into the interstitial spaces and into the alveoli, causing pulmonary congestion and, if untreated, acute pulmonary edema syndrome and death.

[0146] Right ventricular failure

[0147] In one aspect, the invention provides a method of treating right ventricular (RV) failure comprising administering an integrin α5β1 inhibitor (e.g., small molecule compounds and antibodies disclosed herein). In some embodiments, RV failure is caused by RV volume overload. In some embodiments, RV volume overload is caused by septal defects or valvular regurgitation. In some embodiments, RV pressure overload is caused by pulmonary hypertension or other WHO groups of outflow obstruction. In some embodiments, RV failure is caused by pulmonary artery stenosis.

[0148] In one aspect, the invention provides a method of treating RV cardiomyopathy comprising administering an integrin α5β1 inhibitor (eg, small molecule compounds and antibodies disclosed herein). In some embodiments, RV cardiomyopathy is caused by infarction, arrhythmia, or fibrosis.

[0149] In some embodiments, a5b1 inhibition (e.g., using an integrin α5β1 inhibitor) maintains cardiac output. In some embodiments, a5b1 inhibition prevents RV maladaptation. In some embodiments, administration of an α5β1 inhibitor results in improved hypertrophy and / or fibrosis. In some embodiments, administration of an α5β1 inhibitor prevents hypertrophy and / or fibrosis.

[0150] Integrin inhibitors

[0151] Integrins are a family of glycoprotein transmembrane receptors that mediate cell-cell and cell-matrix interactions. Integrins are heterodimers with two different chains, the α subunit and the β subunit. In mammals, eighteen α subunits and eight β subunits have been described.

[0152] Integrin α5β1 is composed of subunits ITGA5 (integrin α5) and integrin β1. Several integrins bind to fibronectin. Integrin α5β1 is selective for fibronectin because it requires the 9th and 10th type II repeats (FNIII-9 and FNIII-10) of fibronectin to interact. α5β1 integrin is mainly expressed in vasculature and connective tissue. Expression is significantly enhanced in tumor blood vessels, but also significantly enhanced in tumor cells themselves of many types of cancer (including colon, breast, ovarian, lung and brain tumors). It is also expressed to varying degrees in many cell types, including fibroblasts, hematopoietic cells, immune cells, smooth muscle cells and epithelial cells. High expression of α5β1 integrin is also observed in fibrotic tissues (such as pulmonary fibrosis).

[0153] In tissues, normal fibroblasts are present in a low population of only 4-5%. However, during fibrosis, they proliferate and can occupy up to 80-90% of the organ mass. Myofibroblasts in fibrotic tissues produce large amounts of extracellular matrix proteins, causing scarring and loss of function of the tissue. Inhibition of myofibroblasts can counteract these processes. Integrins promote cell proliferation, survival, hypertrophic growth, and fibrosis. As described herein, integrin inhibition can modulate these key factors that lead to the progression of pulmonary hypertension (e.g., PAH).

[0154] The present invention provides a method for treating a disease associated with increased expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor.

[0155] In one aspect, the present invention provides an integrin α5β1 inhibitor for use in treating a disease associated with increased expression or activity of integrin α5β1 in a subject in need of treatment, comprising administering the integrin α5β1 inhibitor and a pharmaceutical excipient to the subject.

[0156] In some embodiments, the disease is characterized by a World Health Organization (WHO) panel.

[0157] In some embodiments, the disease is pulmonary hypertension, WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, and WHO Group 5 pulmonary hypertension.

[0158] In some embodiments, the disease is characterized by the World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by the WHO functional classification based on cardiac function. In some embodiments, the disease is pulmonary hypertension, WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension.

[0159] In some embodiments, the disease is heart failure or right ventricular failure.

[0160] In one aspect, the invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject comprising administering an integrin α5β1 inhibitor.

[0161] Small molecule inhibitor of integrin α5β1β

[0162] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

[0163] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5.

[0164] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin β1.

[0165] In some embodiments, the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5β1.

[0166] In some embodiments, the integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0167]

[0168] in:

[0169] R 1 is hydrogen or OMe;

[0170] R 2 is optionally C 1-4 Alkoxy substituted C 1-4 Alkyl; and

[0171] R 3 It is C 1-4 Alkyl or C 3-5 Cycloalkyl.

[0172] In some embodiments, R2 is methyl or ethyl.

[0173] In some embodiments, R3 is methyl, ethyl, isopropyl, or cyclopropyl.

[0174] In some embodiments, R1 is hydrogen.

[0175] In some embodiments, the compound of formula (I) is selected from:

[0176]

[0177]

[0178] In some embodiments, the integrin α5β1 inhibitor is a SMi:

[0179]

[0180] In some embodiments, the integrin α5β1 inhibitor is a dual inhibitor of α5β1 and αvβ1 (eg, MRT).

[0181] Integrin α5β1 antibody inhibitor β

[0182] In some embodiments, the integrin α5β1 inhibitor is a Fab, a single-chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single domain antibody-linked Fab (s-Fab), an antibody, or a combination thereof.

[0183] In some embodiments, the integrin α5β1 inhibitor is an antibody.

[0184] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5.

[0185] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin β1.

[0186] In some embodiments, the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5β1.

[0187] In some embodiments, the antibody is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A.

[0188] In some embodiments, the antibody is the integrin α5β1 antibody volociximab (M200).

[0189] The antibody volociximab (M200) comprises the heavy chain amino acid sequence of SEQ ID NO: 1:

[0190] QVQLKESGPGLVAPSQSLSITCTISGFSLTDYGVHWVRQPPGKGLEWLVVIWSDGSSTYNSALKSRMTIRKDNKSSQVFLIMNSLQTDDSAMYYCARHGTYYGMTTTGDALDY WGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:1)

[0191] The antibody volociximab (M200) comprises the light chain amino acid sequence of SEQ ID NO: 2:

[0192] QIVLTQSPAIMSASLGERVTMTCTASSSVSSNYLHWYQQKPGSAPNLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYLRSPPTTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:2)

[0193] In some embodiments, the antibody is the integrin α5β1 antibody M200. In some embodiments, the antibody is an integrin α5β1 antibody that competes with M200 for integrin binding and / or binds to the same epitope as M200.

[0194] In some embodiments, the antibody is the integrin α5β1 antibody PF- 04605412. In some embodiments, the antibody is an integrin α5β1 antibody that competes with PF-04605412 for integrin binding and / or binds to the same epitope as PF-04605412.

[0195] In some embodiments, the antibody is an integrin α5β1 antibody described in WO2009100110A1, which is incorporated herein by reference. In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding and / or binds to the same epitope as the integrin α5β1 antibody described in WO2009100110A1.

[0196] In some embodiments, the antibody is the integrin α5β1 antibody 22B5. In some embodiments, the antibody is an integrin α5β1 antibody that competes with 22B5 for integrin binding and / or binds to the same epitope as 22B5.

[0197] In some embodiments, the antibody is the integrin α5β1 antibody 24C7. In some embodiments, the antibody is an integrin α5β1 antibody that competes with 24C7 for integrin binding and / or binds to the same epitope as 24C7.

[0198] In some embodiments, the antibody is the integrin α5β1 antibody 1D9. In some embodiments, the antibody is an integrin α5β1 antibody that competes with 1D9 for integrin binding and / or binds to the same epitope as 1D9.

[0199] In some embodiments, the antibody is the integrin α5β1 antibody 2D2. In some embodiments, the antibody is an integrin α5β1 antibody that competes with 2D2 for integrin binding and / or binds to the same epitope as 2D2.

[0200] In some embodiments, the antibody is the integrin α5β1 antibody MINT1526A. In some embodiments, the antibody is the integrin α5β1 antibody 18C12 or an antibody derived from 18C12. In some embodiments, the antibody is the integrin α5β1 antibody h18C12.v6.1.5.

[0201] Exemplary integrin α5β1 antibodies are described in WO2010111254A1, which is incorporated herein by reference. In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding and / or binds to the same epitope as the integrin α5β1 antibody described in WO2010111254A1.

[0202] In some embodiments, the anti-α5β1 antibody comprises: a VL domain comprising a CDR-L1 comprising TL-S / TS / P / TQ / NHF / ST / IYK / TIG / D / S, a CDR-L2 comprising L / IN / TSD / H / SG / SS / L / TH / YN / K / Q / IK / TG / AD / S / V, and a CDR-L3 comprising G / AS / A / YS / YYS / A / YS / Y / T-GY-V / I; and a VH domain comprising a CDR-H1 comprising GFTFS-N / A-RW-I / VY, a CDR-H2 comprising GIKTKP-N / A / TI / R-YAT-E / Q-YADSVKG, and a CDR-H3 comprising L / V-TG-M / KR / K-YFDY.

[0203] In some embodiments, the integrin α5β1 antibody competes for integrin binding and / or binds to the same epitope as an antibody comprising: a VL domain comprising a CDR-L1 comprising TL-S / TS / P / TQ / NHF / ST / IYK / TIG / D / S, a CDR-L2 comprising L / IN / TSD / H / SG / SS / L / TH / YN / K / Q / IK / TG / AD / S / V, a CDR-L3 comprising G / AS / A / YS / YYS / A / YS / Y / T-GY-V / I; and a VH domain comprising a CDR-H1 comprising GFTFS-N / A-RW-I / VY, a CDR-H2 comprising GIKTKP-N / A / TI / R-YAT-E / Q-YADSVKG, and a CDR-H3 comprising L / V-TG-M / KR / K-YFDY.

[0204] In some embodiments, the integrin α5β1 antibody comprises: a VL domain comprising a CDR-L1 comprising TLSSQHSTYTI, a CDR-L2 comprising LNSDSSHNKGSGIPD, and a CDR-L3 comprising AAYYAYGYV; and a VH domain comprising a CDR-H1 comprising GFTFSARWIY, a CDR-H2 comprising GIKTKPAIYATEYADSVKGRFT, and a CDR-H3 comprising LTGMKYFDY.

[0205] In some embodiments, the integrin α5β1 antibody competes for integrin binding and / or binds to the same epitope as an antibody comprising: a VL domain comprising a CDR-L1 comprising TLSSQHSTYTI, a CDR-L2 comprising LNSDSSHNKGSGIPD, and a CDR-L3 comprising AAYYAYGYV; and a VH domain comprising a CDR-H1 comprising GFTFSARWIY, a CDR-H2 comprising GIKTKPAIYATEYADSVKGRFT, and a CDR-H3 comprising LTGMKYFDY.

[0206] In some embodiments, an anti-α5β1 antibody comprises: a VL domain comprising a CDR-L1 comprising TLSSQHSTYTIG, a CDR-L2 comprising LNSDSSHNKGS, and a CDR-L3 comprising AAYYAYGYV; and a VH domain comprising a CDR-H1 comprising residues GFTFSARWIY, a CDR-H2 comprising residues GIKTKPAIYATEYADSVKG, and a CDR-H3 comprising residues LTGMKYFDY.

[0207] In some embodiments, the integrin α5β1 antibody competes for integrin binding and / or binds to the same epitope as an anti-α5β1 antibody comprising: a VL domain comprising a CDR-L1 comprising TLSSQHSTYTIG, a CDR-L2 comprising LNSDSSHNKGS, a CDR-L3 comprising AAYYAYGYV; and a VH domain comprising a CDR-H1 comprising residues GFTFSARWIY, a CDR-H2 comprising residues GIKTKPAIYATEYADSVKG, and a CDR-H3 comprising residues LTGMKYFDY.

[0208] In some embodiments, the integrin α5β1 antibody is selected from the antibodies described in Table 1. In some embodiments, the integrin α5β1 antibody competes with the anti-α5β1 antibodies described in Table 1 for integrin binding and / or binds to the same epitope as them.

[0209]

[0210]

[0211] In some embodiments, the antibody is the integrin α5β1 antibody 3C5 or 5B11. In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from 3C5 and 5B11. 3C5 and 5B11 are integrin α5β1 antibodies described in WO2010072740A2, which is incorporated herein by reference.

[0212] In some embodiments, the antibody is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from the group consisting of volociximab (M200), P1D6, PF-04605412, MINT1526A, BMA5, BMB5, BMC5, HA5, JBS5, LS-C509074, LS-C24758, 1D9, 22B5, 24C7, 2D2, 3C2.2A8, 3C5, 5B11, MOR04055, MOR04624, P8D4, MOR04974, MOR04977, SG / 19, and 18C12 (e.g., clone h18C12.v2.1 or h18C12.v6.1.5).

[0213] In some embodiments, the integrin inhibitor binds to α5β1. In some embodiments, the integrin inhibitor is a broad-spectrum integrin inhibitor. In some embodiments, the integrin inhibitor binds to α5β1 and one or more integrins.

[0214] In some embodiments, the α5β1 integrin inhibitor is selected from Table 2.

[0215] Table 2. Exemplary integrin inhibitors

[0216] molecular Non-antibody inhibitors ATN-161 JSM6427 ATX-107 GLPG0187 SJ749 THR-687 Antibody inhibitors Volociximab (M200) PF-04605412 MINT1526A(RG-7594)

[0217] Treatment

[0218] The present invention provides a method of treating a cardiac or pulmonary disease in a subject comprising administering an integrin α5β1 inhibitor. In one aspect, the present invention provides a method of treating a disease associated with increased expression or activity of integrin α5β1 comprising administering an integrin α5β1 inhibitor. In some embodiments, the disease is characterized by a World Health Organization (WHO) panel.

[0219] In some embodiments, the disease is pulmonary hypertension, WHO Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Group 2 pulmonary hypertension, WHO Group 3 pulmonary hypertension, WHO Group 4 pulmonary hypertension, and WHO Group 5 pulmonary hypertension.

[0220] In some embodiments, the disease is characterized by the World Health Organization (WHO) classification system. In some embodiments, the disease is characterized by the WHO functional classification based on cardiac function. In some embodiments, the disease is pulmonary hypertension, WHO Class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO Class II pulmonary hypertension, WHO Class III pulmonary hypertension, WHO Class IV pulmonary hypertension.

[0221] In some embodiments, the disease is persistent / recurrent chronic thromboembolic pulmonary hypertension (CTEPH) (WHO Group 4). In some embodiments, the disease is pulmonary arterial hypertension (PAH) (WHO Group 1).

[0222] In some embodiments, the patient has persistent / recurrent chronic thromboembolic pulmonary hypertension (CTEPH) (WHO Group 4) or inoperable CTEPH after surgical treatment. In some embodiments, the patient has pulmonary arterial hypertension (PAH) (WHO Group 1).

[0223] In some embodiments, treatment is administered to improve motor ability and WHO functional class. In some embodiments, treatment is administered to improve motor ability, improve WHO functional class, and slow clinical deterioration.

[0224] In some embodiments, the disease is heart failure or right ventricular failure. In some embodiments, the disease is heart failure. In some embodiments, the disease is right ventricular failure.

[0225] In one aspect, the invention provides a method of treating pulmonary arterial hypertension (PAH) in a subject comprising administering an integrin α5β1 inhibitor.

[0226] In some embodiments, the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intrapulmonary.

[0227] Also provided is a method for treating a subject suffering from fibrosis or a fibrosis-related disorder, comprising administering to the subject a therapeutically effective amount of an integrin α5β1 inhibitor according to the invention. As used herein, the term "fibrosis" refers to a disease characterized by deposition of extracellular matrix components in the skin or organs (including the lungs, kidneys, heart, liver, skin, and joints), resulting in scar tissue. The term also refers to the process of scar tissue formation.

[0228] In some embodiments, fibrosis-related disorders are disorders or conditions that may occur due to fibrosis, or disorders or conditions associated with fibrosis. In some embodiments, fibrosis and / or fibrosis-related disorders are diseases or conditions selected from the group consisting of: renal fibrosis, hepatic fibrosis, liver cirrhosis, pulmonary fibrosis, skin fibrosis, bile fibrosis, peritoneal fibrosis, myocardial fibrosis, pancreatic fibrosis, bone marrow and / or myelofibrosis, reperfusion injury after liver or kidney transplantation, interstitial lung disease (ILD), cystic fibrosis (CF), atherosclerosis, systemic sclerosis, osteosclerosis, herniated disc and other spinal cord injuries, fibromatosis, fibromyalgia, arthritis, restenosis. Pulmonary fibrosis includes idiopathic pulmonary fibrosis and scleroderma pulmonary fibrosis.

[0229] Therapeutic dose

[0230] In some embodiments, the method of treating a disease associated with increased expression or activity of integrin α5β1 comprises administering an integrin α5β1 inhibitor at a dose of 1 mg / kg to 1000 mg / kg. In some embodiments, the method of treating a subject's cardiac or pulmonary disease comprises administering an integrin α5β1 inhibitor at a dose of 1 mg / kg to 1000 mg / kg. In some embodiments, the disease is characterized by a World Health Organization (WHO) panel as described above.

[0231] In some embodiments, the dosage is at least 2 mg / kg, at least 4 mg / kg, at least 6 mg / kg, or at least 8 mg / kg. In some embodiments, the dosage is at least 10 mg / kg, at least 20 mg / kg, at least 30 mg / kg, at least 40 mg / kg, at least 50 mg / kg, at least 60 mg / kg, at least 70 mg / kg, at least 80 mg / kg, at least 90 mg / kg, or at least 100 mg / kg. In some embodiments, the dosage is at least 200 mg / kg, at least 300 mg / kg, at least 400 mg / kg, at least 500 mg / kg, at least 600 mg / kg, at least 700 mg / kg, at least 800 mg / kg, at least 900 mg / kg, or at least 1000 mg / kg.

[0232] Cardiac function parameters

[0233] Various endpoint parameters can be assessed to determine the efficacy of the treatment of the present invention, such as A5B1 levels, pulmonary vascular resistance (PVR), mean pulmonary artery pressure (PAP), cardiac index (CI), mean pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), six-minute walk distance (6MWD), brain natriuretic peptide (BNP) levels, lung diffusion capacity (DLCO), and death or survival. See Chung et al., Chest (2010), 138 (6): 1383-1394.

[0234] PVR is often used as an endpoint parameter to determine the therapeutic effect of PAH. A subject's PVR>240dyn·sec / cm5 indicates mild PAH. A subject's PVR of 600-800dyn·sec / cm5 indicates moderate to severe PAH. After treatment with the methods and compositions of the present invention, a decrease in the subject's PVR of 130dyn·sec / cm5 or more indicates that the treatment is effective. For example, administration of an A5B1 inhibitor to a subject with PAH that results in a decrease in PVR of 180-350dyn·sec / cm5 indicates that the treatment is effective.

[0235] Mean pulmonary artery pressure (PAP) is also used as an endpoint parameter to determine the therapeutic effect of PAH. The average PAP of subjects without PAH is about 15-24 mmHg. The average PAP of subjects with mild PAH is about 25-30 mmHg (e.g., >25 mmHg at rest or 30 mmHg during exercise). The PAP of subjects with severe PAH is greater than 30 mmHg, such as 40-70 mmHg or 60-70 mmHg. After treatment, a decrease in PAP greater than 1.5 mmHg indicates that the treatment is effective. In some embodiments, treatment causes a decrease in PAP greater than 5, 10, 20, 40 or 50 mmHg, which indicates that the treatment is effective.

[0236] Cardiac index (CI) is also used as an endpoint parameter to determine the effectiveness of PAH treatment. A low or decreased CI indicates heart failure. For example, a CI of 2.5 L / min / m2 or less indicates PAH or heart failure. After treatment, an increase in CI of more than 0.3 L / min / m2 indicates that the treatment is effective.

[0237] Mean pulmonary capillary wedge pressure (PCWP) can be used as an endpoint parameter to determine the effectiveness of treatment of PAH. A mean PCWP of less than or equal to 18 mmHg (e.g., less than or equal to 10 mmHg) indicates that at least the subject suffers from PAH. After treatment, an increase in mean PCWP greater than 0.5 mmHg indicates that the treatment is effective.

[0238] Right atrial pressure (RAP) is also used as an endpoint parameter to determine the effectiveness of treatment for PAH. A normal RAP for a subject without PAH is 0-8 mmHg. A RAP of 8 mmHg or higher indicates PAH. A subject with severe PAH has a RAP of about 20 mmHg. After treatment, a decrease of more than 0.5 mmHg indicates that the treatment is effective.

[0239] Six-minute walk distance (6MWD) is used as an endpoint parameter to determine the therapeutic effect of PAH. The average 6MWD of CTD-PAH patients is about 300 m. After treatment, an increase of 25 m or more or an increase of more than 10% in 6MWD indicates that the treatment is effective. For example, after treatment, a 6MWD of 1000 m or more indicates that the treatment is effective.

[0240] Brain natriuretic peptide (BNP) is used as an endpoint parameter to determine the therapeutic effect of PAH. BNP is a sensitive marker for worsening heart failure and is also a predictor of mortality in PAH patients. Normal BNP levels are <100pg / mL, such as 30-90pg / mL. Higher BNP levels indicate worsening heart failure. BNP levels of about 100-200pg / mL (e.g., 160pg / mL or higher) indicate early heart failure. BNP levels of about 200-1000pg / mL indicate true heart failure. The average BNP level in CTD-PAH patients is about 430pg / mL. After treatment, any reduction in BNP levels indicates that the treatment is effective.

[0241] N-terminal pro-brain natriuretic peptide (NT-proBNP): A reproducible, non-invasive parameter is useful for the follow-up of patients with PAH. BNP is produced in the ventricles and is elevated in PPH / IPAH. Recent studies have shown that BNP levels are closely associated with functional impairment in patients with PPH / IPAH and parallel changes in pulmonary hemodynamics and the extent of right heart failure. BNP levels are longitudinally correlated with functional assessments performed during the study. Plasma NT-pro-BNP is measured by a sandwich immunoassay using polyclonal antibodies that recognize an epitope located in the N-terminal fragment (1-76) of pro-BNP (1-108) (Elecsys analyzer, Roche Diagnostics, Manheim, Germany).

[0242] The diffusing capacity of the lung (DLCO) or CO diffusing capacity is also used as an endpoint parameter to determine the effectiveness of treatment for PAH. DLCO measures the ability of carbon monoxide (CO) to diffuse through cell membranes. A normal DLCO for a subject without PAH is greater than 80%. An abnormal DLC for a subject with PAH is less than 80%, less than 65%, or less than 45%. After treatment, any increase in DLCO% indicates that the treatment is effective.

[0243] In some embodiments, administration of an integrin α5β1 inhibitor modulates the level of a biomarker in a subject, wherein the biomarker is selected from the group consisting of survivin, PCNA, Ki67, and Annexin V.

[0244] SoC and combination therapy

[0245] As described herein, methods of treating a disease associated with increased expression or activity of integrin α5β1 comprising administering an integrin α5β1 inhibitor may include combination therapy in which an integrin a5b1 inhibitor and one or more drugs approved for the treatment of PH, PAH, heart failure, or right ventricular failure are administered to a patient in need of treatment.

[0246] Approved drugs currently used in the US or the European Union (EU) for the treatment of PH, PAH, heart failure, and right ventricular failure include orally administered PDE-5 inhibitors: sildenafil (Revatio) and tadalafil (Adeirca); dual endothelin-1A receptor antagonists (ERAs): bosentan (Tracleer), ambrisentan (Letairis in the US; Volibris internationally). Patients with more advanced disease are usually treated with a prostacyclin or prostacyclin analog, such as iloprostcycline (Ventavis) or treprostinil (Tyvaso) given as multiple daily inhalations, epoprostenol (Flolan / Veletri) or treprostinil (Remodulin) given as a continuous intravenous infusion, or treprostinil also used as a continuous subcutaneous infusion. Intravenous sildenafil is approved for patients who are currently prescribed but cannot take oral sildenafil for the time being. Inhaled nitric oxide (INOmax) is approved for the neonatal form of PAH, namely persistent pulmonary hypertension of the newborn (PPHN).Thus, according to the present invention, combination therapy of any of these drugs and an integrin a5b1 inhibitor may be used to treat PAH or a condition disclosed herein.

[0247] In some embodiments, the second therapy is selected from the group consisting of anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostanoids, prostanoid receptor agonists, soluble guanylate cyclase stimulators, and / or surgery.

[0248] In some embodiments, the second therapy is oxygen, warfarin, furosemide, bumetanide, bendrofluazide, metolazone, spironolactone, amiloride, digoxin, nifedipine, diltiazem, nicardipine, amlodipine, ambrisentan, bosentan, macitentan, sildenafil, tadalafil, epoprostenol, iloprostcycline, treprostinil, riociguat, selexipag, surgery, pulmonary endarterectomy, and / or atrial septostomy.

[0249] In some embodiments, the second therapy is macitentan and / or tadalafil.

[0250] Flolan (prostacyclin analog) is an approved therapy for PAH, but it is very cumbersome and inconvenient to use (intravenous injection), and has unique safety issues. Therefore, Flolan is usually used for patients with severe functional status or rapidly progressive PAH. Patients must prepare the drug several times a day under sterile conditions. The drug is a lyophilized preparation that needs to be dissolved in an alkaline buffer. Due to its short half-life (3-5min) and stability (8h at room temperature), Flolan must be maintained in a refrigerated state when it is continuously infused via a central venous catheter through a portable pump (CADD pump, Smith's Medical MD, St.Paul, Minn.) worn in a waist bag. In 2008, the FDA also approved a new continuous intravenous injection formulation of epoprostenol, which is stable at room temperature for up to 24h after dilution and can be stored at refrigerator temperature for up to 5 days before use (GeneraMedix Inc., Liberty Corner, NJ). In 2009, GeneraMedix Inc. sold this formulation to Actelion, which began marketing the drug under the trade name Veletri in April 2010. In late 2010, the Veletri label was expanded to allow preparation of the drug at refrigerator temperature up to 7 days or at room temperature up to 48 hours prior to use. Thus, in one embodiment of the invention, the integrin a5b1 inhibitor, in any of its approved forms, is administered in combination with epoprostenol to treat PAH.

[0251] Remodulin, a continuous subcutaneous infusion form of a prostacyclin analog, is not typically used as an initial therapy due to its expense, route of delivery, and limited efficacy. In 2004, the FDA and Health Canada approved an intravenous formulation of Remodulin for use in patients with PAH Class II-IV disease who cannot tolerate the subcutaneous form. In early 2006, the FDA expanded the Remodulin label to include patients who need to transition from Flolan. In 2009, United Therapeutics received FDA approval for an inhaled formulation of Treprostinil (Tyvaso). Thus, in one embodiment of the invention, an integrin a5b1 inhibitor is administered in combination with Treprostinil to treat PAH.

[0252] Ventavis (iloprostacyclin), a prostacyclin analog administered by inhalation, is also marketed in several member states of the EU as Ilomedine for intravenous formulation. In the EU, the labeling for inhaled iloprostacyclin is limited to patients with idiopathic PAH and functional Class III symptoms. In contrast, the US labeling is broader: patients with PAH (regardless of etiology) and Class III or Class IV symptoms. Administration is required 6 to 9 times a day. Therefore, in one embodiment of the invention, an integrin a5b1 inhibitor is administered in combination with iloprostacyclin in any of its approved forms to treat PAH.

[0253] In 2001, the non-selective ERA Tracleer (bosentan) became the first oral PAH therapy and is only available through a special centralized access program in the US due to its significant (reversible) liver damage, teratogenicity, testicular atrophy and male fertility risks. Treatment with Tracleer consists of an initial dose of 62.5 mg twice daily for 4 weeks, followed by a maintenance dose of 125 mg twice daily. Tracleer was initially indicated for patients with PAH and moderate or severe functional status (WHO Class III, IV). In 2008 (EU) and 2009 (US), the label was expanded to patients with mild symptoms (functional Class II). Therefore, in one embodiment of the invention, an integrin a5b1 inhibitor is administered in combination with bosentan in any of its approved forms to treat PAH.

[0254] Ambrisentan is an oral selective ERA receptor antagonist marketed by Gilead Sciences in the US (Letairis) and GlaxoSmithKline in other regions (Volibris) for once-daily treatment of patients with WHO Class II or III symptoms to improve exercise capacity and delay clinical deterioration. Like bosentan, ambrisentan has class effects of teratogenicity, testicular damage, reduced male fertility and anemia. Therefore, in one embodiment of the invention, an integrin a5b1 inhibitor is administered in combination with ambrisentan in any of its approved forms to treat PAH.

[0255] In the US, the oral PDE-5 inhibitor Revatio (sildenafil) is approved for the treatment of PAH (WHO Group I) at a dose of 20 mg three times daily to improve exercise capacity and delay clinical deterioration, regardless of etiology or functional class. The EU label is limited to improving exercise capacity in patients with PAH that is idiopathic or associated with collagen vascular disease and class III functional status. In 2009, the FDA approved the intravenous form of Revatio given as an injection (10 mg, three times a day) for patients who cannot take oral formulations. In May 2010, the EU approved Revatio as an oral suspension (formulated from a 20 mg tablet) for the treatment of patients aged 1 to 17 years with PAH. Therefore, in one embodiment of the present invention, the integrin a5b1 inhibitor is administered in combination with sildenafil in any of its approved forms to treat PAH.

[0256] In the US, the oral PDE-5 inhibitor Adeirca (tadalafil) 40 mg once daily is indicated to improve exercise capacity in patients with PAH (WHO Group I), regardless of etiology or functional class (package insert). EU labeling is limited to patients with functional classes II and III. Tadalafil has a long half-life (35h) in patients with PAH (US package insert), and concomitant use of bosentan may also be beneficial in patients with PAH.

[0257] Thus, the method of treating a patient may include administering at least one additional active agent, i.e., in addition to the integrin a5b1 inhibitor. The additional active agent may be, for example, a vasodilator, such as prostacyclin, epoprostenol, and sildenafil; an endothelin receptor antagonist, such as bosentan; a calcium channel blocker, such as amlodipine, diltiazem, and nifedipine; an anticoagulant, such as warfarin; a diuretic, a prostaglandin (e.g., prostacyclin or PGI2), a drug for treating a disease associated with overactivated B cells or dysfunctional B cells, such as rituximab, and / or a type V phosphodiesterase (PDE5) inhibitor.

[0258] When the methods of the invention involve combination therapy, i.e., where a second agent (such as a vasodilator) is co-administered with an integrin a5b1 inhibitor, the agents may be administered separately at the same or different times of the day, or they may be administered in a single composition. Thus, the invention provides novel pharmaceutical formulations in which an integrin a5b1 inhibitor is combined with one of the above-mentioned active agents, as well as unit dosage forms of these formulations.

[0259] In the combination therapy of the present invention, each agent can be administered in an "immediate release" manner or a "controlled release" manner. When the additional active agent is a vasodilator, for example, any dosage form containing two active agents, i.e., an integrin a5b1 inhibitor and a vasodilator, can provide an immediate release or a controlled release of the vasodilator, and an immediate release or a controlled release of the integrin a5b1 inhibitor.

[0260] As a general example, a combination dosage form of the invention for once-daily administration may contain an integrin a5b1 inhibitor in a controlled release (e.g., sustained release) or immediate release form of the integrin a5b1 inhibitor in the range of about 1 mg to about 1000 mg, and sildenafil in an immediate release or controlled release form, wherein the additional active agent is present in an amount to provide a weight ratio of integrin a5b1 inhibitor to sildenafil or a weight ratio of integrin a5b1 inhibitor to sildenafil as described above. In other formulations of the invention, two or more additional active agents may be present in combination with the integrin a5b1 inhibitor, which additional active agents may or may not be of the same class of drugs (e.g., vasodilators). In such cases, the effective amount of any or each individual additional active agent present will generally be reduced relative to the amount required when only a single added active agent is used.

[0261] As described above, the additional active agent may also be a type V phosphodiesterase inhibitor, administered with an integrin a5b1 inhibitor, or administered with an integrin a5b1 inhibitor and a vasodilator. Examples of type V phosphodiesterase inhibitors include, but are not limited to, avanafil, sildenafil, tadalafil, zaprinast, dipyridamole, vardenafil, and acid adducts or other pharmaceutically acceptable salts thereof. Sildenafil is a good example. In an exemplary embodiment, an integrin a5b1 inhibitor is co-administered with a type V phosphodiesterase inhibitor selected from the group consisting of avanafil, tadalafil, and sildenafil, and the daily dose of the compound of the integrin a5b1 inhibitor is the dose given above for a monotherapy regimen.

[0262] In one embodiment, the vasodilator is selected from sildenafil, avanafil, tadalafil, zaprinast, dipyridamole, vardenafil, bosentan, and pharmaceutically acceptable salts thereof.

[0263] As noted above, the additional active agent may also be an endothelin receptor antagonist, such as bosentan, sitaxsentan, or ambrisentan, with bosentan being an exemplary active agent.

[0264] The pharmaceutical composition of the present invention is a pharmaceutical formulation containing an active agent, which is formulated in a manner compatible with its intended route of administration. A variety of routes are contemplated, including, but not limited to, oral, pulmonary, inhalation, sublingual, intranasal, parenteral, intradermal, transdermal, topical, transmucosal, subcutaneous, intravenous, intramuscular, intraperitoneal, oral, rectal, etc. The term "parenteral" as used herein includes subcutaneous, intravenous and intramuscular injections.

[0265] Typically, the pharmaceutical formulations of the invention are prepared for oral administration and are in an immediate release form suitable for once daily (QD) administration. Certain formulations are suitable for intranasal administration to a patient.

[0266] Certain pharmaceutical formulations of the present invention comprise an integrin a5b1 inhibitor or a salt thereof and one or more pharmaceutically acceptable (approved for use in humans by state or federal regulatory agencies, or listed in the US Pharmacopia, European Pharmacopia) excipients or carriers. As used herein, the term excipient or carrier refers generally to a biologically inactive substance used in combination with the active agent of the formulation. An excipient may be used, for example, as a solubilizer, stabilizer, diluent, inert carrier, preservative, binder, disintegrant, coating agent, flavoring agent, or coloring agent. Preferably, at least one excipient is selected to provide one or more beneficial physical properties to the formulation, such as increasing the stability and / or solubility of the active agent. The integrin a5b1 inhibitor or a salt thereof described herein is an exemplary active agent suitable for use in the formulations of the present invention.

[0267] Examples of suitable excipients include certain inert proteins, such as albumin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as aspartic acid (which may alternatively be referred to as aspartate), glutamic acid (which may alternatively be referred to as glutamate), lysine, arginine, glycine, and histidine; fatty acids and phospholipids, such as alkyl sulfonates and octanoate; surfactants, such as sodium lauryl sulfate and polysorbates; nonionic surfactants, such as or polyethylene glycol (PEG); carbohydrates such as glucose, sucrose, mannose, maltose, trehalose, and dextrins, including cyclodextrins; polyols such as mannitol and sorbitol; chelating agents such as EDTA; and salt-forming counterions such as sodium.

[0268] The solution or suspension for delivery may include the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, polysorbate, tocopherol polyethylene glycol succinate (TPGS) or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl paraben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate, and a tonicity modifier such as sodium chloride or glucose. The pH value can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. These preparations can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0269] In some embodiments, the pharmaceutical formulation of the present invention contains a plurality of liposomes or microparticles containing an integrin a5b1 inhibitor active agent. In various embodiments, the pharmaceutical formulation of the integrin a5b1 inhibitor is a powder containing solid particles (e.g., liposomes or microparticles) suitable for administration by inhalation. The solid particles contain an active agent, a carrier, optionally a surfactant, and optionally an additional receptor. The powder can be prepared by any convenient method. One example of a preparation method is to spray dry a solution containing the active agent (and other components) onto a powder containing a carrier compound. Another example is to freeze dry a solution containing all the components of the final powder.

[0270] Liposomes suitable for use in the formulations of the present invention are known in the art. For example, suitable liposomes include cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and PEG-DSPE, wherein the weight ratio is about 5:10:1. In some embodiments, the liposome formulation comprises about 0.1-25%, for example 0.1%, 1%, 5%, 10% or 20% (w / w) of phospholipids such as dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the liposome formulation comprises about 0.5-20%, for example 1%, 5% or 10% (w / w) of a hydrophilic polymer such as polyvinylpyrrolidone (PVP). In some embodiments, the liposome formulation comprises about 10-35% of an amino acid such as L-leucine.

[0271] Microparticles suitable for use in the formulations of the present invention are known in the art. For example, the microparticles are formed from one or more hydrophilic polymers, such as polyvinyl pyrrolidone (e.g., PVP-10), polyvinyl alcohol (e.g., PVA-30), polyvinyl acetate, or poloxamer (e.g., poloxamer-188). In some embodiments, the microparticle formulation comprises about 70-85% by weight polyvinyl alcohol (e.g., PVA-30), about 5-15% PVP (e.g., PVP-10), 1-5% poloxamer (e.g., poloxamer-188), 0-10% L-leucine, and about 0.5-10% integrin a5b1 inhibitor compound (e.g., 5%). In some embodiments, the formulation is suitable for administration through the respiratory tract.

[0272] Pharmaceutical formulations of integrin a5b1 inhibitors useful in the methods of the invention can be prepared in liquid or solid form, such as powders, tablets, pills or capsules for oral administration. Liquid formulations of the invention can take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending agents, stabilizers and / or dispersants. In one embodiment, the formulation is an aqueous solution. In another embodiment, the final formulation is lyophilized. In some embodiments, the integrin a5b1 inhibitor is formulated for inhalation.

[0273] In various embodiments, the formulations of the invention comprise an integrin a5b1 inhibitor at a concentration of 0.25% to 100%, or 0.25% to 50%, or 0.8% to 25%, or 1% to 10%, or 1.5% to 5%. In certain embodiments, the integrin a5b1 inhibitor compound is formulated at a concentration of about 0.5% to about 5%. In certain embodiments, the integrin a5b1 inhibitor compound is formulated at a concentration of about 0.25% to about 10%.

[0274] The present invention also provides a pharmaceutical package or kit comprising one or more containers filled with a solid or liquid formulation of an integrin a5b1 inhibitor. In a specific embodiment, the formulation is a powder formulation of an integrin a5b1 inhibitor. In various embodiments, the integrin a5b1 inhibitor is formulated at a concentration of at least about 0.5% by weight, and the formulation is suitable for administration to a human by inhalation.

[0275] The invention also provides for use of a formulation of an integrin a5bl inhibitor in the manufacture of a medicament for treating PAH or a disorder disclosed herein in a subject in need thereof. Typically, the pharmaceutical formulation is sterile.

[0276] Typically, the dosage form (e.g., inhalable dosage form) provides a sustained release (i.e., gradual release) of the compound of the invention (e.g., integrin a5b1 inhibitor) from the dosage form into the patient's body over an extended period of time, typically providing a substantially constant blood level of the drug over a period of time in the range of about 4 to about 12 hours, typically in the range of about 6 to about 10 hours. In a particularly preferred embodiment, after nasal administration of a dosage form containing a compound of the invention (e.g., integrin a5b1 inhibitor), there is a very slow increase in the blood level of the drug, such that the peak blood level is not reached until at least 4-6 hours later, wherein the rate of increase in the blood level of the drug is approximately linear. In addition, in a preferred embodiment, at the end of the sustained release period, the blood level also gradually decreases.

[0277] Although the pharmaceutical compositions of the present invention are preferably formulated for inhalation, for example as saline solutions, dry powders or aerosols, other modes of administration are also suitable. For example, administration may be sublingual, oral, parenteral, transdermal, via an implanted reservoir, transmucosal, for example rectal or vaginal, preferably using suppositories containing excipients (such as suppository waxes) in addition to the active agent. Transmucosal administration also encompasses transurethral administration, as described in U.S. Pat. Nos. 5,242,391, 5,474,535 and 5,773,020 to Place et al.

[0278] Depending on the intended mode of administration, the pharmaceutical formulation can be solid, semisolid or liquid, such as, for example, tablets, capsules, caplets, liquids, suspensions, emulsions, suppositories, granules, pills, beads, powders, etc., preferably unit dosage forms suitable for single administration of precise doses. Suitable pharmaceutical compositions and dosage forms can be prepared using conventional methods known to those skilled in the art of pharmaceutical formulations and described in relevant texts and literature, such as Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995). For those compounds with oral activity, oral dosage forms are generally preferred and include tablets, capsules, caplets, solutions, suspensions and syrups, and may also include a number of granules, beads, powders or pills that may be encapsulated or not. Preferred oral dosage forms are tablets and capsules.

[0279] In an embodiment, it may be particularly advantageous to formulate the composition of the present invention into a unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" as used herein refers to a physically discrete unit suitable as a single dose for an individual to be treated. That is, the composition is formulated into discrete dosage units, each dosage unit containing a predetermined "unit dose" amount of active agent, which is calculated together with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the unit dosage form of the present invention depend on the unique characteristics of the active agent to be delivered. The dosage can also be determined by reference to the usual dosage and mode of administration of the ingredients. It should be noted that in some cases, two or more separate dosage units in combination provide a therapeutically effective amount of the active agent, for example, two tablets or two capsules taken together can provide a therapeutically effective dose of an integrin a5b1 inhibitor, so that the unit dose in each tablet or capsule is about 50% of the therapeutically effective amount.

[0280] Tablets can be made using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred. In addition to the active agent, tablets generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, colorants, etc.

[0281] Capsules are another oral dosage form for those compounds of the invention (e.g., integrin a5b1 inhibitors) that are orally active, in which case the composition containing the active agent may be encapsulated in liquid or solid (including particulate, such as granules, beads, powders or pills) form. Suitable capsules may be hard or soft and are typically made of gelatin, starch or cellulose materials, preferably gelatin capsules. Two pieces of hard gelatin capsules are preferably sealed, such as with gelatin tape or the like. See, for example, Remington: The Science and Practice of Pharmacy cited above for a description of materials and methods for preparing encapsulated drugs.

[0282] If desired, the oral dosage form, whether tablet, capsule, caplet or microparticle, can be formulated to provide controlled release of the compound of the invention (eg, integrin a5b1 inhibitor), and in preferred embodiments, the invention formulation is a controlled release oral dosage form.

[0283] Typically, as understood by those of ordinary skill in the art, sustained-release dosage forms are formulated by dispersing the active agent in a matrix of a gradually hydrolyzable material, such as a hydrophilic polymer, or by coating a drug-containing solid dosage form with such a material. Hydrophilic polymers that can be used to provide a sustained-release coating or matrix include, for example, cellulosic polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, cellulose acetate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, alkyl acrylates, alkyl methacrylates, and the like, such as copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate; and vinyl polymers and copolymers, such as polyvinyl pyrrolidone, polyvinyl acetate, and ethylene-vinyl acetate copolymers.

[0284] Preparations for parenteral administration according to the present invention include sterile aqueous solutions and non-aqueous solutions, suspensions and emulsions. Injectable aqueous solutions contain active agents in water-soluble form. Examples of non-aqueous solvents or vehicles include fatty oils, such as olive oil and corn oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, low molecular weight alcohols, such as propylene glycol, synthetic hydrophilic polymers, such as polyethylene glycol, liposomes, etc. Parenteral formulations may also contain adjuvants, such as solubilizers, preservatives, wetting agents, emulsifiers, dispersants and stabilizers, and aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol and dextran. Injectable formulations may be sterile by incorporating sterilizing agents, filtering through bacteria-retaining filters, irradiating or heating. They may also be manufactured with sterile injection media. Active agents may also be dry, such as lyophilized forms, which may be rehydrated with a suitable vehicle immediately before administration by injection.

[0285] Active agent can also use conventional transdermal drug delivery system to be applied by skin, wherein active agent is included in the layered structure used as the drug delivery device fixed on the skin.In this structure, pharmaceutical composition is included in the layer below the upper backing layer or in " reservoir ".Layered structure can contain single reservoir, or it can contain multiple reservoirs.In one embodiment, reservoir comprises the polymer matrix of pharmaceutically acceptable contact adhesive material, and it is used for fixing system on the skin during drug delivery.Alternately, drug-containing reservoir and skin contact adhesive exist as separate and different layers, wherein adhesive is below reservoir, in this case, reservoir can be polymer matrix as above, or it can be liquid or hydrogel reservoir, or can take some other forms.Transdermal drug delivery system can also contain skin penetration enhancer.

[0286] In addition to the formulations described above, the active agent can also be formulated as a long-acting preparation for controlled release of the active agent, preferably sustained release over an extended period of time. These sustained release dosage forms are usually administered by implantation (e.g., subcutaneously or intramuscularly or by intramuscular injection).

[0287] Certain compounds or active agents of the present invention are capable of further forming salts. All of these forms are also within the scope of the present invention.

[0288] The compounds of the present invention can also be prepared as esters, such as pharmaceutically acceptable esters. For example, the carboxylic acid functional group in the compound can be converted into its corresponding ester, such as methyl ester, ethyl ester or other ester. In addition, the alcohol group in the compound can be converted into its corresponding ester, such as acetate, propionate or other ester.

[0289] Some compounds of the present invention can also be prepared as prodrugs, such as pharmaceutically acceptable prodrugs. The terms "prodrug" and "prodrug" are used interchangeably herein, and refer to any compound that releases an active parent drug in vivo. Because it is known that prodrugs can improve many desired qualities of drugs (e.g., solubility, bioavailability, manufacturing, etc.), the compounds of the present invention can be administered in the form of prodrugs. Therefore, the present invention is intended to cover the prodrugs of the compounds currently claimed, their delivery methods, and compositions comprising them. "Prodrug" is intended to include any covalently bonded carrier, and when this prodrug is applied to a subject, it releases the active parent drug of the present invention in vivo. Prodrugs in the present invention are prepared by modifying the functional groups present in the compound, and the modification method is to modify and cleave into parent compounds in conventional operations or in vivo. Prodrugs include compounds of the present invention, wherein hydroxyl, amino, sulfhydryl, carboxyl or carbonyl are combined with any group that can be cleaved in vivo to form free hydroxyl, free amino, free sulfhydryl, free carboxyl or free carbonyl, respectively.

[0290] Examples of prodrugs include, but are not limited to, esters of hydroxyl functional groups (e.g., acetate, dialkylaminoacetate, formates, phosphates, sulfates and benzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl), esters of carboxyl functional groups (e.g., ethyl esters, morpholinoethanol esters), N-acyl derivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases of amino functional groups and enaminones, oximes, acetals, ketals and enol esters of ketone and aldehyde functional groups in the compounds of the present invention, and the like, see Bundegaard, H., Design of Prodrugs, pp. 1-92, Elesevier, New York-Oxford (1985).

[0291] The dosage regimen utilizing the compound is selected based on a variety of factors, including the type, species, age, weight, sex, and physical condition of the patient; the severity of the condition to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progression of the condition.

[0292] In some embodiments, the composition is suitable for inhalation.In one embodiment, the composition is an inhalable formulation for treating PAH or a disorder disclosed herein.

[0293] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an integrin a5b1 inhibitor and a plurality of particles, wherein the plurality of particles are a plurality of liposomes comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE) or a plurality of microparticles comprising a hydrophilic polymer. In one embodiment, the composition is suitable for inhalation, and in one embodiment, the composition is an inhalable formulation for treating PAH or a condition disclosed herein.

[0294] Composition

[0295] The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering pharmaceutical compositions by oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal and intracranial methods, the pharmaceutical compositions comprising a peptide or polymer according to the invention, preferably a peptide according to the invention, and comprising a pharmaceutically acceptable carrier. For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions.

[0296] The pharmaceutical composition according to the present invention comprises at least one pharmaceutically acceptable carrier, diluent or excipient. Examples of suitable carriers include, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA) and ovalbumin. In some embodiments, the suitable carrier is a solution, such as saline. Examples of excipients that can be incorporated into tablets, capsules, etc. are as follows: binders, such as gum tragacanth, gum arabic, corn starch or gelatin; excipients, such as microcrystalline cellulose; disintegrants, such as corn starch, pregelatinized starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; flavorings, such as mint, wintergreen oil or cherry. When the dosage unit form is a capsule, in addition to the above-mentioned types of materials, it may also contain a liquid carrier, such as a fatty oil. Various other materials may exist as a coating, or otherwise change the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.The pharmaceutical compositions according to the invention are preferably suitable for human use.

[0297] Sterile compositions for injection can be prepared according to conventional pharmaceutical practices by dissolving or suspending the integrin a5b1 inhibitor of the present invention in an injection vehicle (such as water or natural vegetable oils, such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc.) or a synthetic fatty vehicle (such as ethyl oleate, etc.). Buffers, preservatives, antioxidants, etc. may also be added.

[0298] Compositions for topical application can also be prepared according to conventional pharmaceutical practice. "Topical application" as used herein refers to application to body surface (such as skin or mucous membrane) to treat the condition caused by microorganism or parasitic infection in a local manner. Examples of formulations suitable for topical application include but are not limited to creams, gels, ointments, lotions, foams, suspensions, sprays, aerosols, powder aerosols. Topicals can be epidermal, which means that they are directly applied to the skin. Topicals can also be inhalable, for example, applied to the mucous epithelium of the respiratory tract, or applied to tissue surfaces other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear. The pharmaceutical composition prepared for topical application preferably comprises at least one pharmaceutical excipient suitable for topical application, such as emulsifiers, diluents, wetting agents, preservatives, pH adjusting agents and / or water.

[0299] Example

[0300] The following examples describe some preferred modes for making and implementing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0301] Example 1: Integrin α5β1 inhibitor impairs hPASMC proliferation on fibronectin

[0302] This example demonstrates the in vitro effects of integrin α5β1 inhibitors on hPASMC in vitro. The RNA expression of human integrins expressed in pulmonary artery smooth muscle cells from PAH patients was measured using NanoString detection of integrin targets ( Figure 1 ).

[0303] Antibody integrin α5β1 inhibitors were evaluated to determine whether integrin α5β1 inhibitors (e.g., P1D6 antibody or M200 antibody) would impair proliferation. Cells were seeded at 3000 cells / well in 96-well plates coated with fibronectin. Integrin α5β1 inhibitor treatment was added the next day and proliferation was monitored for 5 days. Figure 2A ) and P1D6( Figure 2B )-treated hPASMCs expressing integrins showed reduced proliferation. Selective inhibition of a5b1 resulted in approximately a 70% reduction in hPASMC proliferation.

[0304] like Figure 3 As shown in , the proliferation of hPASMCs treated with P1D6 was reduced only on fibronectin-coated plates, indicating that the proliferation inhibition by blocking a5b1 is fibronectin-dependent.

[0305] A dose response experiment was performed for P1D6 to determine the cell specificity range of P1D6. PAH-PASMCs were treated with P1D6 at doses of 10 μg / ml, 20 μg / ml, and 40 μg / ml on fibronectin-coated plates. The medium was changed every 48 hours and efficacy markers were assessed at 48, 72, and 120 hours. Figures 4A-4C Figure 2 shows the expression of p-FAK in PAH-PASMCs after exposure to P1D6 for 72 hours on fibronectin-coated plates. Figure 4B ) and PCNA( Figure 4C ) are shown in Figure 2.

[0306] Proliferation and apoptosis of integrin α5β1 inhibitors (MRT, SMi, P1D6 or M200) were assessed on Fb-coated plates. Figures 5A-5C Exemplary proliferation (Ki67) and apoptosis (Annexin V) of PAH-PASMCs treated with integrin α5β1 inhibitors (MRT, SMi, P1D6, or M200) are shown.

[0307] PAH-PASMCs were treated with 0.25 μM, 1 μM, and 4 μM of the integrin α5β1 inhibitor SMi on Fb-coated plates. Cells were evaluated by Western blot analysis to determine the expression levels of ITGα5, ITGαV, ITGβ1, p-FAK, MCM2, PLK1, p-ERK, ERK, PCNA, and survivin in hPASMCs ( Figure 6A-6B ).like Figures 7A-7C As shown in , SMi treatment impaired proliferation and increased apoptosis in a dose-dependent manner.

[0308] Primary rat cardiomyocytes were treated with 50 μM, 100 μM or 200 μM phenylephrine (PE). The expression of ITGα5, ITGαV, ITGβ1, ITGβ3, and ITGβ5 was determined by Western blotting ( Fig.21A ). Cells treated with 100 μM PE were simultaneously exposed to 0.25 μM, 1 μM, and 4 μM of the integrin α5β1 inhibitor SMi. Fig.21B and Fig. 21C As shown in , inhibition of α5β1 prevents PE-induced hypertrophy in adult rat cardiomyocytes.

[0309] The expression of fibronectin-binding integrins was evaluated in PAH patient samples. Figures 20A-20D As shown in , the expression of certain integrins was significantly altered in dissected PA, isolated PASMC, isolated PAEC, and decompensated RV from PAH patients compared with controls. Human PAH-RVFb were treated with 0.25 μM, 1 μM, and 4 μM of the integrin α5β1 inhibitor SMi. Pharmacological inhibition with SMi demonstrated that integrin α5β1 reduced the proliferation and activation of PAH-RVFb in a dose-dependent manner. ( Fig.22A and Fig. 22B ).

[0310] Example 2: In vivo efficacy of integrin α5β1 inhibitor in the Sugen hypoxia animal model

[0311] This example demonstrates the efficacy of integrin α5β1 inhibitors alone and in combination with standard of care (SoC) therapy in the Sugen / hypoxia (SuHx)-induced PAH rat model. Broad-spectrum pharmacological inhibition of α5β1 improves hemodynamics and vascular remodeling in Su / Hx rats with established PAH, alone or in combination with standard of care.

[0312] Animals were injected with 20 mg / kg Sugen 5416 on day 0 and subjected to hypoxic conditions (10% oxygen) for 3 weeks. Echocardiograms were performed at week 3 and week 5 of integrin α5β1 inhibitor treatment. Right heart catheterization was performed at the end of week 5. Tissues were collected for analysis.

[0313] like Figure 8 As shown in, SMi demonstrated vascular remodeling in Sugen / hypoxia (SuHx)-induced PAH rats by exemplary histological staining of EVG, αSMA, PCNA, and C3C. Treatment with SMi and / or vasodilators SoC (e.g., macitentan (Maci) and tadalafil (Tada)) improved cardiac output ( Fig.9A ), stroke volume ( Fig. 9B ), the thickness of the cell wall of the culture medium ( Fig. 9C ), vascular remodeling ( Fig.9D ),proliferation( Fig.9E ) and apoptosis ( Fig.9F ). Dashed lines indicate disease windows.

[0314] like Figures 10A-10C As shown in , treatment with SMi and / or SoC (eg, macitentan (Maci) and tadalafil (Tada)) reduced hypertrophy and fibrosis in SuHx rats.

[0315] Integrin α5β1 inhibition improves cardiac function in SuHx animals

[0316] To determine the selectivity of SMi for α5β1, SuHx animals were prepared as described above and treated with a dual α5β1 / αvβ1 inhibitor (MRT). The animals were divided into the following groups:

[0317] 1) Non-SuHx media PO BID (n=5)

[0318] 2) SuHx+vehicle PO BID (PBS) (n=10)

[0319] 3)SuHx+MRT 60mpk PO BID(n=10)

[0320] 4) SuHx + macitentan 1 mg / Kg + tadalafil 10 mg / Kg, daily (n=10)

[0321] 5) SuHx + MRT 60 mpk PO BID + macitentan 1 mg / Kg + tadalafil 10 mg / Kg, daily (n=10)

[0322] MRT-treated SuHx rats were evaluated for improved vascular remodeling in cardiac function, as demonstrated by EVG staining, cultured cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular fractional area change.

[0323] Integrin α5β1 antibody inhibitor in the Sugen / hypoxia (SuHx) mouse model of pulmonary arterial hypertension (PAH)

[0324] SuHx-treated mice were prepared as described above and treated with an integrin α5β1 antibody inhibitor (e.g., anti-mouse integrin α5β1 clone 339.1) or SMi anti-mouse integrin α5β1 clone 339.1 (described in Bhaskar, V., Zhang, D., Fox, M. et al. A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo. J Transl Med 5, 61 (2007). doi.org / 10.1186 / 1479-5876-5-61 ), which is incorporated herein by reference.

[0325] Mice were injected with 20 mg / kg Sugen 5416 (vascular endothelial growth factor receptor inhibitor) on days 0, 7, and 14 and subjected to hypoxic conditions (10% oxygen) for 3 weeks. Echocardiography was performed at the end of the 3rd week (e.g., day 21) and the 5th week of treatment with the integrin α5β1 inhibitor. Right heart catheterization was performed at the end of the 5th week. Tissues were harvested for analysis, such as Fig.24A as outlined in .

[0326] Cardiac function in SuHx rats treated with anti-integrin α5β1 antibody was evaluated for improved vascular remodeling as demonstrated by EVG staining, endocardial cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular fractional area change. Cardiac function was measured, including improved cardiac output and stroke volume. Other characteristics, including endocardial cell wall thickness, vascular remodeling, proliferation, and apoptosis, were also measured.

[0327] Divide the mice into the following treatment and control groups:

[0328] Control group (n=5)

[0329] · Vehicle PO BID (n=10)

[0330] ·SMi 100mpk PO BID (n=10)

[0331] · Macitentan 15 mpk PO BID (n=15)

[0332] IgG isotype control 10 mpk IP (3x weeks) (n=15)

[0333] 339.1 10mpk IP (3x weeks) (n=15)

[0334] 339.1 10 mpk IP + macitentan 15 mpk PO BID (n=15)

[0335] like Figures 24B-24G As shown in , an anti-α5β1 mouse antibody (antibody 339.1) phenocopies the cardiac and vascular improvements of SMi. These results confirm that α5β1 plays a key role in PAH and that integrin α5β1 inhibitors can be used to treat PAH.

[0336] The potent effects of a5b1 inhibitor in the right ventricle of SuHx animals were demonstrated by a reduction in RVSP and TAPSE, an improvement in cardiomyocyte hypertrophy, and a reduction in cardiac fibrosis. Treatment with a5b1 inhibitor resulted in a significant increase in cardiac output, indicating a functional improvement in the right heart.

[0337] Example 3: In vivo efficacy of integrin α5β1 inhibitors in MCT studies

[0338] This example demonstrates the efficacy of Compound SMi treatment in the monocrotaline (MCT) rat model.

[0339] The MCT animals were divided into the following study groups:

[0340] (1) Non-MCT vehicle, oral twice daily (BID) (n=6)

[0341] (2) MCT + PBS vehicle, oral administration twice daily (BID) (n = 8)

[0342] (3) MCT + SMi, 100 mpk orally, BID (n = 8)

[0343] MCT (60 mg / kg) was administered by subcutaneous injection. Animals were exposed to vehicle or SMi treatment for 2 weeks. Blood, RV, and lung analyses were performed. Cells were evaluated for expression of EVG, αSMA, PCNA, and C3C. Fig.11 As shown in , animals treated with SMi showed improved vascular remodeling in the monocrotaline (MCT) rat model of pulmonary hypertension. Figures 12A-12D Exemplary results of treatment with SMi in MCT rats are shown. Occlusion scores ( Fig. 12A ), vascular remodeling ( Fig. 12B ), apoptosis ( Fig. 12C ) and proliferation ( Fig.12D).

[0344] To test the effect of SMi on cardiac function, MCT animals with heart defects were treated with SMi or sildenafil as follows: (1) non-MCT vehicle PO BID; (2) MCT + vehicle PO BID; (3) MCT + SMi 100 mg / Kg PO BID; (4) MCT + sildenafil 10 mg / Kg PO BID; (5) MCT + sildenafil 30 mg / Kg PO BID; (6) MCT + sildenafil 100 mg / Kg PO BID. Pulmonary hypertension phenotype was not induced in this study (MCT causes vascular and cardiac damage). From day 14 to day 27, SMi, vehicle or sildenafil treatment was administered twice a day. All animals were monitored for disease progression by echocardiography on day 0, day 14 and the day of surgery (day 28). Blood samples were collected on day 0, day 14 and day 28.

[0345] Determine the maximum velocity of the pulmonary artery, cardiac output, stroke volume, right ventricular anterior wall thickness and pulmonary artery acceleration time. Use an echocardiograph (Vivid E9 model with XDclear Ultrasound, GE Healthcare, Illinois, United States) connected to a 13.0MHz linear transducer to measure the maximum velocity of the pulmonary artery (Vmax), arterial pulmonary velocity time integral (VTI), pulmonary artery diameter, heart rate, right ventricular anterior wall thickness and pulmonary artery acceleration time. Data are used to calculate cardiac output and stroke volume as follows:

[0346] Cardiac output was calculated using the following formula (JF Lewis et al., 1984):

[0347] Cardiac output = heart rate × VTI ((pulmonary artery diameter 2 ×3.1416) / 4)

[0348] Calculate stroke volume using the following formula:

[0349] Stroke volume = cardiac output / heart rate

[0350] The significant increase in cardiac output observed with SMi in this MCT model suggests that the cardiac benefits are dissociated from effects on pulmonary resistance. Treatment with SMi leads to direct cardiac improvements via a5b1 inhibition that are independent of its reverse remodeling effects on pulmonary arterioles.

[0351] Combination therapy of integrin α5β1 inhibitor and SoC in MCT animals

[0352] MCT animals were treated with SMi alone or in combination with SoC therapy (Macitentan (1 mg / kg) + Tadalafil (10 mg / kg), daily). MCT animals were divided into the following study groups:

[0353] (1) Non-MCT vehicle, oral twice daily (BID) (n=6)

[0354] (2) MCT + PBS vehicle, oral administration twice daily (BID) (n = 8)

[0355] (3) MCT + SMi, 100 mpk orally, BID (n = 8)

[0356] (4) MCT+SMi, 100 mpk orally, BID+SoC (macitentan (1 mg / kg)+tadalafil (10 mg / kg), daily) (n=8).

[0357] MCT (60 mg / kg) was administered by subcutaneous injection on day 0. Starting from week 3, animals were exposed to vehicle or SMi treatment for 2 weeks. Fig.23A ) were analyzed in blood, RV, and lung. Exemplary histological and tissue analyses demonstrated that the use of SMi alone or in combination with SoC improved hypertrophy and right ventricular (RV) fibrosis ( Figures 15A-15C Right heart catheterization was performed and the animals were evaluated for RVSP (mmHg), mPAP (mmHg), and CO (ml / min) ( Fig. 23B ).like Fig.23C As shown in , MCT PH animals treated with SMi alone or in combination with macitentan (1 mg / kg) + tadalafil (10 mg / kg) showed improved vascular remodeling.

[0358] Integrin α5β1 inhibition improves cardiac function in MCT animals

[0359] To determine the selectivity of SMi for α5β1, MCT animals were prepared as described above and treated with SMi or a dual inhibitor of α5β1 / αvβ1 (MRT).

[0360] like Figures 14A-14E As shown in Figure 2, both SMi- and MRT-treated MCT rats showed improved cardiac function and vascular remodeling as evidenced by EVG staining ( Fig.14A ), the thickness of the cell wall of the culture medium ( Fig. 14B ), mean pulmonary artery pressure ( Fig. 14C ), cardiac output ( Fig.14D ) and right ventricular area fraction change ( Fig.14E ) to confirm. Fig.13Exemplary pharmacokinetics of SMi and MRT are shown. These data suggest that integrin α5β1 inhibition is responsible for the improved cardiac function in MCT animals.

[0361] Measuring the protein levels of a5b1 integrin in human and mouse tissues confirmed that a5b1 is highly expressed in human and mouse hearts ( Fig.25A and Fig.25B ). In cultured cardiomyocytes stimulated by phenylephrine, an alpha-1 adrenergic receptor agonist, blocking A5B1 attenuated cardiomyocyte hypertrophy. Furthermore, in patient-derived cardiac fibroblasts, A5B1 blockade reduced fibroblast activation. These experiments in an in vitro system further confirmed a direct pharmacological effect of A5B1 inhibition in cardiac cells.

[0362] Integrin α5β1 antibody inhibitor in MCT animals

[0363] MCT animals were prepared as described above and treated with integrin α5β1 antibody inhibitor. Anti-integrin α5β1 antibody treated MCT rats were evaluated for improvement in cardiac function vascular remodeling as confirmed by EVG staining, culture cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular area fractional change. Cardiac function parameters including improved cardiac output, stroke volume, culture cell wall thickness, vascular remodeling, proliferation, and apoptosis were measured.

[0364] Example 4: In vivo efficacy of integrin α5β1 inhibitors in PAB

[0365] This example demonstrates the efficacy of compound SMi in the right ventricle of a pulmonary artery ligation (PAB) rat model. The animals were divided into three study groups as follows:

[0366] (1) Control group / sham operation group (n=5)

[0367] (2) PAB + PBS vehicle, oral administration twice daily (BID) (n = 10)

[0368] (3) PAB + SMi, 100 mpk orally, BID (n = 10)

[0369] Rats underwent pulmonary artery constriction surgery on day 0. Starting at week 4 post-surgery, animals were exposed to vehicle or SMi treatment. Echocardiograms were performed weekly from the start of surgery to week 10. After a total of 10 weeks, animals underwent RV catheterization. Blood, RV, and lung analyses were performed. Exemplary histological and tissue analyses demonstrated improvements in hypertrophy and right ventricular (RV) fibrosis in the pulmonary artery ligation (PAB) rat model treated with SMi ( Figures 16A-16D ). Fig.17Representative echocardiographic images of (PAB) rats treated with SMi are shown in .

[0370] Hemodynamic and tissue collection analyses revealed that inhibition of integrin α5β1 by SMi directly improved cardiac function over time compared with vehicle controls in the RV PAB model. Fig.18A ), stroke volume ( Fig.18B ), tricuspid annular plane systolic excursion (TAPSE) ( Fig. 18C )、S wave( Fig.18D ), right ventricular fractional area change (RVFAC) ( Fig.18E )、RVEDD( Fig.18F )、cardiac index (CI) ( Figure 18G ), pressure gradient (PG) ( Fig.18H ).

[0371] Right heart catheterization

[0372] PAB rats underwent right heart catheterization (RHC) using standard techniques with subjects in a stable state. Figures 19A-19E Exemplary cardiac parameters from right heart catheterization of PAB rats treated with SMi are shown in. Treatment with the integrin α5β1 inhibitor SMi improved cardiac output, stroke volume, RVSP, and RVEPD.

[0373] Integrin α5β1 antibody inhibitor in PAB animals

[0374] PAB rats were prepared as described above and treated with integrin α5β1 antibody inhibitor. PAB rats treated with anti-integrin α5β1 antibody were evaluated for improvement in cardiac function vascular remodeling as confirmed by EVG staining, culture cell wall thickness, mean pulmonary artery pressure, cardiac output, and right ventricular fractional area change. Cardiac function parameters including improved cardiac output, stroke volume, culture cell wall thickness, vascular remodeling, proliferation, and apoptosis were measured.

[0375] Example 5: Characterization of Integrin α5β1 Inhibitors

[0376] This example illustrates methods for screening and characterizing integrin α5β1 inhibitors.

[0377] Cell Adhesion Assay (CAA)

[0378] 96-well assay plates were coated with 0.625 μg / ml purified recombinant human fibronectin, which consisted of domain 9 and domain 10, fused with glutathione S-transferase, and subsequently blocked with 1% bovine serum albumin. In 96-well deep-well plates, cells expressing rat α5β1 were incubated with test samples prepared in a serial dilution series. 0.1 ml assay mixture consisted of 100,000 cells, antibody samples, 50 mM HEPES pH 7.3, 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM calcium chloride, 1% bovine serum albumin, and 10 mM glucose. After 15 minutes of pre-incubation time, 0.1 ml of assay mixture was transferred to a 96-well assay plate and the plate was incubated at room temperature for 1 hour. Non-adherent cells were removed using a BlueWasher (Blue Cat Bio) instrument, and the amount of adherent cells was quantified using CellTiter Glo (Promega). Concentration-response curves were analyzed for IC50 values ​​using 4-parameter nonlinear regression analysis.

[0379] As shown in Table 3, a5b1 antibody inhibitors (eg, P1D6 and M200) exhibited nanomolar range affinities in blocking cell adhesion.

[0380] Table 3:

[0381]

[0382]

[0383] Solid phase (SP) assay

[0384] By the binding competition with the purified fibronectin separated from rat plasma, the antibody activity is measured using solid phase (SP) assay. 384-well assay plates are coated with 2 μg / ml fibronectin and subsequently blocked with 1% bovine serum albumin. In a separate 384-well plate, the rat α5β1 protein labeled with 2nM His is incubated together with the test samples in 50mM HEPES pH 7.3, 150mM sodium chloride, 0.5% bovine serum albumin, 1mM magnesium chloride, 1mM calcium chloride and 0.05% Tween 20 at room temperature for 1 hour. The assay mixture of 20 μl is transferred to the analysis plate coated with fibronectin and incubated at room temperature for 1 hour. Unbound components are removed by 3 rounds of washing using BioTek 405 / TS plate washer. 20 μl of anti-6XHistag antibody conjugated with horseradish peroxide is added, incubated at room temperature for 1 hour and unbound antibodies are removed by 3 rounds of washing. The amount of His-tagged rat α5β1 protein bound to the anti-6X Histag antibody was quantified using Quantablue fluorescent substrate (Thermo Fisher) and the IC50 values ​​of the concentration-response curves were analyzed using 4-parameter nonlinear regression analysis.

[0385] Ligand Binding Assay (LBA)

[0386] To measure the potency of samples against α5β1 in a cell-based ligand binding assay (LBA), cells expressing rat α5β1 were incubated with a volume of 10 μl of the test sample at room temperature in a buffer containing 50 mM HEPES pH 7.3, 150 mM sodium chloride, 1% bovine serum albumin, 2 mM magnesium chloride, 2 mM calcium chloride, 15 mM glucose, 1.5% dimethyl sulfoxide and 0.025% e780 fixable active dye for 15 minutes. 5 μl of 75 nM fibronectin separated from human plasma and fluorescently labeled with Dylight 650 in 50 mM HEPES pH 7.3, 150 mM sodium chloride and 1% bovine serum albumin was added to the cells. The samples were incubated at room temperature for 45 minutes, fixed with 0.8% formaldehyde for 30 minutes at room temperature, and washed with 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA and 1% bovine serum albumin. The fluorescence intensity of each cell was measured by flow cytometry. Dead cells were excluded from further analysis based on staining with 780 fixable vital dyes. The median fluorescence intensity of Dylight 650 was determined for each sample, and the concentration-response curves for IC50 values ​​were analyzed using 4-parameter nonlinear regression analysis.

[0387] Table 4: Ligand binding of integrin α5β1 antibody inhibitors

[0388]

[0389]

[0390] List of implementation plans

[0391] Embodiment 1: A method of treating pulmonary arterial hypertension (PAH) in a subject comprising administering an integrin α5β1 inhibitor.

[0392] Embodiment 2: A method for treating a disease associated with increased expression or activity of integrin α5β1, comprising administering an integrin α5β1 inhibitor.

[0393] Embodiment 3: A method of treating a heart or lung disease comprising administering an integrin α5β1 inhibitor.

[0394] Embodiment 4: A method as described in embodiments 1 to 3, wherein the disease is pulmonary hypertension WHO group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO group 2 pulmonary hypertension, WHO group 3 pulmonary hypertension, WHO group 4 pulmonary hypertension, WHO group 5 pulmonary hypertension, WHO class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO class II pulmonary hypertension, WHO class III pulmonary hypertension, WHO class IV pulmonary hypertension.

[0395] Embodiment 5: The method of embodiments 1 to 3, the method of claim 3, wherein the disease is cardiac fibrosis, heart failure or right ventricular failure.

[0396] Embodiment 6: The method of embodiment 5, wherein the disease is cardiac fibrosis.

[0397] Embodiment 7: The method of embodiment 5, wherein the disease is heart failure.

[0398] Embodiment 8: The method of embodiment 5, wherein the disease is right ventricular failure.

[0399] Embodiment 9: The method of Embodiment 3, wherein the disease is heart disease.

[0400] Embodiment 10: The method of embodiment 3, wherein the disease is a lung disease.

[0401] Embodiment 11: A method as described in any of the preceding embodiments, wherein the integrin α5β1 inhibitor is Fab, single-chain Fv (scFv), single domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single domain antibody-linked Fab (s-Fab), antibody or a combination thereof.

[0402] Embodiment 12: The method of any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is an antibody.

[0403] Embodiment 13: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5.

[0404] Embodiment 14: The method of any one of Embodiments 1 to 12, wherein the integrin α5β1 inhibitor is an antibody that specifically binds to integrin β1.

[0405] Embodiment 15: The method according to any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is an antibody that specifically binds to integrin α5β1.

[0406] Embodiment 16: The method of embodiment 15, wherein the antibody is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A.

[0407] Embodiment 17: The method of Embodiment 16, wherein the antibody is volociximab (M200).

[0408] Embodiment 18: The method of embodiment 16, wherein the antibody is PF-04605412.

[0409] Embodiment 19: A method as described in Embodiment 16, wherein the antibody is MINT1526A.

[0410] Embodiment 20: A method as described in embodiment 11, wherein the integrin α5β1 inhibitor is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from the group consisting of: volociximab (M200), P1D6, PF-04605412, MINT1526A, BMA5, BMB5, BMC5, HA5, JBS5, LS-C509074, LS-C24758, 1D9, 22B5, 24C7, 2D2, 3C2.2A8, 3C5, 5B11, MOR04055, MOR04624, P8D4, MOR04974, MOR04977, SG / 19 and 18C12.

[0411] Embodiment 21: The method of any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

[0412] Embodiment 22: The method of any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5.

[0413] Embodiment 23: The method of any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin β1.

[0414] Embodiment 24: The method of any one of Embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a small molecule compound that specifically binds to integrin α5β1.

[0415] Embodiment 25: The method according to any one of embodiments 1 to 10, wherein the integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0416]

[0417] in:

[0418] R 1 is hydrogen or OMe;

[0419] R 2 is optionally C 1-4 Alkoxy substituted C 1-4 Alkyl; and

[0420] R 3 It is C 1-4 Alkyl or C 3-5 Cycloalkyl.

[0421] Embodiment 26: A method as described in Embodiment 25, wherein R2 is methyl or ethyl.

[0422] Embodiment 27: A method as described in Embodiment 25 or 26, wherein R3 is methyl, ethyl, isopropyl or cyclopropyl.

[0423] Embodiment 28: A method as described in any one of Embodiments 25 to 27, wherein R1 is hydrogen.

[0424] Embodiment 29: A method as described in Embodiment 25, wherein the compound of formula (I) is selected from:

[0425]

[0426] as well as

[0427]

[0428] Embodiment 30: The method of any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intrapulmonary.

[0429] Embodiment 31: The method of any of the preceding embodiments, further comprising administering a second therapy to the subject.

[0430] Embodiment 32: A method as described in any of the preceding embodiments, wherein the second therapy is selected from the group consisting of: anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostaglandins, prostaglandin receptor agonists, soluble guanylate cyclase stimulators and / or surgery.

[0431] Embodiment 33: The method of embodiment 32, wherein the second therapy is a prostaglandin.

[0432] Embodiment 34: The method of embodiment 33, wherein the prostaglandin is epoprostenol or treprostinil.

[0433] Embodiment 35: The method of embodiment 32, wherein the second therapy is an endothelin receptor antagonist.

[0434] Embodiment 36: The method of embodiment 35, wherein the endothelin receptor is bosentan, ambrisentan, or macitentan.

[0435] Embodiment 37: The method of embodiment 32, wherein the second therapy is a phosphodiesterase type 5 (PDE-5) inhibitor.

[0436] Embodiment 38: The method of embodiment 37, wherein the phosphodiesterase type 5 (PDE-5) inhibitor is sildenafil or tadalafil.

[0437] Embodiment 39: The method of embodiment 32, wherein the second therapy is a soluble guanylate cyclase (sGC) stimulator.

[0438] Embodiment 40: A method as described in Embodiment 39, wherein the soluble guanylate cyclase (sGC) stimulator is riociguat.

[0439] Embodiment 41: The method of embodiment 31 or 32, wherein the second therapy is oxygen, warfarin, furosemide, bumetanide, bendrofluazide, metolazone, spironolactone, amiloride, digoxin, nifedipine, diltiazem, nicardipine, amlodipine, ambrisentan, bosentan, macitentan, sildenafil, tadalafil, epoprostenol, iloprost, treprostinil, riociguat, selexipag, surgery, pulmonary endarterectomy and / or atrial septostomy.

[0440] Embodiment 42: The method of embodiment 31, wherein the second therapy is macitentan and / or tadalafil.

[0441] Embodiment 43: The method of any of the preceding Embodiments, wherein the subject has previously received therapy for pulmonary hypertension.

[0442] Embodiment 44: A method as described in Embodiment 43, wherein the previously received pulmonary hypertension therapy is selected from the group consisting of: anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostaglandins, prostaglandin receptor agonists, soluble guanylate cyclase stimulators and / or surgery.

[0443] Embodiment 45: A method as described in any of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor reduces the proliferation and / or survival of PASMCs, fibroblasts, right ventricular fibroblasts (RVFb), vascular fibroblasts, epicardial fibroblasts, cardiomyocytes and / or endothelial cells.

[0444] Embodiment 46: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in an improvement in mean pulmonary arterial pressure (mPAP).

[0445] Embodiment 47: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in improvement in pulmonary vascular resistance (PVR).

[0446] Embodiment 48: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in improved systemic vascular resistance (SVR).

[0447] Embodiment 49: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in improvement in right atrial pressure (RAP).

[0448] Embodiment 50: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in improved cardiac output (CO).

[0449] Embodiment 51: The method of any one of the preceding embodiments, wherein administration of the integrin α5β1 inhibitor results in improved heart rate (HR).

[0450] Embodiment 52: The method of any one of the preceding embodiments, wherein the α5β1 inhibitor is administered to improve motor ability and delay clinical worsening of the disease.

[0451] Embodiment 53: The method of any of the preceding embodiments, wherein the α5β1 inhibitor and the second therapy are administered to improve motor ability and delay clinical worsening of the disease.

[0452] Embodiment 54: A method as described in any of the preceding embodiments, comprising administering the integrin α5β1 inhibitor to modulate the level of a biomarker in the subject, wherein the biomarker is brain natriuretic peptide (BNP) or the N-terminal fragment (NT) of BNP precursor (NT-proBNP), TNFα, IFNγ, IL-6, IL-8 or IL-10.

[0453] Embodiment 55: The method of any of the preceding embodiments, further comprising administering the integrin α5β1 inhibitor at a dose of 1 mg to 1000 mg.

[0454] Embodiment 56: The method of any one of the preceding embodiments, wherein the integrin α5β1 inhibitor is administered daily.

[0455] Embodiment 57: An integrin α5β1 inhibitor for use in treating pulmonary arterial hypertension (PAH) in a subject in need of such treatment, comprising administering to the subject an integrin α5β1 inhibitor and a pharmaceutical excipient.

[0456] Equivalents and scope

[0457] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the following appended claims.

Claims

1. A method of treating pulmonary arterial hypertension (PAH) in a subject comprising administering an integrin α5β1 inhibitor.

2. A method of treating a cardiac or pulmonary disease in a subject comprising administering an integrin α5β1 inhibitor.

3. The method of claim 2, wherein the disease is pulmonary hypertension WHO group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO group 2 pulmonary hypertension, WHO group 3 pulmonary hypertension, WHO group 4 pulmonary hypertension, WHO group 5 pulmonary hypertension, WHO class I pulmonary hypertension or pulmonary arterial hypertension (PAH), WHO class II pulmonary hypertension, WHO class III pulmonary hypertension, WHO class IV pulmonary hypertension.

4. The method of claim 2, wherein the disease is cardiac fibrosis, heart failure or right ventricular failure.

5. The method of any of the preceding claims, wherein the integrin α5β1 inhibitor is a Fab, a single-chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibody (bsFab), a single domain antibody-linked Fab (s-Fab), an antibody, or a combination thereof.

6. The method of claim 5, wherein the integrin α5β1 inhibitor is an integrin α5β1 antibody selected from the group consisting of volociximab (M200), PF-04605412, and MINT1526A.

7. The method of claim 5, wherein the integrin α5β1 inhibitor is an integrin α5β1 antibody that competes for integrin binding with an antibody selected from the group consisting of volociximab (M200), P1D6, PF-04605412, MINT1526A, BMA5, BMB5, BMC5, HA5, JBS5, LS-C509074, LS-C24758, 1D9, 22B5, 24C7, 2D2, 3C2.2A8, 3C5, 5B11, MOR04055, MOR04624, P8D4, MOR04974, MOR04977, SG / 19 and 18C12.

8. The method of any one of claims 1 to 4, wherein the integrin α5β1 inhibitor is a small molecule compound that binds to integrin α5β1.

9. The method of claim 8, wherein the integrin α5β1 inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof, in: R 1 is hydrogen or OMe; R 2 is optionally C 1-4 Alkoxy substituted C 1-4 Alkyl; and R 3 It is C 1-4 Alkyl or C 3-5 Cycloalkyl.

10. The method of claim 9, wherein R2 is methyl or ethyl.

11. The method of claim 9 or 10, wherein R3 is methyl, ethyl, isopropyl or cyclopropyl.

12. The method of claim 9, wherein the compound of formula (I) is selected from: as well as 13. The method of any one of the preceding claims, wherein the integrin α5β1 inhibitor is administered orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, or intrapulmonary.

14. The method of any one of claims 1 to 13, further comprising administering to the subject a second therapy.

15. The method of claim 14, wherein the second therapy is selected from the group consisting of anticoagulants, diuretics, digitalis glycosides, calcium channel blockers, endothelin receptor antagonists, phosphodiesterase 5 (PDE5) inhibitors, prostaglandins, prostaglandin receptor agonists, soluble guanylate cyclase stimulators and / or surgery.

16. The method of claim 15, wherein the second therapy is oxygen, warfarin, furosemide, bumetanide, bendrofluazide, metolazone, spironolactone, amiloride, digoxin, nifedipine, diltiazem, nicardipine, amlodipine, ambrisentan, bosentan, macitentan, sildenafil, tadalafil, epoprostenol, iloprostcycline, treprostinil, riociguat, selexipag, surgery, pulmonary endarterectomy, and / or atrial septostomy.

17. The method of any of the preceding claims, wherein administration of the integrin α5β1 inhibitor reduces proliferation and / or survival of PASMCs, fibroblasts, right ventricular fibroblasts (RVFb), vascular fibroblasts, adventitial fibroblasts, cardiomyocytes, and / or endothelial cells.

18. The method of any of the preceding claims, comprising administering the integrin α5β1 inhibitor to modulate the level of a biomarker in the subject, wherein the biomarker is brain natriuretic peptide (BNP) or the N-terminal fragment (NT) of the BNP precursor (NT-proBNP), TNFα, IFNγ, IL-6, IL-8, or IL-10.

19. The method of any one of the preceding claims, comprising administering the integrin α5β1 inhibitor at a dose of 1 mg to 1000 mg.

20. An integrin α5β1 inhibitor for use in treating pulmonary arterial hypertension (PAH) in a subject in need of such treatment, comprising administering to the subject an integrin α5β1 inhibitor and a pharmaceutical excipient.

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